ModernaTX, Inc. et al v. Pfizer Inc. et al
Filing
1
COMPLAINT against BioNTech US Inc., BioNTech SE, Pfizer Inc., BioNTech Manufacturing GmbH Filing fee: $ 402, receipt number AMADC-9469742 (Fee Status: Filing Fee paid), filed by Moderna US, Inc., ModernaTX, Inc.. (Attachments: #1 Exhibit 1, #2 Exhibit 2, #3 Exhibit 3, #4 Exhibit 4, #5 Exhibit 5, #6 Exhibit 6, #7 Exhibit 7, #8 Exhibit 8, #9 Exhibit 9, #10 Civil Cover Sheet, #11 Category Form)(Lee, William)
EXHIBIT 2
US010702600B1
(12) United States Patent
( 10 ) Patent No.: US 10,702,600 B1
(45 ) Date of Patent:
Jul. 7 , 2020
Ciaramella et al.
( 54 ) BETACORONAVIRUS MRNA VACCINE
(71) Applicant: Moderna TX , Inc. , Cambridge, MA
(58) Field of Classification Search
None
See application file for complete search history .
(72 ) Inventors: Giuseppe Ciaramella , Sudbury , MA
(US ); Sunny Himansu , Winchester,
MA (US)
( 73 ) Assignee : ModernaTX , Inc., Cambridge, MA
(56 )
(US)
U.S. PATENT DOCUMENTS
5,169,628 A
5,427,782 A
6,225,091 B1
6,500,419 B1
Subject to any disclaimer , the term of this
patent is extended or adjusted under 35
Filed :
Feb. 28 , 2020
Related U.S. Application Data
(63) Continuation of application No. 16 /368,270, filed on
Mar. 28 , 2019, which is a continuation of application
No. 16 /040,981, filed on Jul. 20 , 2018 , now Pat. No.
10,272,150, which is a continuation of application
No. 15 /674,599, filed on Aug. 11, 2017 , now Pat. No.
10,064,934 , which is a continuation of application
No. PCT /US2016 /058327 , filed on Oct. 21 , 2016 .
(60 ) Provisional application No. 62 /247,362, filed on Oct.
28 , 2015 , provisional application No. 62/247,394 ,
filed on Oct. 28 , 2015 , provisional application No.
62/ 247,483 , filed on Oct. 28, 2015 , provisional
application No. 62 /247,297 , filed on Oct. 28 , 2015 ,
provisional application No. 62 /244,802, filed on Oct.
22, 2015 , provisional application No. 62/244,946 ,
filed on Oct. 22 , 2015 , provisional application No.
62/ 244,813 , filed on Oct. 22, 2015 , provisional
application No. 62 /244,837 , filed on Oct. 22, 2015 ,
provisional application No. 62 /245,031, filed on Oct.
22, 2015 .
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FOREIGN PATENT DOCUMENTS
2473135
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(Continued )
Int. Cl.
A61P
A61K
A61K
A61K
11/00
39/12
39/215
39/155
COZK 16/10
A61K 39/00
(52 ) U.S. CI.
CPC
(2006.01 )
( 2006.01 )
( 2006.01)
(2006.01)
( 2006.01)
(2006.01 )
A61K 39/155 (2013.01); A61K 39/12
(2013.01); A61K 39/215 (2013.01); A61P
11/00 (2018.01); C07K 16/10 (2013.01) ; CO7K
16/1027 ( 2013.01 ); A61K 2039/53 (2013.01 );
A61K 2039/55511 (2013.01) ; A61K
2039/55555 (2013.01) ; A61K 2039/6018
(2013.01); A61K 2039/70 (2013.01) ; CO7K
2317/76 (2013.01); C12N 2760/18034
( 2013.01 ); C12N 2760/18334 (2013.01 ); C12N
2760/18434 (2013.01); C12N 2760/18534
( 2013.01) ; CI2N 2760/18634 ( 2013.01 ); C12N
2770/20034 (2013.01); YO2A 50/381
(2018.01) ; YO2A 50/39 (2018.01)
OTHER PUBLICATIONS
U.S. Appl. No. 16 /036,318 , filed Jul. 16 , 2018 , Ciaramella et al.
(Continued )
Primary Examiner Nicole Kinsey White
(74 ) Attorney, Agent, or Firm Wolf, Greenfield &
Sacks, P.C.
ABSTRACT
(57)
The disclosure relates to respiratory virus ribonucleic acid
(RNA ) vaccines and combination vaccines , as well as meth
ods of using the vaccines and compositions comprising the
vaccines .
26 Claims, 24 Drawing Sheets
Specification includes a Sequence Listing .
US 10,702,600 B1
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BETACORONAVIRUS MRNA VACCINE
respectively ), also like hMPV, are second only to RSV as
important causes of viral LRI in young children .
RSV , too , is a negative -sense, single- stranded RNA virus
of the genus Pneumovirinae and of the family Paramyxo
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. 5 viridae . Symptoms in adults typically resemble a sinus
No. 16 / 368,270 , filed Mar. 28 , 2019 , which is a continuation infection or the common cold , although the infection may be
of Ser. No. 16 /040,981 , filed Jul. 20 , 2018 ,now U.S. Pat. No. asymptomatic. In older adults (e.g., >60 years), RSV infec
10,272,150 , which is a continuation of U.S. application Ser. tion may progress to bronchiolitis or pneumonia . Symptoms
No. 15/674,599 , filed Aug. 11, 2017 , now U.S. Pat. No. in children are often more severe , including bronchiolitis
10,064,934, which is a continuation of International appli- 10 and pneumonia. It is estimated that in the United States,
cation number PCT/US2016 /058327 , filed Oct. 21, 2016 , most children are infected with RSV by the age of three . The
which claims the benefit under 35 U.S.C. $ 119 (e ) of U.S. RSV virion consists of an internal nucleocapsid comprised
provisional application No.62 /244,802 , filed Oct. 22 , 2015 ,
U.S. provisional application No. 62 /247,297, filed Oct. 28 , of the viral RNA bound to nucleoprotein (N ), phosphopro
2015, U.S. provisional application No. 62/244,946 , filed 15 teinsurrounded
(P ), and large
polymerase protein (L ). The nucleocapsid
by matrix protein (M ) and is encapsulated by
Oct. 22, 2015, U.S. provisional application No. 62 /247,362, ais lipid
bilayer into which the viral fusion (F ) and attachment
filed Oct. 28, 2015 , U.S. provisional application No.62/244 , (G ) proteins
as well as the small hydrophobic protein (SH )
813, filed Oct. 22 , 2015 , U.S. provisional application No. are
incorporated
. The viral genome also encodes two non
62 /247,394, filed Oct. 28 , 2015, U.S. provisional application
No. 62/244,837 , filed Oct. 22 , 2015, U.S. provisional appli- 20 structural proteins (NS1 and NS2), which inhibit type I
cation No. 62/247,483 , filed Oct. 28 , 2015 , and U.S. provi
sional application No. 62/245,031, filed Oct. 22 , 2015, each
ofwhich is incorporated by reference herein in its entirety .
interferon activity as well as the M -2 protein .
The continuing health problems associated with hMPV,
PIV3 and RSV are of concern internationally , reinforcing the
importance of developing effective and safe vaccine candi
BACKGROUND
25 dates against these virus.
Despite decades of research , no vaccines currently exist
Respiratory disease is a medical term that encompasses ( Sato and Wright, Pediatr. Infect. Dis. J. 2008 ; 27 ( 10 Suppl):
pathological conditions affecting the organs and tissues that S123-5 ). Recombinant technology , however, has been used
make gas exchange possible in higher organisms, and to target the formation of vaccines for hPIV -1 , 2 and 3
includes conditions of the upper respiratory tract, trachea , 30 serotypes, for example, and has taken the form of several
bronchi, bronchioles, alveoli , pleura and pleural cavity, and live -attenuated intranasal vaccines. Two vaccines in particu
the nerves and muscles of breathing . Respiratory diseases lar were found to be immunogenic and well tolerated against
range from mild and self-limiting, such as the common cold , hPIV - 3 in phase I trials. hPIV1 and hPIV2 vaccine candi
to life- threatening entities like bacterialpneumonia, pulmo dates remain less advanced (Durbin and Karron, Clinical
nary embolism , acute asthma and lung cancer. Respiratory 35 infectious diseases: an official publication of the Infectious
disease is a common and significant cause of illness and Diseases Society of America 2003 ; 37 ( 12 ): 1668-77) .
death around the world . In the US, approximately 1 billion
Measles virus (MeV ) , like hMPV , PIV3 and RSV, is a
“ common colds” occur each year. Respiratory conditions are negative -sense, single- stranded RNA virus that is the cause
among the most frequent reasons for hospital stays among ofmeasles, an infection of the respiratory system . MeV is of
children .
40 the genus Morbillivirus within the family Paramyxoviridae .
The human metapneumovirus (hMPV) is a negative Humans are the natural hosts of the virus; no animal
sense, single -stranded RNA virus of the genus Pneumoviri reservoirs are known to exist. Symptoms ofmeasles include
nae and of the family Paramyxoviridae and is closely related fever, cough , runny nose , red eyes and a generalized ,macu
to the avian metapneumovirus ( AMPV ) subgroup C. It was lopapular, erythematous rash . The virus is highly contagious
isolated for the first time in 2001 in the Netherlands by using 45 and is spread by coughing
the RAP- PCR ( RNA arbitrarily primed PCR ) technique for
In additional to hMPV, PIV , RSV and MeV, betacorona
identification of unknown viruses growing in cultured cells. viruses are known to cause respiratory illnesses. Betacoro
hPMV is second only to RSV as an important cause of viral naviruses (BetaCoVs ) are one of four genera of coronavi
lower respiratory tract illness (LRI) in young children . The ruses of the subfamily Coronavirinae in the family
seasonal epidemiology of hMPV appears to be similar to that 50 Coronaviridae , of the order Nidovirales. They are envel
of RSV, but the incidence of infection and illness appears to oped , positive -sense, single -stranded RNA viruses of
be substantially lower.
zoonotic origin . The coronavirus genera are each composed
Parainfluenza virus type 3 ( PIV3 ), like hMPV, is also a of varying viral lineages, with the betacoronavirus genus
negative -sense , single-stranded sense RNA virus of the containing four such lineages . The BetaCoVs of the greatest
genus Pneumovirinae and of the family Paramyxoviridae 55 clinical importance concerning humans are OC43 and
and is a major cause of ubiquitous acute respiratory infec
HKU1 of the A lineage , SARS- CoV of the B lineage, and
tions of infancy and early childhood . Its incidence peaks MERS -CoV of the C lineage. MERS-CoV is the first beta
around 4-12 months of age , and the virus is responsible for coronavirus belonging to lineage C that is known to infect
3-10 % of hospitalizations, mainly for bronchiolitis and humans.
pneumonia . PIV3 can be fatal, and in some instances is 60 The Middle East respiratory syndrome coronavirus
associated with neurologic diseases, such as febrile seizures . (MERS- CoV ), or EMC /2012 (HCOV-EMC/2012), initially
It can also result in airway remodeling, a significant cause of
morbidity . In developing regions of the world , infants and
young children are at the highest risk of mortality, either
from primary PIV3 viral infection or a secondary conse- 65
quences, such as bacterial infections . Human parainfluenza
viruses (hPIV ) types 1 , 2 and 3 (hPIV1, hPIV2 and hPIV3 ,
referred to as novel coronavirus 2012 or simply novel
coronavirus, was first reported in 2012 after genome
sequencing of a virus isolated from sputum samples from a
person who fell ill during a 2012 outbreak of a new flu . As
of July 2015 , MERS -CoV cases have been reported in over
21 countries . The outbreaks of MERS -CoV have raised
US 10,702,600 B1
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serious concerns world-wide, reinforcing the importance of
developing effective and safe vaccine candidates against
MERS -CoV .
Severe acute respiratory syndrome (SARS) emerged in
In some aspects the invention is a respiratory virus
vaccine, comprising at least one RNA polynucleotide having
an open reading frame encoding at least one respiratory
virus antigenic polypeptide, formulated in a cationic lipid
China in 2002 and spread to other countries before brought 5 nanoparticle
.
Surprisingly, in some aspects , it has also been shown that
under control. Because of a concern for reemergence or a
efficacy of mRNA vaccines can be significantly enhanced
deliberate release of the SARS coronavirus, vaccine devel when
combined with a flagellin adjuvant, in particular , when
opment was initiated.
one
or
more antigen -encoding mRNAs is combined with an
Deoxyribonucleic acid (DNA ) vaccination is one tech
encoding flagellin .
nique used to stimulate humoral and cellular immune 10 mRNA
RNA
(e.g., mRNA ) vaccines combined with the flagellin
responses to foreign antigens, such as hMPV antigens and /or
( e.g., mRNA -encoded flagellin adjuvant) have
PIV antigens and/or RSV antigens. The direct injection of adjuvant
superior
properties
in that they may produce much larger
genetically engineered DNA (e.g., naked plasmid DNA ) into antibody titers and produce
responses earlier than commer
a living host results in a small number of its cells directly 15 cially available vaccine formulations. While not wishing to
producing an antigen , resulting in a protective immunologi be bound by theory, it believed that the RNA ( e.g.,
cal response . With this technique, however, comes potential mRNA ) vaccines, for example , as mRNA polynucleotides,
problems, including the possibility of insertionalmutagen are better designed to produce the appropriate protein con
esis, which could lead to the activation of oncogenes or the formation upon translation , for both the antigen and the
inhibition of tumor suppressor genes.
20 adjuvant, as the RNA (e.g., mRNA ) vaccines co -opt natural
cellular machinery. Unlike traditional vaccines, which are
SUMMARY
manufactured ex vivo and may trigger unwanted cellular
responses, RNA (e.g., mRNA ) vaccines are presented to the
Provided herein are ribonucleic acid (RNA ) vaccines that cellular system in a more native fashion .
build on the knowledge that RNA ( e.g., messenger RNA 25 Some embodiments of the present disclosure provide
(mRNA )) can safely direct the body's cellular machinery to RNA (e.g., mRNA) vaccines that include at least one RNA
produce nearly any protein of interest, from native proteins ( e.g., mRNA ) polynucleotide having an open reading frame
to antibodies and other entirely novel protein constructs that encoding at least one antigenic polypeptide or an immuno
can have therapeutic activity inside and outside of cells . The genic fragment thereof ( e.g., an immunogenic fragment
RNA (e.g., mRNA ) vaccines of the present disclosure may 30 capable of inducing an immune response to the antigenic
be used to induce a balanced immune response against polypeptide ) and at least one RNA ( e.g., mRNA polynucle
hMPV, PIV , RSV , MeV, and/or BetaCoV (e.g. ,MERS- COV, otide ) having an open reading frame encoding a flagellin
SARS -CoV, HCOV -OC43, HCOV - 2291 HCOV -NL63, adjuvant.
HCOV -NL , HCOV -NH and/or HCOV -HKU1), or any com
In some embodiments, at least one flagellin polypeptide
bination of two or more of the foregoing viruses, comprising 35 (e.g., encoded flagellin polypeptide ) is a flagellin protein . In
both cellular and humoral immunity , without risking the some embodiments, at least one flagellin polypeptide (e.g.,
possibility of insertional mutagenesis , for example. hMPV ,
PIV , RSV, MeV, BetaCoV (e.g., MERS- CoV, SARS - COV ,
HCOV -OC43 , HCOV -229E , HCOV -NL63 , HCOV -NL ,
encoded flagellin polypeptide ) is an immunogenic flagellin
fragment. In some embodiments, at least one flagellin poly
peptide and at least one antigenic polypeptide are encoded
referred to herein as “ respiratory viruses .” Thus, the term
“ respiratory virus RNA vaccines” encompasses hMPV RNA
vaccines , PIV RNA vaccines, RSV RNA vaccines, MeV
RNA vaccines , BetaCOV RNA vaccines , and any combina
embodiments , at least one flagellin polypeptide and at least
one antigenic polypeptide are each encoded by a different
RNA polynucleotide.
In some embodiments at least one flagellin polypeptide
mRNA ) vaccines may be utilized to treat and /or prevent a
hMPV, PIV , RSV , MeV , a BetaCoV ( e.g., MERS - COV,
having an open reading frame encoding at least one (e.g. , at
least 2 , 3 , 4 or 5) hMPV,PIV , RSV ,MeV , or a BetaCoV ( e.g.,
types, strains, and isolates . TheRNA (e.g.,mRNA ) vaccines
have superior properties in that they produce much larger
antibody titers and produce responses earlier than commer
cially available anti- viral therapeutic treatments. While not
foregoing antigenic polypeptides . Herein , use of the term
" antigenic polypeptide” encompasses immunogenic frag
ments of the antigenic polypeptide (an immunogenic frag
ment that is induces (or is capable of inducing) an immune
(e.g., mRNA ) vaccines, as mRNA polynucleotides, are
better designed to produce the appropriate protein confor
mation upon translation as the RNA ( e.g., mRNA ) vaccines
co -opt natural cellular machinery . Unlike traditional vac
cines , which are manufactured ex vivo and may trigger 65
unwanted cellular responses, RNA (e.g., mRNA ) vaccines
otherwise stated .
Also provided herein , in some embodiments , is a RNA
(e.g.,mRNA ) vaccine comprising at least one (e.g., at least
2 , 3 , 4 or 5 ) RNA polynucleotide having an open reading
frame encoding at least one ( e.g., at least 2 , 3, 4 or 5 ) hMPV,
PIV , RSV , MeV , and /or a BetaCoV (e.g., MERS -COV,
SARS -CoV, HCV-OC43 , HCV- 229E , HCOV -NL63 ,
HCOV-NH and HCOV- HKU1) and combinations thereof are 40 by a single RNA (e.g., mRNA ) polynucleotide. In other
tion of two or more of hMPV RNA vaccines, PIV RNA 45 has at least 80 % , at least 85 % , at least 90 % , or at least 95 %
vaccines , RSV RNA vaccines , MeV RNA vaccines , and identity to a flagellin polypeptide having a sequence iden
BetaCOV RNA vaccines .
tified by any one of SEQ ID NO : 54-56 .
The RNA (e.g., mRNA ) vaccines may be utilized in
Provided herein , in some embodiments, is a ribonucleic
various settings depending on the prevalence of the infection acid (RNA ) ( e.g.,mRNA ) vaccine , comprising at least one
or the degree or level of unmetmedicalneed . The RNA (e.g. 50 (e.g., at least 2 , 3 , 4 or 5 ) RNA (e.g., mRNA ) polynucleotide
SARS -COV, HCV-OC43 , HCOV -229E , HCOV-NL63, MERS -CoV, SARS- CoV, HCOV -OC43 , HCOV -229E ,
HCOV -NL , HCOV-NH , HCOV -HKU1), or any combination HCOV-NL63, HCOV-NL , HCOV-NH , HCOV-HKU1) anti
of two or more of the foregoing viruses, of various geno- 55 genic polypeptide, or any combination of two ormore of the
wishing to be bound by theory, it is believed that the RNA 60 response to hMPV , PIV , RSV , MeV, or a BetaCoV ), unless
are presented to the cellular system in a more native fashion .
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HCOV-NL, HCOV- NH , HCOV-HKU1) antigenic polypep
In some embodiments , at least one PIV3 antigenic poly
tide or an immunogenic fragment thereof, linked to a signal peptide comprises hemagglutinin - neuraminidase , Fusion (F )
peptide.
glycoprotein , matrix protein (M ), nucleocapsid protein (N ),
Further provided herein , in some embodiments, is a viral replicase (L ), non -structural V protein , or an immuno
nucleic acid (e.g., DNA ) encoding at least one (e.g., at least 5 genic fragment thereof.
2 , 3 , 4 or 5 ) hMPV, PIV , RSV ,MeV, and /or a BetaCoV (e.g.,
In some embodiments, at least one PIV3 antigenic poly
MERS -CoV , SARS - CoV, HCOV -OC43 , HCOV - 229E , peptide comprises an amino acid sequence identified by any
HCOV -NL63, HCOV -NL , HCOV -NH , HCOV -HKU1) RNA one of SEQ ID NO : 12-13 (Table 6 ; see also amino acid
( e.g., mRNA ) polynucleotide .
sequences of Table 7 ). In some embodiments , the amino acid
Further still, provided herein , in some embodiments , is a 10 sequence of the PIV3 antigenic polypeptide is , or is a
method of inducing an immune response in a subject, the fragment of, or is a homolog or variant having at least 80 %
method comprising administering to the subject a vaccine ( e.g. , 85 % , 90 % , 95 % , 98 % , 99 % ) identity to , the amino acid
comprising at least one (e.g. , at least 2 , 3 , 4 or 5 ) RNA ( e.g., sequence identified by any one of SEQ ID NO : 12-13 ( Table
mRNA ) polynucleotide having an open reading frame 6 ; see also amino acid sequences of Table 7 ).
encoding at least one (e.g., at least 2, 3, 4 or 5 ) hMPV, PIV, 15 In some embodiments , at least one PIV3 antigenic poly
RSV , MeV , and /or a BetaCoV ( e.g., MERS -CoV, SARS
COV, HCV-OC43, HCOV- 229E , HCOV -NL63 , HCOV-NL ,
HCOV-NH , HCOV -HKU1) antigenic polypeptide, or any
combination of two or more of the foregoing antigenic
peptide is encoded by a nucleic acid sequence identified by
any one of SEQ ID NO : 9-12 ( Table 5 ; see also nucleic acid
sequences of Table 7 ).
In some embodiments, at least one PIV3 RNA (e.g.,
polypeptides.
20 mRNA ) polynucleotide is encoded by a nucleic acid
hMPV /PIV3 /RSV
sequence , or a fragment of a nucleotide sequence , identified
In some embodiments, a RNA (e.g., mRNA ) vaccine by any one of SEQ ID NO : 9-12 ( Table 5 ; see also nucleic
comprises at least one RNA ( e.g., mRNA ) polynucleotide acid sequences of Table 7 ). In some embodiments, at least
having an open reading frame encoding at least one hMPV, one PIV3 RNA (e.g.,mRNA) polynucleotide comprises a
PIV3 or RSV antigenic polypeptide. In some embodiments , 25 nucleic acid sequence , or a fragment of a nucleotide
at least one antigenic polypeptide is a hMPV , PIV3 or RSV sequence , identified by any one of SEQ ID NO :61-64 ( Table
polyprotein . In some embodiments, at least one antigenic 5 ) .
polypeptide is major surface glycoprotein G or an immu
In some embodiments , at least one antigenic polypeptide
nogenic fragment thereof. In some embodiments, at least is obtained from PIV3 strain HPIV3/Homo sapiens/PER /
one antigenic polypeptide is Fusion (F ) glycoprotein (e.g., 30 FLA4815/2008.
Fusion glycoprotein F0 , F1 or F2) or an immunogenic
In some embodiments , at least one RSV antigenic poly
fragment thereof. In some embodiments , at least one anti peptide comprises at least one antigenic polypeptide that
genic polypeptide is major surface glycoprotein G or an comprises glycoprotein G , glycoprotein F, or an immuno
immunogenic fragment thereof and F glycoprotein or an genic fragment thereof. In some embodiments, at least one
immunogenic fragment thereof. In some embodiments , the 35 RSV antigenic polypeptide comprises at least one antigenic
antigenic polypeptide is nucleoprotein (N ) or an immuno polypeptide that comprises glycoprotein F and at least one or
genic fragment thereof, phosphoprotein (P ) or an immuno at least two antigenic polypeptide selected from G , M , N , P ,
genic fragment thereof, large polymerase protein (L ) or an L , SH , M2, NS1 and NS2 .
immunogenic fragment thereof, matrix protein (M ) or an
MeV
immunogenic fragment thereof, small hydrophobic protein 40 In some embodiments, a RNA (e.g., mRNA) vaccine
(SH ) or an immunogenic fragment thereof nonstructural comprises at least one RNA (e.g., mRNA ) polynucleotide
proteinl (NSi ) or an immunogenic fragment thereof, or having an open reading frame encoding at least one MeV
nonstructural protein 2 (NS2 ) and an immunogenic fragment antigenic polypeptide . In some embodiments, at least one
thereof.
antigenic polypeptide is a hemagglutinin (HA ) protein or an
In some embodiments , at least one hMPV antigenic 45 immunogenic fragment thereof. The HA protein may be
polypeptide comprises an amino acid sequence identified by from MeV strain D3 or B8, for example. In some embodi
any one of SEQ ID NO : 5-8 ( Table 3 ; see also amino acid ments, at least one antigenic polypeptide is a Fusion ( F )
sequences of Table 4 ). In some embodiments , the amino acid protein or an immunogenic fragment thereof. The F protein
sequence of the hMPV antigenic polypeptide is, or is a may be from MeV strain D3 or B8, for example . In some
fragment of, or is a homolog or variant having at least 80 % 50 embodiments, a MeV RNA (e.g., mRNA ) vaccines com
( e.g., 85 % , 90 % , 95 % , 98 % , 99 % ) identity to , the amino acid prises a least one RNA polynucleotide encoding a HA
sequence identified by any one of SEQ ID NO : 5-8 ( Table protein and a F protein . The HA and F proteins may be from
MeV strain D3 or B8 , for example .
3 ; see also amino acid sequences of Table 4 ).
In some embodiments, at least one hMPV antigenic
In some embodiments , at least one MeV antigenic poly
polypeptide is encoded by a nucleic acid sequence identified 55 peptide comprises an amino acid sequence identified by any
by any one of SEQ ID NO : 1-4 ( Table 2 ).
one of SEQ ID NO : 47-50 ( Table 14 ). In some embodiments,
In some embodiments, at least one hMPV RNA ( e.g., the amino acid sequence of the MeV antigenic polypeptide
mRNA ) polynucleotide is encoded by a nucleic acid is , or is a fragment of, or is a homolog or variant having at
sequence, or a fragment of a nucleotide sequence , identified least 80 % ( e.g., 85 % , 90 % , 95 % , 98 % , 99 % ) identity to , the
by any one of SEQ ID NO : 1-4 ( Table 2 ). In some embodi- 60 amino acid sequence identified by any one of SEQ ID NO :
ments , at least one hMPV RNA (e.g., mRNA ) polynucle
47-50 ( Table 14 ).
otide comprises a nucleic acid sequence , or a fragment of a
In some embodiments , at least one MeV antigenic poly
nucleotide sequence , identified by any one of SEQ ID NO : peptide is encoded by a nucleic acid sequence of SEQ ID
NO : 35-46 ( Table 13).
57-60 ( Table 2 ).
In some embodiments , at least one antigenic polypeptide 65 In some embodiments, at least one MeV RNA ( e.g.,
is obtained from hMPV strain CAN98-75 (CAN75 ) or the mRNA) polynucleotide is encoded by a nucleic acid
sequence , or a fragment of a nucleotide sequence , identified
hMPV strain CAN97-83 (CAN83 ).
US 10,702,600 B1
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by any one of SEQ ID NO : 35-46 ( Table 13 ). In some
HCOV -NL63 , HCOV-NL , HCOV -NH and HCOV-HKU1.
embodiments , at least one MeV RNA ( e.g., mRNA ) poly
Other betacoronaviruses are encompassed by the present
nucleotide comprises a nucleic acid sequence, or a fragment disclosure .
In some embodiments , at least one antigenic polypeptide
of a nucleotide sequence , identified by any one of SEQ ID
5 is a MERS -CoV structural protein . For example, a MERS
NO : 69-80 ( Table 13 ).
In some embodiments , at least one antigenic polypeptide COV structural protein may be spike protein (S ), envelope
is obtained from MeV strain B3/B3.1, C2, D4 , D6 , D7, D8 , protein ( E ), nucleocapsid protein (N ) , membrane protein
(M ) or an immunogenic fragment thereof. In some embodi
G3, H1, Moraten , Rubeovax, MVi/New Jersey.USA /45.05 , ments
- CoV structural protein is a spike protein
MVi/Texas.USA/4.07 , AIK -C , MVi/New York.USA / 26.09 / 10 (S ) (see, ,thee.g.MERS
Coleman C M et al . Vaccine 2014 ; 32:3169-74 ,
3 , MVi/ California.USA/ 16.03, MVi/Virginia .USA/ 15.09 , incorporated, herein
by reference ). In some embodiments,
MVi/California.USA / 8.04 , or MVi/Pennsylvania.USA /
the MERS - CoV structural protein is a S1 subunit or a S2
20.09 .
subunit of spike protein (S ) or an immunogenic fragment
BetaCoV
thereof
(Li J et al. Viral Immunol 2013 ; 26 (2 ): 126-32 ; He Y
In some embodiments , a RNA (e.g., mRNA ) vaccine 15 et al. Biochem
Res Commun 2004 ; 324 ( 2 ): 773-81,
comprises at least one RNA (e.g., mRNA ) polynucleotide each ofwhich isBiophys
incorporated
herein by reference).
having an open reading frame encoding at least one Beta
In some embodiments, at least one MERS -CoV antigenic
CoV antigenic polypeptide. In some embodiments , the Beta polypeptide comprises an amino acid sequence identified by
COV is MERS -CoV . In some embodiments , the BetaCoV is any one of SEQ ID NO : 24-28 or 33 ( Table 11 ). In some
SARS- CoV. In some embodiments, the BetaCoV is HCOV- 20 embodiments, the amino acid sequence of the MERS-CoV
OC43 . In some embodiments, the BetaCoV is HCOV - 229E . antigenic polypeptide is , or is a fragment of, or is a homolog
In some embodiments , the BetaCoV is HCOV-NL63. In or variant having at least 80 % (e.g., 85 % , 90 % , 95 % , 98 % ,
some embodiments, the BetaCoV is HCOV -HKU1. In some 99 % ) identity to , the amino acid sequence identified by any
embodiments, at least one antigenic polypeptide is a beta
one of SEQ ID NO : 24-28 or 33 ( Table 11 ).
coronavirus structural protein . For example , a betacorona- 25 In some embodiments, at least one MERS -CoV antigenic
virus structural protein may be spike protein (S ), envelope polypeptide is encoded by a nucleic acid sequence identified
protein ( E ), nucleocapsid protein (N ), membrane protein by any one of SEQ ID NO : 20-23 ( Table 10 ).
In some embodiments, at least one MERS -CoV RNA
( M ) or an immunogenic fragment thereof. In some embodi
(e.g., mRNA ) polynucleotide is encoded by a nucleic acid
ments , a betacoronavirus structural protein is a spike protein 30 sequence
, or a fragment of a nucleotide sequence, identified
(S ). In some embodiments, a betacoronavirus structural by any one
ID NO : 20-23 ( Table 10 ). In some
protein is a S1 subunit or a S2 subunit of spike protein (S ) embodiments,ofatSEQ
least one MERS -CoV RNA ( e.g.,mRNA )
or an immunogenic fragment thereof.
comprises a nucleic acid sequence, or a
BetaCOV RNA ( e.g., mRNA ) polynucleotides of the vac polynucleotide
fragment of a nucleotide sequence, identified by any one of
cines provided herein may encode viral protein components 35 SEQ ID NO :65-68 ( Table 10 ).
of betacoronaviruses, for example, accessory proteins, rep
In some embodiments , at least one antigenic polypeptide
is obtained from MERS- CoV strain Riyadh_14_2013 ,
disclosure . RNA (e.g., mRNA ) vaccines may include RNA 2cEMC /2012 , or Hasa_1_2013 .
polynucleotides encoding at least one accessory protein
In some embodiments , at least one antigenic polypeptide
( e.g., protein 3 ,protein 4a, protein 4b , protein 5 ), at least one 40 is a SARS-CoV structural protein . For example, a SARS
replicase protein (e.g., protein 1a, protein 1b ), or a combi CoV structural protein may be spike protein (S ), envelope
nation of at least one accessory protein and at least one protein (E ), nucleocapsid protein (N ), membrane protein
replicase protein . The present disclosure also encompasses (M ) or an immunogenic fragment thereof. In some embodi
RNA (e.g.,mRNA ) vaccines comprising RNA (e.g. ,mRNA ) ments , the SARS -CoV structural protein is a spike protein
polynucleotides encoding an accessory protein and / or a 45 (S ). In some embodiments , the SARS - CoV structural protein
licase proteins and the like are encompassed by the present
replicase protein in combination with at least one structural
protein . Due to their surface expression properties, vaccines
featuring RNA polynucleotides encoding structural proteins
are believed to have preferred immunogenic activity and,
hence , may be most suitable for use in the vaccines of the 50
present disclosure .
Some embodiments of the present disclosure provide
betacoronavirus ( e.g. , MERS -CoV , SARS- CoV, HCOV
OC43, HCOV - 229E , HCOV-NL63, HCOV-NL , HCOV-NH ,
HCOV-HKU1 or a combination thereof) vaccines that 55
include at least one RNA ( e.g. , mRNA ) polynucleotide
having an open reading frame encoding at least one beta
coronavirus (e.g., MERS- CoV , SARS -CoV, HCV-OC43,
HCoV -229E, HCoV- NL63, HCoV- NL, HCoV- NH, HCOV
is a S1 subunit or a S2 subunit of spike protein ( S ) or an
immunogenic fragment thereof.
In some embodiments , at least one SARS -CoV antigenic
polypeptide comprises an amino acid sequence identified by
any one of SEQ ID NO : 29 , 32 or 34 ( Table 11). In some
embodiments, the amino acid sequence of the SARS -CoV
antigenic polypeptide is , or is a fragment of,or is a homolog
or variant having at least 80 % (e.g., 85 % , 90 % , 95 % , 98 % ,
99 % ) identity to , the amino acid sequence identified by any
one of SEQ ID NO : 29, 32 or 34 ( Table 11 ).
In some embodiments, at least one antigenic polypeptide
is a HCV-OC43 structural protein . For example , a HCOV
OC43 structural protein may be spike protein (S ), envelope
protein ( E ), nucleocapsid protein (N ), membrane protein
cine comprising a RNA (e.g.,mRNA ) polynucleotide having
an open reading frame encoding any one, two, three or four
of MERS - COV, SARS - CoV, HCV-OC43, HCOV - 229E ,
HCOV -NL63 , and HCOV-HKU1, for example, may be effec- 65
tive against any one of, any combination of, or all of,
MERS -CoV, SARS -CoV, HCV-OC43, HCOV - 229E ,
(S ). In some embodiments , the HCOV -OC43 structural pro
tein is a S1 subunit or a S2 subunit of spike protein (S ) or
an immunogenic fragment thereof.
In some embodiments , at least one HCOV -OC43 antigenic
polypeptide comprises an amino acid sequence identified by
HKU1) antigenic polypeptide. Also provided herein are 60 (M ) or an immunogenic fragment thereof. In some embodi
pan -betacoronavirus vaccines . Thus, a betacoronavirus vac ments, the HCV-OC43 structural protein is a spike protein
any one of SEQ ID NO : 30 ( Table 11 ). In some embodi
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ments , the amino acid sequence of the HCOV - OC43 anti identified by any one of SEQ ID NO : 1-4 , 9-12 , 20-23 , or
genic polypeptide is , or is a fragment of, or is a homolog or 35-46 (Tables 2 , 5 , 10 and 13; see also nucleic acid
variant having at least 80 % (e.g., 85 % , 90 % , 95 % , 98 % , sequences of Table 7 ) and having less than 40-90 % ,50-90 % ,
99 % ) identity to , the amino acid sequence identified by any 60-90 % , 30-90 % , 70-90 % , 75-90 % , 80-90 % , or 85-90 %
5 identity to wild -type mRNA sequence .
one of SEQ ID NO : 30 ( Table 11 ).
In some embodiments, an antigenic polypeptide is a
In some embodiments, at least one RNA polynucleotide
HCOV -HKU1 structural protein . For example, a HCOV
encodes at least one antigenic polypeptide having an amino
HKU1 structural protein may be spike protein (S ), envelope acid sequence identified by any one of SEQ ID NO : 5-8 ,
protein (E ), nucleocapsid protein (N ), membrane protein 12-13 , 24-34 , or 47-50 ( Tables 3 , 6 , 11 and 14 ; see also
(M ) or an immunogenic fragment thereof. In some embodi- 10 amino acid sequences of Tables 4 , 7 , 12 and 15 ) and having
ments , the HCOV -HKU1 structural protein is a spike protein at least 80 % (e.g., 85 % , 90 % , 95 % , 98 % , 99 % ) identity to
(S ). In some embodiments, the HCOV -HKU1 structural wild -type mRNA sequence, but does not include wild -type
protein is a S1 subunit or a S2 subunit of spike protein (S ) mRNA sequence .
or an immunogenic fragment thereof.
In some embodiments , at least one RNA polynucleotide
In some embodiments, at least one HCOV -HKU1 anti- 15 encodes at least one antigenic polypeptide having an amino
genic polypeptide comprises an amino acid sequence iden
acid sequence identified by any one of SEQ ID NO : 5-8 ,
tified by any one of SEQ ID NO : 31 ( Table 11 ). In some 12-13 , 24-34 , or 47-50 ( Tables 3 , 6 , 11 and 14 ; see also
embodiments, the amino acid sequence of the HCOV -HKU1 amino acid sequences of Tables 4 , 7, 12 and 15 ) and has less
antigenic polypeptide is, or is a fragment of, or is a homolog than 95 % , 90 % , 85 % , 80 % or 75 % identity to wild - type
or variant having at least 80 % (e.g., 85 % , 90 % , 95 % , 98 % , 20 mRNA sequence .
99 % ) identity to , the amino acid sequence identified by any
In some embodiments , at least one RNA polynucleotide
encodes at least one antigenic polypeptide having an amino
one of SEQ ID NO : 31 ( Table 11) .
In some embodiments, an open reading frame of a RNA acid sequence identified by any one of SEQ ID NO : 5-8 ,
(e.g.,mRNA ) vaccine is codon -optimized . In some embodi 12-13 , 24-34 , or 47-50 ( Tables 3 , 6 , 11 and 14 ; see also
ments, at least one RNA polynucleotide encodes at least one 25 amino acid sequences of Tables 4 , 7 , 12 and 15 ) and has
antigenic polypeptide having an amino acid sequence iden
tified by any one of SEQ ID NO : 5-8 , 12-13 , 24-34 , or 47-50
( Tables 3 , 6 , 11 and 14 ; see also amino acid sequences of
30-80 % , 40-80 % , 50-80 % , 60-80 % , 70-80 % , 75-80 % or
78-80 % , 30-85 % , 40-85 % , 50-805 % , 60-85 % , 70-85 % ,
75-85 % or 78-85 % , 30-90 % , 40-90 % , 50-90 % , 60-90 % ,
Tables 4, 7 , 12 and 15 ) and is codon optimized mRNA .
70-90 % , 75-90 % , 80-90 % or 85-90 % identity to wild -type
In some embodiments , a RNA ( e.g., mRNA ) vaccine 30 mRNA sequence.
further comprising an adjuvant.
In some embodiments , at least one RNA polynucleotide
Tables 4 , 7, 12 and 15 provide National Center for encodes at least one antigenic polypeptide having at least
Biotechnology Information (NCBI) accession numbers of
0 % , least 95 % , at least 96 % , at least 97 % , at least 98 % ,
interest. It should be understood that the phrase " an amino or at least 99 % identity to an amino acid sequence identified
acid sequence of Tables 4 , 7 , 12 and 15 ” refers to an amino 35 by any one of SEQ ID NO : 5-8 , 12-13 , 24-34 , or 47-50
acid sequence identified by one or more NCBI accession ( Tables 3, 6 , 11 and 14 ; see also amino acid sequences of
numbers listed in Tables 4 , 7 , 12 and 15. Each of the amino
Tables 4 , 7 , 12 and 15 ). In some embodiments , at least one
acid sequences , and variants having greater than 95 % iden
RNA polynucleotide encodes at least one antigenic poly
tity or greater than 98 % identity to each of the amino acid peptide having 95 % -99 % identity to an amino acid sequence
sequences encompassed by the accession numbers of Tables 40 identified by any one of SEQ ID NO : 5-8 , 12-13 , 24-34 , or
4 , 7 , 12 and 15 are included within the constructs (poly
47-50 ( Tables 3 , 6 , 11 and 14 ; see also amino acid sequences
nucleotides/polypeptides) of the present disclosure .
of Tables 4 , 7 , 12 and 15 ).
In some embodiments, at least onemRNA polynucleotide
In some embodiments , at least one RNA polynucleotide
is encoded by a nucleic acid having a sequence identified by encodes at least one antigenic polypeptide having at least
any one of SEQ ID NO : 1-4 , 9-12 , 20-23 , or 35-46 ( Tables 45 90 % , at least 95 % , at least 96 % , at least 97 % , at least 98 % ,
2 , 5 , 10 and 13 ; see also nucleic acid sequences of Table 7 ) or at least 99 % identity to an amino acid sequence identified
and having less than 80 % identity to wild - type mRNA by any one of SEQ ID NO : 5-8 , 12-13 , 24-34 , or 47-50
sequence. In some embodiments , at least one mRNA poly
( Tables 3 , 6 , 11 and 14 ; see also amino acid sequences of
nucleotide is encoded by a nucleic acid having a sequence Tables 4 , 7, 12 and 15 ) and havingmembrane fusion activity .
identified by any one of SEQ ID NO : 1-4 , 9-12 , 20-23 , or 50 In some embodiments , at least one RNA polynucleotide
35-46 ( Tables 2 , 5, 10 and 13 ; see also nucleic acid encodes at least one antigenic polypeptide having 95 % -99 %
sequences of Table 7 ) and having less than 75 % , 85 % or identity to an amino acid sequence identified by any one of
95 % identity to a wild -type mRNA sequence . In some SEQ ID NO : 5-8 , 12-13 , 24-34 , or 47-50 ( Tables 3, 6 , 11 and
embodiments, at least onemRNA polynucleotide is encoded 14 ; see also amino acid sequences of Tables 4 , 7, 12 and 15 )
by a nucleic acid having a sequence identified by any one of 55 and having membrane fusion activity .
SEQ ID NO : 1-4 , 9-12 , 20-23 , or 35-46 ( Tables 2 , 5, 10 and
In some embodiments , at least one RNA polynucleotide
13 ; see also nucleic acid sequences of Table 7) and having encodes at least one antigenic polypeptide (e.g., at least one
less than 50-80 % , 60-80 % , 40-80 % , 30-80 % , 70-80 % , hMPV antigenic polypeptide , at least one PIV3 antigenic
75-80 % or 78-80 % identity to wild -type mRNA sequence . polypeptide, at least one RSV antigenic polypeptide, at least
In some embodiments, at least one mRNA polynucleotide is 60 one MeV antigenic polypeptide , or at least one BetaCOV
encoded by a nucleic acid having a sequence identified by antigenic polypeptide , e.g., selected from MERS-CoV,
any one of SEQ ID NO : 1-4 , 9-12 , 20-23 , or 35-46 ( Tables SARS -CoV, HCV-OC43 , HCOV-229E , HCOV-NL63,
2 , 5 , 10 and 13 ; see also nucleic acid sequences of Table 7 ) HCOV -NL , HCOV-NH and HCOV-HKU1, or any combina
and having less than 40-85 % , 50-85 % , 60-85 % , 30-85 % , tion of two or more of the foregoing antigenic polypeptides)
70-85 % , 75-85 % or 80-85 % identity to wild - type mRNA 65 that attaches to cell receptors.
sequence . In some embodiments, at least one mRNA poly
In some embodiments , at least one RNA polynucleotide
nucleotide is encoded by a nucleic acid having a sequence encodes at least one antigenic polypeptide ( e.g., at least one
US 10,702,600 B1
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11
hMPV antigenic polypeptide , at least one PIV3 antigenic a cationic lipid is selected from the group consisting of
polypeptide , at least one RSV antigenic polypeptide, at least 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ]-dioxolane
one MeV antigenic polypeptide, or at least one BetaCoV (DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu
antigenic polypeptide, e.g., selected from MERS- CoV, tyrate (DLin -MC3 -DMA), di ((Z )-non - 2-en -1 -yl) 9 -((4-(di
SARS - CoV, HCV-OC43 , HCOV -229E , HCOV -NL63 , 5 methylamino )butanoyl )oxy )heptadecanedioate (L319),
HCOV -NL , HCOV-NH and HCOV-HKU1, or any combina
(127,152 ) N , N -dimethyl- 2 - nonylhenicosa - 12,15 -dien - 1
tion of two or more of the foregoing antigenic polypeptides ) amine (L608 ), and N ,N - dimethyl-1- (18,2R )-2 -octylcyclo
propyl]heptadecan - 8 -amine (L530 ).
that causes fusion of viral and cellular membranes.
In some embodiments , at least one RNA polynucleotide
encodes at least one antigenic polypeptide ( e.g., at least one
hMPV antigenic polypeptide , at least one PIV3 antigenic
10
In some embodiments , the lipid is (L608 ).
In some embodiments , the lipid is
(L608 )
polypeptide, at least one RSV antigenic polypeptide, at least 25
(L530 )
antigenic polypeptide , e.g., selected from MERS- CoV,
SARS -COV, HCV-OC43, HCV-229E , HCOV-NL63 ,
HCOV-NL , HCOV-NH and HCOV-HKU1, or any combina
tion of two or more of the foregoing antigenic polypeptides) 30
that is responsible for binding of the virus to a cell being
infected .
Some embodiments of the present disclosure provide a
In some embodiments , a lipid nanoparticle comprises
vaccine that includes at least one ribonucleic acid (RNA )
(e.g.,mRNA ) polynucleotide having an open reading frame 35 below
compounds
of Formula (1) and/or Formula (II ), discussed
.
encoding at least one antigenic polypeptide (e.g. , at least one
In some embodiments , a repiratory virus RNA ( e.g.,
hMPV antigenic polypeptide , at least one PIV3 antigenic
polypeptide, at least one RSV antigenic polypeptide, at least mRNA ) vaccine is formulated in a lipid nanoparticle that
one MeV antigenic polypeptide, or at least one BetaCOV comprises a compound selected from Compounds 3 , 18 , 20 ,
one MeV antigenic polypeptide , or at least one BetaCoV
antigenic polypeptide , e.g. , selected from MERS -CoV, 4025 , 26 , 29 , 30 , 60 , 108-112 and 122, described below .
SARS-CoV, HCOV-OC43, HCOV - 229E , HCOV -NL63,
Some embodiments of the present disclosure provide a
HCOV-NL , HCOV-NH and HCOV -HKU1, or any combina
vaccine that includes at least one RNA ( e.g.,mRNA ) poly
tion of two or more of the foregoing antigenic polypeptides ), nucleotide having an open reading frame encoding at least
at least one 5 ' terminal cap and at least one chemical one antigenic polypeptide (e.g., at least one hMPV antigenic
modification, formulated within a lipid nanoparticle .
45 polypeptide, at least one PIV3 antigenic polypeptide, at least
In some embodiments , a 5' terminal cap is 7mG (5 ')ppp one RSV antigenic polypeptide , at least one MeV antigenic
(5 ')NlmpNp.
polypeptide, or at least one BetaCoV antigenic polypeptide,
In some embodiments , at least one chemical modification e.g., selected from MERS -CoV, SARS -COV, HCOV -OC43 ,
is selected from pseudouridine, Nl-methylpseudouridine , HCOV- 229E, HCOV-NL63 , HCOV-NL , HCOV -NH and
N1- ethylpseudouridine , 2 -thiouridine , 4'- thiouridine, 50 HCOV -HKU1, or any combination of two or more of the
5 -methylcytosine , 5 -methyluridine , 2 -thio - 1-methyl- 1 foregoing antigenic polypeptides), wherein at least 80 %
deaza -pseudouridine ,
2 -thio - 1-methyl-pseudouridine , ( e.g., 85 % , 90 % , 95 % , 98 % , 99 % ) of the uracil in the open
2 - thio -5 -aza -uridine , 2-thio -dihydropseudouridine , 2-thio
reading frame have a chemical modification , optionally
dihydrouridine, 2 - thio -pseudouridine, 4 -methoxy -2 - thio wherein the vaccine is formulated in a lipid nanoparticle
pseudouridine , 4 -methoxy -pseudouridine, 4 -thio -1-methyl- 55 (e.g., a lipid nanoparticle comprises a cationic lipid , a
pseudouridine, 4 -thio - pseudouridine, 5 -aza -uridine, PEG -modified lipid, a sterol and a non -cationic lipid ).
dihydropseudouridine, 5-methoxyuridine and 2 -O -methyl
uridine . In some embodiments , the chemical modification is
In some embodiments, 100 % of the uracil in the open
reading frame have a chemical modification . In some
in the 5 -position of the uracil. In some embodiments, the embodiments, a chemical modification is in the 5 - position of
chemical modification is a N1-methylpseudouridine. In 60 the uracil. In some embodiments, a chemicalmodification is
some embodiments, the chemical modification is a N1- eth
a N1-methylpseudouridine. In some embodiments, 100 % of
ylpseudouridine.
the uracil in the open reading frame have a N1-methyl
In some embodiments, a lipid nanoparticle comprises a pseudouridine in the 5 -position of the uracil.
cationic lipid , a PEG -modified lipid , a sterol and a non
In some embodiments , an open reading frame of a RNA
cationic lipid . In some embodiments , a cationic lipid is an 65 (e.g., mRNA ) polynucleotide encodes at least two antigenic
ionizable cationic lipid and the non -cationic lipid is a neutral
lipid , and the sterol is a cholesterol. In some embodiments ,
polypeptides (e.g., at least two hMPV antigenic polypep
tides , at least two PIV3 antigenic polypeptides, at least two
US 10,702,600 B1
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14
RSV antigenic polypeptides, at least two MeV antigenic BetaCoV vaccine , e.g., selected from MERS - CoV, SARS
COV, HCOV -OC43, HCOV- 229E , HCOV -NL63, HCOV-NL ,
tides, e.g., selected from MERS- CoV , SARS- CoV , HCOV HCOV-NH and HCOV-HKU1, or any combination of two or
OC43 , HCV-229E , HCOV-NL63, HCOV -NL , HCOV -NH more of the foregoing vaccines ), formulated in a nanopar
and HCOV -HKU1, or any combination of two ormore of the 5ticle ( e.g., a lipid nanoparticle ).
foregoing antigenic polypeptides ). In some embodiments ,
In some embodiments, the nanoparticle has a mean diam
the open reading frame encodes at least five or at least ten eter of 50-200 nm . In some embodiments, the nanoparticle
antigenic polypeptides. In some embodiments, the open is a lipid nanoparticle . In some embodiments , the lipid
reading frame encodes at least 100 antigenic polypeptides. nanoparticle comprises a cationic lipid, a PEG -modified
In some embodiments, the open reading frame encodes 10 lipid , a sterol and a non -cationic lipid . In some embodi
2-100 antigenic polypeptides.
ments, the lipid nanoparticle comprises a molar ratio of
In some embodiments , a vaccine comprises at least two about 20-60 % cationic lipid , 0.5-15 % PEG -modified lipid ,
RNA ( e.g., mRNA ) polynucleotides , each having an open 25-55 % sterol , and 25 % non -cationic lipid . In some embodi
reading frame encoding at least one antigenic polypeptide ments, the cationic lipid is an ionizable cationic lipid and the
polypeptides, or at least two BetaCoV antigenic polypep
(e.g. , at least one hMPV antigenic polypeptide, at least one 15 non -cationic lipid is a neutral lipid, and the sterol is a
PIV3 antigenic polypeptide, at least one RSV antigenic cholesterol . In some embodiments , the cationic lipid is
polypeptide, at least one MeV antigenic polypeptide , or at selected from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [1,3 ]-di
least one BetaCoV antigenic polypeptide, e.g., selected from
MERS -CoV, SARS -CoV, HCV-OC43, HCOV -229E ,
HCOV-NL63 , HCOV-NL, HCOV-NH and HCOV-HKU1, or 20
any combination of two or more of the foregoing antigenic
polypeptides). In some embodiments, the vaccine comprises
at least five or at least ten RNA (e.g., mRNA ) polynucle
otides, each having an open reading frame encoding at least
one antigenic polypeptide or an immunogenic fragment 25
thereof. In some embodiments, the vaccine comprises at
least 100 RNA (e.g. , mRNA ) polynucleotides , each having
oxolane (DLin -KC2-DMA ), dilinoleyl-methyl-4 - dimethyl
aminobutyrate (DLin -MC3 -DMA ), and di(( Z ) -non- 2 -en -1
yl) 9-((4-(dimethylamino )butanoyl)oxy)heptadecanedioate
(L319 ) .
In some embodiments , a lipid nanoparticle comprises
compounds of Formula (I) and /or Formula (II), as discussed
below .
In some embodiments , a lipid nanoparticle comprises
Compounds 3, 18 , 20 , 25 , 26 , 29, 30 , 60, 108-112 , or 122 ,
as discussed below .
In some embodiments, the nanoparticle has a polydisper
sity value of less than 0.4 ( e.g., less than 0.3 , 0.2 or 0.1).
In some embodim ts, at least one antigenic polypeptide
( e.g., at least one hMPV antigenic polypeptide, at least one
multivalent.
Some embodiments of the present disclosure provide
polypeptide, at least one MeV antigenic polypeptide, or at
a subject, comprising administering to the subject any of the
an open reading frame encoding at least one antigenic
polypeptide. In some embodiments , the vaccine comprises
2-100 RNA (e.g., mRNA ) polynucleotides , each having an 30 In some embodiments , the nanoparticle has a net neutral
open reading frame encoding at least one antigenic poly charge at a neutral pH value.
peptide.
In some embodiments , the respiratory virus vaccine is
PIV3 antigenic polypeptide, at least one RSV antigenic 35 methods of inducing an antigen specific immune response in
least one BetaCoV antigenic polypeptide, e.g., selected from
RNA (e.g.,mRNA ) vaccine as provided herein in an amount
MERS-CoV, SARS - CoV, HCOV -OC43 , HCOV - 229E , effective to produce an antigen -specific immune response . In
HCOV -NL63, HCOV -NL , HCOV-NH and HCOV -HKU1, or some embodiments , the RNA (e.g., mRNA ) vaccine is a
any combination of two or more of the foregoing antigenic 40 hMPV vaccine, a PIV3 vaccine, a RSV vaccine , a MeV
polypeptides ) is fused to a signal peptide. In some embodi vaccine, or a BetaCoV vaccine, e.g., selected from MERS
ments , the signal peptide is selected from : a HulgGk signal COV, SARS -COV, HCV-OC43 , HCOV-229E , HCOV -NL63,
peptide (METPAQLLFLLLLWLPDTTG ; SEQ ID NO : 15); HCOV -NL , HCOV -NH and HCOV-HKU1 vaccines . In some
IgE heavy chain epsilon - 1 signal peptide (MDWTWIL
embodiments, the RNA (e.g. ,mRNA ) vaccine is a combi
FLVAAATRVHS; SEQ ID NO : 16 ); Japanese encephalitis 45 nation vaccine comprising a combination ofany two ormore
PRM signal sequence (MLGSNSGQRVVFTILLLLVA of the foregoing vaccines .
PAYS; SEQ ID NO : 17 ), VSVg protein signal sequence
In some embodiments, an antigen -specific immune
(MKCLLYLAFLFIGVNCA; SEQ ID NO : 18 ) and Japanese response comprises a T cell response or a B cell response .
encephalitis JEV signal sequence (MWLVSLAIVTA
In some embodiments, a method of producing an antigen
50 specific immune response comprises administering to a
CAGA ; SEQ ID NO : 19 ).
In some embodiments , the signal peptide is fused to the subject a single dose (no booster dose ) of a RNA ( e.g.,
N -terminus of at least one antigenic polypeptide. In some mRNA ) vaccine of the present disclosure. In some embodi
embodiments, a signal peptide is fused to the C -terminus of ments , the RNA (e.g. , mRNA ) vaccine is a hMPV vaccine ,
at least one antigenic polypeptide .
a PIV3 vaccine, a RSV vaccine , a MeV vaccine, or a
In some embodiments , at least one antigenic polypeptide 55 BetaCoV vaccine , e.g., selected from MERS -COV , SARS
(e.g., at least one hMPV antigenic polypeptide , at least one COV, HCOV -OC43, HCOV- 229E , HCOV -NL63, HCOV -NL ,
PIV3 antigenic polypeptide , at least one RSV antigenic HCOV -NH and HCOV -HKU1 vaccines . In some embodi
polypeptide, at least one MeV antigenic polypeptide, or at ments , the RNA ( e.g. , mRNA ) vaccine is a combination
least one BetaCoV antigenic polypeptide , e.g., selected from
vaccine comprising a combination of any two ormore of the
MERS -CoV, SARS- CoV, HCOV -OC43 , HCOV-229E , 60 foregoing vaccines.
HCOV-NL63, HCOV-NL , HCOV-NH and HCOV -HKU1, or
In some embodiments, a method further comprises
any combination of two or more of the foregoing antigenic administering to the subject a second (booster ) dose of a
polypeptides) comprises a mutated N - linked glycosylation RNA (e.g., mRNA ) vaccine. Additional doses of a RNA
site .
(e.g., mRNA ) vaccine may be administered .
Also provided herein is a RNA (e.g., mRNA ) vaccine of 65 In some embodiments , the subjects exhibit a seroconver
any one of the foregoing paragraphs ( e.g., a hMPV vaccine, sion rate of at least 80 % (e.g. , at least 85 % , at least 90 % , or
a PIV3 vaccine, a RSV vaccine , a MeV vaccine, or a
at least 95 % ) following the first dose or the second (booster )
US 10,702,600 B1
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16
dose of the vaccine. Seroconversion is the time period
during which a specific antibody develops and becomes
detectable in the blood . After seroconversion has occurred ,
a virus can be detected in blood tests for the antibody.
During an infection or immunization , antigens enter the 5
anti-antigenic polypeptide antibody titer produced in a con
trol subject administered the standard of care dose of a
recombinant hMPV , PIV3, RSV, MeV and /or BetaCoV
protein vaccine , a purified hMPV , PIV3, RSV, MeV and /or
BetaCoV protein vaccine, a live attenuated hMPV, PIV3,
infection .
In some embodiments , a RNA (e.g., mRNA ) vaccine is
administered to a subject by intradermal or intramuscular
protein vaccine, wherein the anti-antigenic polypeptide anti
body titer produced in the subject is equivalent to an
anti- antigenic polypeptide antibody titer produced in a con
vaccines of the present disclosure . In some embodiments,
and / or BetaCoV .
antibody titer produced in a subject is increased at least 2
times relative to a control. In some embodiments, the
In some embod ents, the effective amount is a dose of 25
ug administered to the subject a total of two times . In some
subject is increased at least 5 times relative to a control. In
some embodiments , the anti-antigenic polypeptide antibody
titer produced in the subject is increased at least 10 times
administered to the subject a total of two times. In some
embodiments, the effective amount is a dose of 400 ug
administered to the subject a total of two times. In some
polypeptide antibody titer produced in a subject who has not
embodiments, the RNA (e.g., mRNA ) polynucleotide of the
blood , and the immune system begins to produce antibodies RSV, MeV and/ or BetaCoV vaccine, an inactivated hMPV,
in response . Before seroconversion , the antigen itself may or PIV3, RSV , MeV and /or BetaCoV vaccine , or a hMPV,
may not be detectable , but antibodies are considered absent. PIV3, RSV, MeV and/or BetaCoV VLP vaccine . In some
During seroconversion , antibodies are present but not yet embodiments, the effective amount is a dose equivalent to
detectable . Any time after seroconversion , the antibodies can 10 2-1000 - fold reduction in the standard of care dose of a
be detected in the blood , indicating a prior or current recombinant hMPV , PIV3, RSV, MeV and /or BetaCoV
injection .
15 trol subject administered the standard of care dose of a
Some embodiments, of the present disclosure provide recombinant hMPV , PIV3 , RSV , MeV and/or BetaCOV
methods of inducing an antigen specific immune response in protein vaccine, a purified hMPV , PIV3 , RSV , MeV and /or
a subject , including administering to a subject a RNA (e.g. , BetaCoV protein vaccine, a live attenuated hMPV , PIV3,
mRNA ) vaccine in an effective amount to produce an RSV, MeV and/or BetaCoV vaccine, an inactivated hMPV ,
antigen specific immune response in a subject. Antigen- 20 PIV3 , RSV, MeV and/or BetaCoV vaccine , or a HMPV,
specific immune responses in a subjectmay be determined , PIV3, RSV, MeV and/or BetaCOV VLP vaccine.
in some embodiments , by assaying for antibody titer ( for
In some embodiments , the control is an anti -antigenic
titer of an antibody that binds to a hMPV , PIV3, RSV, MeV polypeptide antibody titer produced in a subject who has
and /or BetaCoV antigenic polypeptide) following adminis been administered a virus-like particle (VLP ) vaccine com
tration to the subject of any of the RNA (e.g. , mRNA) 25 prising structural proteins of hMPV, PIV3, RSV, MeV
the anti -antigenic polypeptide antibody titer produced in the
In some embodiments , the RNA ( e.g., mRNA ) vaccine is
subject is increased by at least 1 log relative to a control. In formulated in an effective amount to produce an antigen
some embodiments , the anti -antigenic polypeptide antibody specific immune response in a subject .
titer produced in the subject is increased by 1-3 log relative 30 In some embodiments, the effective amount is a total dose
to a control.
of 25 ug to 1000 ug , or 50 ug to 1000 ug . In some
In some embodiments , the anti-antigenic polypeptide embodiments, the effective amount is a total dose of 100 ug.
anti -antigenic polypeptide antibody titer produced in the 35 embodiments, the effective amount is a dose of 100 ug
relative to a control. In some embodiments , the anti- anti
embodiments, the effective amount is a dose of 500 ug
genic polypeptide antibody titer produced in the subject is 40 administered to the subject a total of two times.
increased 2-10 times relative to a control.
In some embodiments, the efficacy (or effectiveness ) of a
In some embodiments, the control is an anti -antigenic RNA ( e.g., mRNA ) vaccine is greater than 60 % . In some
been administered a RNA ( e.g. , mRNA ) vaccine of the vaccine at least one hMPV antigenic polypeptide, at least
present disclosure . In some embodiments , the control is an 45 One PIV3 antigenic polypeptide , at least one RSV antigenic
anti-antigenic polypeptide antibody titer produced in a sub polypeptide, at least one MeV antigenic polypeptide , at least
ject who has been administered a live attenuated or inacti one BetaCoV antigenic polypeptide, e.g., selected from
MERS -CoV, SARS -CoV , HCV-OC43, HCOV -229E ,
(see , e.g., Ren J. et al. J of Gen. Virol. 2015; 96 : 1515-1520 ), HCOV-NL63, HCOV-NL , HCOV-NH and HCOV-HKU1, or
or wherein the control is an anti -antigenic polypeptide 50 any combination of two or more of the foregoing antigenic
antibody titer produced in a subject who has been adminis polypeptides.
tered a recombinant or purified hMPV, PIV3, RSV, MeV
Vaccine efficacy may be assessed using standard analyses
and /or BetaCoV protein vaccine. In some embodiments , the (see, e.g. , Weinberg et al ., J Infect Dis. 2010 Jun . 1 ;
control is an anti-antigenic polypeptide antibody titer pro
201 ( 11 ): 1607-10 ). For example , vaccine efficacy may be
duced in a subject who has been administered a hMPV, 55 measured by double- blind, randomized , clinical controlled
PIV3, RSV,MeV and /or BetaCoV virus -like particle (VLP) trials . Vaccine efficacy may be expressed as a proportionate
vaccine (see, e.g., Cox R G et al., J Virol. 2014 June; 88 ( 11 ): reduction in disease attack rate (AR ) between the unvacci
6368-6379 ).
nated (ARU ) and vaccinated (ARV ) study cohorts and can
ARNA (e.g., mRNA ) vaccine of the present disclosure is be calculated from the relative risk (RR ) of disease among
administered to a subject in an effective amount ( an amount 60 the vaccinated group with use of the following formulas:
effective to induce an immune response ). In some embodi
Efficacy = (ARU - ARV )/ARUx100 ; and
ments, the effective amount is a dose equivalent to an at least
vated hMPV, PIV3, RSV , MeV and /or BetaCOV vaccine
2 - fold , at least 4 -fold , at least 10 - fold , at least 100 - fold , at
least 1000 - fold reduction in the standard of care dose of a
Efficacy = ( 1 - RR )x100 .
recombinant hMPV , PIV3, RSV , MeV and/or BetaCoV 65 Likewise , vaccine effectiveness may be assessed using
protein vaccine , wherein the anti-antigenic polypeptide anti standard analyses (see , e.g., Weinberg et al., J Infect Dis.
body titer produced in the subject is equivalent to an 2010 Jun . 1 ; 201 ( 11 ): 1607-10 ). Vaccine effectiveness is an
US 10,702,600 B1
17
assessment of how a vaccine (which may have already
18
In some embodiments , the subject is a young adult
proven to have high vaccine efficacy ) reduces disease in a between the ages of about 20 years and about 50 years ( e.g.,
population . This measure can assess the net balance of about 20 , 25 , 30 , 35 , 40 , 45 or 50 years old ).
In some embodiments, the subject is an elderly subject
benefits and adverse effects of a vaccination program , not
just the vaccine itself, under natural field conditions rather 5 about 60 years old , about 70 years old , or older (e.g., about
60, 65 , 70, 75 , 80, 85 or 90 years old ).
than in a controlled clinical trial. Vaccine effectiveness is
In some embodiments , the subject is has a chronic pul
proportional to vaccine efficacy (potency ) but is also
disease (e.g., chronic obstructive pulmonary disease
affected by how well target groups in the population are monary
(
COPD
)
asthma). Two forms of COPD include chronic
immunized , as well as by other non -vaccine -related factors 10 bronchitisor, which
involves a long-term cough with mucus ,
that influence the “ real-world outcomes of hospitalizations , and emphysema, which
involves damage to the lungs over
ambulatory visits , or costs . For example, a retrospective case time. Thus , a subject administered
(e.g. , mRNA )
control analysis may be used , in which the rates of vacci vaccine may have chronic bronchitis a orRNA
emphysema
.
nation among a set of infected cases and appropriate controls
In
some
embodiments
,
the
subject
has
been
exposed
are compared . Vaccine effectivenessmay be expressed as a 15 hMPV , PIV3, RSV , MeV , BetaCoV (e.g., selected fromto
rate difference, with use of the odds ratio (OR ) for devel MERS -CoV, SARS - CoV , HCOV -OC43 , HCV- 229E ,
oping infection despite vaccination :
HCOV-NL63 , HCOV-NL , HCOV-NH and HCOV-HKU1), or
any
combination of two ormore of the foregoing viruses ; the
Effectiveness = ( 1 -OR )x100 .
subject is infected with hMPV , PIV3, RSV, MeV , BetaCoV
In some embodiments, the efficacy (or effectiveness) of a 20 HCOV
(e.g., selected
from MERS-CoV, SARS- CoV,HCOV-OC43 ,
- 229E , HCOV-NL63, HCOV -NL , HCOV -NH and
RNA ( e.g., mRNA ) vaccine is at least 65 % , at least 70 % , at
least 75 % , at least 80 % , at least 85 % , or at least 90 % .
In some embodiments, the vaccine immunizes the subject
HCOV-HKU1), or any combination of two or more of the
foregoing viruses; or subject is at risk of infection by hMPV ,
PIV3, RSV ,MeV, BetaCoV ( e.g., selected from MERS- CoV,
against hMPV , PIV3, RSV, MeV, BetaCoV ( e.g., selected 25 SARS -CoV, HCV-OC43 , HCOV-229E , HCOV -NL63 ,
from MERS -CoV, SARS -CoV, HCV-OC43, HCOV - 229E ,
-NL , HCOV-NH and HCOV -HKU1), or any combina
HCOV -NL63 , HCOV -NL , HCOV -NH and HCOV-HKU1), or HCOV
tion
of
two or more of the foregoing viruses .
any combination of two or more of the foregoing viruses for
In
some
, the subject is immunocompro
up to 2 years . In some embodiments, the vaccine immunizes mised (has anembodiments
impaired immune system , e.g., has an immune
the subject against hMPV , PIV3, RSV ,MeV, BetaCoV (e.g., 30 disorder or autoimmune disorder).
selected from MERS - COV, SARS -CoV , HCOV -OC43 ,
In some embodiments the nucleic acid vaccines described
HCOV-229E , HCOV -NL63, HCOV -NL , HCOV -NH and
herein are chemically modified . In other embodiments the
foregoing viruses for more than 2 years,more than 3 years ,
Yet other aspects provide compositions for and methods
35 of vaccinating a subject comprising administering to the
more than 4 years , or for 5-10 years.
In some embodiments, the subject is about 5 years old or subject a nucleic acid vaccine comprising one ormore RNA
younger. For example , the subject may be between the ages polynucleotides having an open reading frame encoding a
of about 1 year and about 5 years ( e.g., about 1, 2 , 3 , 5 or first respiratory virus antigenic polypeptide , wherein the
5 years ), or between the ages of about 6 months and about RNA polynucleotide does not include a stabilization ele
HCOV -HKU1), or any combination of two or more of the
nucleic acid vaccines are unmodified .
1 year (e.g., about 6 , 7 , 8, 9, 10 , 11 or 12 months). In some 40 ment, and wherein an adjuvant is not coformulated or
embodiments , the subject is about 12 months or younger co -administered with the vaccine .
( e.g., 12 , 11, 10 , 9 , 8, 7 , 6 , 5 , 4 , 3 , 2 months or 1 month ). In
In other aspects the invention is a composition for or
some embodiments, the subject is about 6 months or method ofvaccinating a subject comprising administering to
younger .
the subject a nucleic acid vaccine comprising one or more
In some embodiments , the subject was born full term 45 RNA polynucleotides having an open reading frame encod
( e.g., about 37-42 weeks). In some embodiments, the subject ing a first antigenic polypeptide wherein a dosage of
was born prematurely , for example , at about 36 weeks of between 10 ug/kg and 400 ug /kg of the nucleic acid vaccine
gestation or earlier ( e.g., about 36 , 35 , 34 , 33 , 32 , 31, 30, 29, is administered to the subject. In some embodiments the
28 , 27 , 26 or 25 weeks). For example , the subjectmay have dosage of the RNA polynucleotide is 1-5 ug , 5-10 ug, 10-15
been born at about 32 weeks of gestation or earlier. In some 50 ug, 15-20 ug, 10-25 ug , 20-25 ug, 20-50 ug , 30-50 ug, 40-50
embodiments, the subject was born prematurely between ug , 40-60 ug , 60-80 ug , 60-100 ug , 50-100 ug , 80-120 ug ,
about 32 weeks and about 36 weeks of gestation . In such 40-120 ug, 40-150 ug , 50-150 ug, 50-200 ug , 80-200 ug,
subjects , a RNA (e.g., mRNA ) vaccine may be administered 100-200 ug, 120-250 ug, 150-250 ug, 180-280 ug , 200-300
later in life , for example , at the age of about 6 months to ug, 50-300 ug , 80-300 ug, 100-300 ug , 40-300 ug , 50-350
about 5 years, or older.
55 ug, 100-350 ug , 200-350 ug, 300-350 ug, 320-400 ug ,
In some embodiments , the subject is pregnant (e.g., in the 40-380 ug , 40-100 ug, 100-400 ug, 200-400 ug, or 300-400
first, second or third trimester ) when administered an RNA ug per dose. In some embodiments, the nucleic acid vaccine
(e.g., mRNA ) vaccine. Viruses such as hMPV, PIV3 and is administered to the subject by intradermal or intramus
RSV causes infections of the lower respiratory tract, mainly cular injection . In some embodiments, the nucleic acid
in infants and young children . One - third of RSV related 60 vaccine is administered to the subject on day zero . In some
deaths, for example, occur in the first year of life , with 99 embodiments , a second dose of the nucleic acid vaccine is
percent of these deaths occurring in low -resource countries . administered to the subject on day twenty one.
It's so widespread in the United States that nearly all
In some embodiments, a dosage of 25 micrograms of the
children become infected with the virus before their second RNA polynucleotide is included in the nucleic acid vaccine
birthdays. Thus, the present disclosure provides RNA (e.g., 65 administered to the subject. In some embodiments, a dosage
mRNA ) vaccines for maternal immunization to improve of 100 microgramsof the RNA polynucleotide is included in
mother - to -child transmission of protection against the virus. the nucleic acid vaccine administered to the subject. In some
US 10,702,600 B1
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20
embodiments, a dosage of 50 micrograms of the RNA ing a first antigenic polypeptide, wherein the RNA poly
polynucleotide is included in the nucleic acid vaccine nucleotide is present in the formulation for in vivo admin
administered to the subject. In some embodiments, a dosage istration to a host such that the level of antigen expression
of 75 microgramsof the RNA polynucleotide is included in in the host significantly exceeds a level of antigen expres
the nucleic acid vaccine administered to the subject. In some 5 sion produced by an mRNA vaccine having a stabilizing
embodiments, a dosage of 150 micrograms of the RNA element or formulated with an adjuvant and encoding the
polynucleotide is included in the nucleic acid vaccine first antigenic polypeptide .
administered to the subject. In some embodiments , a dosage
Other aspects provide nucleic acid vaccines comprising
of 400 micrograms of the RNA polynucleotide is included in one or more RNA polynucleotides having an open reading
the nucleic acid vaccine administered to the subject. In some 10 frame comprising at least one chemical modification or
embodiments, a dosage of 200 micrograms of the RNA optionally no nucleotide modification , the open reading
polynucleotide is included in the nucleic acid vaccine frame encoding a first antigenic polypeptide , wherein the
administered to the subject. In some embodiments, the RNA vaccine has at least 10 fold less RNA polynucleotide than is
polynucleotide accumulates at a 100 fold higher level in the required for an unmodified mRNA vaccine to produce an
local lymph node in comparison with the distal lymph node. 15 equivalent antibody titer. In some embodiments , the RNA
In other embodiments the nucleic acid vaccine is chemically polynucleotide is present in a dosage of 25-100 micrograms.
modified and in other embodiments the nucleic acid vaccine
Aspects of the invention also provide a unit of use
is not chemically modified .
vaccine, comprising between 10 ug and 400 ug of one or
Aspects of the invention provide a nucleic acid vaccine more RNA polynucleotides having an open reading frame
comprising one or more RNA polynucleotides having an 20 comprising at least one chemicalmodification or optionally
open reading frame encoding a first antigenic polypeptide ,
wherein the RNA polynucleotide does not include a stabi
lization element, and a pharmaceutically acceptable carrier
or excipient, wherein an adjuvant is not included in the
vaccine . In some embodiments, the stabilization element is 25
a histone stem -loop . In some embodiments, the stabilization
element is a nucleic acid sequence having increased GC
content relative to wild type sequence .
Aspects of the invention provide nucleic acid vaccines
comprising one or more RNA polynucleotides having an 30
open reading frame encoding a first antigenic polypeptide,
wherein the RNA polynucleotide is present in the formula
tion for in vivo administration to a host, which confers an
antibody titer superior to the criterion for seroprotection for
the first antigen for an acceptable percentage of human 35
subjects . In some embodiments , the antibody titer produced
by the mRNA vaccines of the invention is a neutralizing
antibody titer. In some embodiments the neutralizing anti
body titer is greater than a protein vaccine . In other embodi
ments the neutralizing antibody titer produced by themRNA 40
vaccines of the invention is greater than an adjuvanted
protein vaccine . In yet other embodiments the neutralizing
antibody titer produced by the mRNA vaccines of the
invention is 1,000-10,000 , 1,200-10,000 , 1,400-10,000 ,
no nucleotide modification , the open reading frame encod
ing a first antigenic polypeptide , and a pharmaceutically
acceptable carrier or excipient, formulated for delivery to a
human subject. In some embodiments , the vaccine further
comprises a cationic lipid nanoparticle .
Aspects of the invention provide methods of creating,
maintaining or restoring antigenic memory to a respiratory
virus strain in an individual or population of individuals
comprising administering to said individual or population an
antigenic memory booster nucleic acid vaccine comprising
( a ) at least one RNA polynucleotide, said polynucleotide
comprising at least one chemical modification or optionally
no nucleotide modification and wo or more codon -opti
mized open reading frames, said open reading frames encod
ing a set of reference antigenic polypeptides, and (b ) option
ally a pharmaceutically acceptable carrier or excipient. In
some embodiments, the vaccine is administered to the
individual via a route selected from the group consisting of
intramuscular administration , intradermal administration
and subcutaneous administration . In some embodiments , the
administering step comprises contacting a muscle tissue of
the subject with a device suitable for injection of the
composition . In some embodiments , the administering step
comprises contacting a muscle tissue of the subject with a
1,500-10,000 , 1,000-5,000 , 1,000-4,000, 1,800-10,000 , 45 device suitable for injection of the composition in combi
2000-10,000 , 2,000-5,000 , 2,000-3,000 , 2,000-4,000 , 3,000
nation with electroporation .
5,000 , 3,000-4,000 , or 2,000-2,500 . A neutralization titer is
Aspects of the invention provide methods of vaccinating
typically expressed as the highest serum dilution required to a subject comprising administering to the subject a single
dosage of between 25 ug/kg and 400 ug /kg of a nucleic acid
achieve a 50 % reduction in the number of plaques.
Also provided are nucleic acid vaccines comprising one 50 vaccine comprising one ormore RNA polynucleotides hav
or more RNA polynucleotides having an open reading frame ing an open reading frame encoding a first antigenic poly
encoding a first antigenic polypeptide, wherein the RNA peptide in an effective amount to vaccinate the subject.
polynucleotide is present in a formulation for in vivo admin
Other aspects provide nucleic acid vaccines comprising
istration to a host for eliciting a longer lasting high antibody one or more RNA polynucleotides having an open reading
titer than an antibody titer elicited by an mRNA vaccine 55 frame comprising at least one chemical modification , the
having a stabilizing element or formulated with an adjuvant open reading frame encoding a first antigenic polypeptide ,
and encoding the first antigenic polypeptide. In some wherein the vaccine has at least 10 fold less RNA poly
embodiments , the RNA polynucleotide is formulated to nucleotide than is required for an unmodified mRNA vac
produce a neutralizing antibodies within one week of a cine to produce an equivalent antibody titer. In some
single administration . In some embodiments, the adjuvant is 60 embodiments , the RNA polynucleotide is present in a dos
selected from a cationic peptide and an immunostimulatory age of 25-100 micrograms.
nucleic acid . In some embodiments , the cationic peptide is
Other aspects provide nucleic acid vaccines comprising
protamine.
an LNP formulated RNA polynucleotide having an open
Aspects provide nucleic acid vaccines comprising one or reading frame comprising no nucleotide modifications (un
more RNA polynucleotides having an open reading frame 65 modified ), the open reading frame encoding a first antigenic
comprising at least one chemicalmodification or optionally polypeptide, wherein the vaccine has at least 10 fold less
no nucleotide modification , the open reading frame encod RNA polynucleotide than is required for an unmodified
US 10,702,600 B1
21
22
mRNA vaccine not formulated in a LNP to produce an
equivalent antibody titer. In some embodiments, the RNA
polynucleotide is present in a dosage of 25-100 micrograms.
modifications, or is unmodified . In yet other embodiments
the at least one RNA polynucleotide encodes an antigenic
protein of any of SEQ ID NO : 5-8 , 12-13, 24-34, and 47-50
The data presented in the Examples demonstrate signifi
and includes at least one chemical modification . In other
cant enhanced immune responses using the formulations of 5 embodiments the RNA polynucleotide encodes an antigenic
the invention . Both chemically modified and unmodified protein of any of SEQ ID NO : 5-8 , 12-13, 24-34 , and 47-50
RNA vaccines are useful according to the invention . Sur and does not include any nucleotide modifications, or is
prisingly, in contrast to prior art reports that it was preferable unmodified .
to use chemically unmodified mRNA formulated in a carrier
preferred aspects, vaccines of the invention (e.g.,
for the production of vaccines, it is described herein that 10 LNPIn -encapsulated
mRNA vaccines ) produce prophylacti
chemically modified mRNA -LNP vaccines required a much cally- and /or therapeutically
- efficacious levels, concentra
lower effective mRNA dose than unmodified mRNA , i.e., tions and /or titers of antigen -specific
antibodies in the blood
tenfold less than unmodified mRNA when formulated in
or
serum
of
a
vaccinated
subject
.
As
defined
herein , the term
carriers other than LNP. Both the chemically modified and
unmodified RNA vaccines of the invention produce better 15 body
antibodyproduces
titer refers
to
the
amount
of
antigen
in s subject, e.g., a human-specific
subjectanti
. In
immune responses than mRNA vaccines formulated in a
exemplary
embodiments
,
antibody
titer
is
expressed
as
the
different lipid carrier.
In other aspects the invention encompasses a method of inverse of the greatest dilution (in a serial dilution ) that still
treating an elderly subject age 60 years or older comprising gives a positive result . In exemplary embodiments, antibody
ing one or more RNA polynucleotides having an open nosorbent assay (ELISA ). In exemplary embodiments, anti
reading frame encoding a respiratory virus antigenic poly
body titer is determined or measured by neutralization assay,
peptide in an effective amount to vaccinate the subject.
e.g., by microneutralization assay. In certain aspects, anti
In other aspects the invention encompasses a method of body titermeasurement is expressed as a ratio , such as 1:40,
treating a young subject age 17 years or younger comprising 25 1:100 , etc. In exemplary embodiments of the invention, an
administering to the subject a nucleic acid vaccine compris efficacious vaccine produces an antibody titer of greater than
ing one or more RNA polynucleotides having an open 1:40 , greater that 1 : 100 , greater than 1: 400 , greater than
reading frame encoding a respiratory virus antigenic poly 1 :1000 , greater than 1:2000 , greater than 1:3000 , greater
peptide in an effective amount to vaccinate the subject.
than 1 :4000 , greater than 1:500 , greater than 1 :6000 , greater
In
other
aspects
the
invention
encompasses
a
method
of
30
than
, greater than 1:10000. In exemplary embodi
treating an adult subject comprising administering to the ments1,:7500
the
antibody
titer is produced or reached by 10 days
subject a nucleic acid vaccine comprising one or more RNA
vaccination , by 20 days following vaccination , by
polynucleotides having an open reading frame encoding a following
respiratory virus antigenic polypeptide in an effective 30 days following vaccination , by 40 days following vac
35 cination , or by 50 or more days following vaccination . In
amount to vaccinate the subject.
embodiments, the titer is produced or reached
In some aspects the invention is a method of vaccinating exemplary
following
a
single dose of vaccine administered to the
a subject with a combination vaccine including at least two
nucleic acid sequences encoding respiratory antigens subject. In other embodiments , the titer is produced or
wherein the dosage for the vaccine is a combined therapeutic reached following multiple doses, e.g., following a first and
dosage wherein the dosage of each individual nucleic acid 40 a second dose (e.g.,a booster dose.) In exemplary aspects of
encoding an antigen is a sub therapeutic dosage. In some the invention , antigen -specific antibodies are measured in
embodiments, the combined dosage is 25 microgramsof the units of ug /ml or aremeasured in units of IU /L ( International
RNA polynucleotide in the nucleic acid vaccine adminis Units per liter) ormIU /ml (milli International Units per ml).
tered to the subject. In some embodiments, the combined In exemplary embodiments of the invention , an efficacious
dosage is 100 micrograms of the RNA polynucleotide in the 45 vaccine produces > 0.5 ug/ml, >0.1 ug/ml, >0.2 ug/ml, > 0.35
nucleic acid vaccine administered to the subject. In some ug/ml, >0.5 ug /ml, > 1 ug/ml, > 2 ug /ml, > 5 ug /ml or > 10
embodiments the combined dosage 50 microgramsof the ug/ml. In exemplary embodiments of the invention , an
RNA polynucleotide in the nucleic acid vaccine adminis efficacious vaccine produces > 10 mIU /ml, > 20 mIU /ml, > 50
tered to the subject. In some embodiments , the combined mIU /ml, > 100 mIU /ml, > 200 mIU /ml, > 500 mIU /ml or
dosage
is 75 micrograms of the RNA polynucleotide in the 50 > 1000 mIU /ml. In exemplary embodiments, the antibody
nucleic acid vaccine administered to the subject. In some level or concentration is produced or reached by 10 days
embodiments, the combined dosage is 150 micrograms of following vaccination , by 20 days following vaccination , by
the RNA polynucleotide in the nucleic acid vaccine admin 30
days following vaccination , by 40 days following vac
istered to the subject. In some embodiments, the combined cination
, or by 50 or more days following vaccination . In
dosage is 400 micrograms of the RNA polynucleotide in the 55 exemplary
embodiments , the level or concentration is pro
nucleic acid vaccine administered to the subject. In some duced or reached
following a single dose of vaccine admin
embodiments, the sub therapeutic dosage of each individual
nucleic acid encoding an antigen is 1, 2 , 3 , 4 , 5, 6 , 7 , 8 , 9 , istered to the subject. In other embodiments , the level or
10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , or 20 micrograms. In concentration is produced or reached following multiple
administering to the subject a nucleic acid vaccine compris- 20 titer is determined or measured by enzyme-linked immu
other embodiments the nucleic acid vaccine is chemically 60 doses
, e.g., following a first and a second dose ( e.g., a
booster dose.) In exemplary embodiments , antibody level or
modified and in other embodiments the nucleic acid vaccine
is not chemically modified .
The RNA polynucleotide is one of SEQ ID NO : 1-4 , 9-12 ,
20-23 , 35-46, 57-61, and 64-80 and includes at least one
chemical modification . In other embodiments the RNA 65
polynucleotide is one of SEQ ID NO : 1-4 , 9-12 , 20-23 ,
35-46 , 57-61, and 64-80 and does not include any nucleotide
concentration is determined or measured by enzyme-linked
immunosorbent assay (ELISA ). In exemplary embodiments,
antibody level or concentration is determined or measured
by neutralization assay, e.g., by microneutralization assay .
The details of various embodiments of the disclosure are
set forth in the description below . Other features, objects,
US 10,702,600 B1
23
and advantages of the disclosure will be apparent from the
description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
24
IL - 10 ( FIG . 8A ), TNF- a (FIG . 8B ), IL4 (FIG . 8C ), IL -5
(FIG . 8D ) and IL -6 (FIG . 8E ).
FIGS. 9A -9B are graphs showing the results of cotton rat
challenge experiments. Two different doses of the hMPV
5 mRNA vaccines were used (2 ug or 10 ug doses ) to
The foregoing and other objects , features and advantages immunize the cotton rats before challenge. The hMPV
will be apparent from the following description of particular mRNA vaccines reduced the viral titer in the lung and nose
embodiments of the disclosure, as illustrated in the accom of the cotton rat, with the 10 ug dose being more effective
panying drawings in which like reference characters refer to in reducing viral titer. Use of a 10 ug dose resulted in 100 %
the sameparts throughout the different views. The drawings 10 titer
protectionofina the
lung and a ~ 2 log reduction in nose viral
2 ug dose resulted in a 1 log reduction in lung
are not necessarily to scale , emphasis instead being placed vital. Use
titer and no reduction in nose viral titer. The vaccine
upon illustrating the principles of various embodiments of was administered
on Day 0 , and a boost was administered on
the disclosure .
FIG . 1 shows a schematic of one example of a RNA ( e.g. 15 DayFIG21.. 10 is a graph showing the lung histopathology of
mRNA) vaccine construct of the present disclosure . The
cotton rats that received hMPV mRNA vaccines. Pathology
construct depicts a human metapneumovirus and human associated
with vaccine -enhanced disease was not observed
respiratory syncytial virus full length fusion protein obtained in immunized
groups.
from wild - type strains ( The Journal of General Virology .
FIG . 11 is a graph showing hMPV neutralization antibody
2008 ; 89 (Pt 12 ):3113-3118 , incorporated herein by refer- 20 titers in cotton rats that received hMPV mRNA vaccines (2
ence ).
ug or 10 ug doses) on days 35 and 42 post immunization .
FIGS. 2A - 2C are graphs showing the levels of anti-hMPV
FIG . 12 is a graph showing the lung and nose viral load
fusion protein -specific antibodies in the serum of mice in cotton rats challenged with a hMPV /A2 strain after
immunized with hMPV mRNA vaccines on day 0 ( FIG . 2A ), immunization with the indicated mRNA vaccines (hMPV
day 14 (FIG . 2B ) and day 35 (FIG . 2C ) post immunization . 25 mRNA vaccine or hMPV /PIV mRNA combination vaccine ).
The mice were immunized with a single dose ( 2 ug or 10 ug) Vaccinated cotton rats showed reduced lung and nose viral
on day 0 and were given a boost dose ( 2 ug or 10 ug) on day loads after challenge, compared to control.
21. hMPV fusion protein -specific antibodies were detected
FIG . 13 is a graph showing the lung and nose viral load
at up to 1: 10000 dilution of serum on day 35 for both doses . in cotton rats challenged with PIV3 strain after immuniza
FIGS. 3A -3C are graphs showing the result of IgG 30 tion with indicated mRNA vaccines ( PIV mRNA vaccine or
isotyping in the serum of mice immunized with hMPV hMPV /PIV combination vaccine). Vaccinated cotton rats
mRNA vaccines. The levels of hMPV fusion protein -specific
IgG2a (FIG . 3 . and IgG1 (FIG . 3B ) antibodies in the serum
are measured by ELISA . FIG . 3C shows that hMPV fusion
showed reduced lung and nose viral loads after challenge ,
control.
FIG . 14 is a graph showing hMPV neutralizing antibody
protein mRNA vaccine induced a mixed Th1/ Th2 cytokine 35 titers in cotton rats that received different dosages of hMPV
response with a Th1 bias.
mRNA vaccines or hMPV / PIV combination mRNA vac
FIG . 4 is a graph showing in vitro neutralization of a cines on day 42 post immunization . The dosages of the
hMPV B2 strain (TN /91-316 ) using the sera ofmice immu
vaccine are indicated in Table 9 .
nized with a mRNA vaccine encoding hMPV fusion protein .
FIG . 15 is a graph showing PIV3 neutralizing antibody
Mouse serum obtained from mice receiving a 10 ug or a 2 40 titers in cotton rats that received different dosages of PIV
ug dose contained hMPV -neutralizing antibodies.
mRNA vaccines or hMPV / PIV combination mRNA vac
FIGS.5A -5C are graphs showing a Th1 cytokine response cines on day 42 post immunization . The dosages of the
induced by a hMPV fusion peptide pool (15 -mers -50 (over vaccine are indicated in Table 9 .
lap )) in splenocytes isolated from mice immunized with the
FIG . 16 is a graph showing the lung histopathology score
compared
hMPV mRNA vaccines . Virus - free media was used as a 45 of cotton rats immunized with hMPV mRNA vaccines, PIV
negative control and Concanavalin A (ConA , a positive
control for splenocyte stimulation ) was included . The cytok
mRNA vaccines or hMPV /PIV combination mRNA vac
cines as indicated in Table 9. Low occurrence of alevolitis
ines tested included IFN -Y (FIG . 5A ), IL - 2 (FIG . 5B ) and and interstitialpneumonia was observed , indicating no anti
body -dependent enhancement (ADE ) of hMPV associated
IL12 (FIG . 5C ).
FIGS . 6A -6E are graphs showing the Th2 cytokine 50 diseases.
response induced by a hMPV fusion peptide pool ( 15 -mers
FIG . 17 is a graph showing the reciprocal MERS - CoV
50 ) in splenocytes isolated from mice immunized with the neutralizing antibody titers in mice immunized with beta
hMPV mRNA vaccines . Virus - free media was used as a coronavirus mRNA vaccine encoding the MERS - CoV full
negative control and Concanavalin Awas also included . The length Spike protein , on days 0 , 21 , 42 , and 56 post
cytokines tested included IL - 10 (FIG . 6A ), TNF - a (FIG . 55 immunization .
6B ), IL4 (FIG . 6C ), IL -5 (FIG . 6D ) and IL -6 (FIG . 6E ).
FIG . 18 is a graph showing the reciprocal MERS -CoV
FIGS. 7A -7C are graphs showing the Thl response neutralizing antibody titers in mice immunized with beta
induced by inactivated hMPV virus in splenocytes isolated coronavirus mRNA vaccine encoding either theMERS-CoV
from mice immunized with hMPV mRNA vaccines . Virus
full- length Spike protein , or the S2 subunit of the Spike
free media was used as a negative control and Concanavalin 60 protein . The full length spike protein induced a stronger
A was included . The cytokines tested included IFN - Y ( FIG .
7A ), IL - 2 (FIG . 7B ) and IL 12 (FIG . 7C ).
FIGS. 8A -8E are graphs showing the Th2 response
induced by inactivated hMPV virus in splenocytes isolated
from mice immunized with the hMPV mRNA vaccines. 65
Virus - free media was used as a negative control and Con
canavalin A was included . The cytokines tested include
immune response compared to the S2 subunit alone .
FIGS. 19A - 19C are graphs showing the viral load in the
nose and throat, the bronchoalveolar lavage (BAL ), or the
lungs of New Zealand white rabbits 4 days post challenge
with MERS -CoV. The New Zealand white rabbits were
immunized with one 20 ug -dose (on day 0 ) or two 20
ug -doses ( on day 0 and 21) ofMERS-CoV mRNA vaccine
US 10,702,600 B1
25
26
encoding the full-length Spike protein before challenge.
(e.g., mRNA) vaccines . In some embodiments , a RNA ( e.g.,
FIG . 19A shows that two doses of MERS -CoV mRNA
mRNA ) vaccine comprises an adjuvant, such as a flagellin
vaccine resulted in a 3 log reduction of viral load in the nose
adjuvant, as provided herein .
viral load in the BAL of theNew Zealand white rabbits . FIG .
19C show one dose ofMERS -CoV mRNA vaccine resulted
in a 2 log reduction of viral load, while two doses of
MERS -CoV mRNA vaccine resulted in an over 4 log
balanced immune response, comprising both cellular and
humoral immunity , without many of the risks associated
with DNA vaccination .
The entire contents of International Application No. PCT/
and led to complete protection in the throat of the New
The RNA ( e.g., mRNA ) vaccines (e.g., hMPV , PIV3,
Zealand white rabbits . FIG . 19B shows that two doses of 5 RSV, MeV , BetaCOV RNA vaccines and combinations
MERS -CoV mRNA vaccine resulted in a 4 log reduction of thereof), in some embodiments , may be used to induce a
10
reduction of viral load in the lungs of the New Zealand white
rabbits .
FIGS . 20A - 20B are images and graphs showing viral load
US2015 /02740 is incorporated herein by reference .
Human Metapneumovirus (HMPV )
hMPV shares substantial homology with respiratory syn
Zealand white rabbits 4 days post challenge with MERS
protein ( F ) is related to other paramyxovirus fusion proteins
or replicating virus detected by PCR in the lungs of New 15 cytial virus (RSV) in its surface glycoproteins. hMPV fusion
COV . The New Zealand white rabbits were immunized with
and appears to have homologous regions that may have
a single 20 ug dose (on day 0 , Group 1a ) of MERS- CoV similar functions. The hMPV fusion protein amino acid
mRNA vaccine encoding the full -length Spike protein , two sequence contains features characteristic of other paramyxo
20 ug doses (on day 0 and 21, Group 1b ) of MERS -CoV 20 virus F proteins, including a putative cleavage site and
mRNA vaccine encoding the full -length Spike protein , or potential N - linked glycosylation sites. Paramyxovirus
placebo (Group 2 ) before challenge . FIG . 20A shows that fusion proteins are synthesized as inactive precursors (FO )
two doses of 20 ug a MERS-CoV mRNA vaccine reduced that are cleaved host cell proteases into the biologically
over 99 % (2 log ) of viruses in the lungs of New Zealand fusion -active F1 and F2 domains (see , e.g., Cseke G. et al.
white rabbits. FIG . 20B shows that the groupug of New 25 Journal of Virology 2007 ; 81(2):698-707, incorporated
Zealand white rabbits that received 2 doses of 20 MERS
herein by reference). hMPV has one putative cleavage site,
CoV mRNA vaccine did not have any detectable replicating
MERS -CoV virus in their lungs .
FIG . 21 is a graph showing the MERS - CoV neutralizing
antibody titers in New Zealand white rabbits immunized
in contrast to the two sites established for RSV F , and only
shares 34 % amino acid sequence identity with RSV F. F2 is
extracellular and disulfide linked to F1. Fusion proteins are
type I glycoproteins existing as trimers ,with two 4-3 heptad
30
with MERS- CoV mRNA vaccine encoding the full -length
repeat domains at the N- and C - terminal regions of the
Spike protein . Immunization of the in New Zealand white
rabbits were carried out as described in FIGS. 21A -21C . The
results show that two doses of 20 ug MERS-CoV mRNA
vaccine induced a significant amount of neutralizing anti
bodies against MERS -CoV (EC50 between 500-1000 ). The
MERS-CoV mRNA vaccine induced antibody titer is 3-5
fold better than any other vaccines tested in the samemodel.
DETAILED DESCRIPTION
protein (HR1 and HR2), which form coiled - coil alpha
35
helices. These coiled coils becomeapposed in an antiparallel
fashion when the protein undergoes a conformational
change into the fusogenic state. There is a hydrophobic
fusion peptide N proximal to the N -terminal heptad repeat ,
which is thought to insert into the target cell membrane,
while the association of the heptad repeats brings the trans
40 membrane domain into close proximity , inducing membrane
fusion (see , e.g., Baker, K A et al. Mol. Cell 1999 ; 3: 309
319 ). This mechanism has been proposed for a number of
The present disclosure provides, in some embodiments , different viruses , including RSV, influenza virus, and human
vaccines that comprise RNA (e.g.,mRNA polynucleotides immunodeficiency virus . Fusion proteins are major anti
encoding a human metapneumovirus (hMPV ) antigenic 45 genic determinants for all known paramyxoviruses and for
polypeptide , a parainfluenza virus type 3 ( PIV3) antigenic other viruses that possess similar fusion proteins such as
polypeptide, a respiratory syncytial virus (RSV ) antigenic human immunodeficiency virus, influenza virus , and Ebola
polypeptide, a measles virus (MeV ) antigenic polypeptide, virus .
or a betacoronavirus antigenic polypeptide (e.g., Middle
In some embodiments , a hMPV vaccine of the present
East respiratory syndrome coronavirus (MERS -CoV ), 50 disclosure comprises a RNA (e.g. ,mRNA ) polynucleotide
SARS -CoV, human coronavirus (HCOV )-OC43 , HCOV
encoding hMPV fusion protein (F ). In some embodiments,
229E , HCOV -NL63, HCOV -NL , HCOV-NH (New Haven )
and HCOV -HKU1) (see , e.g., Esper F. et al. Emerging
Infectious Diseases, 12 (5 ), 2006 ; and Pyrc K. et al. Journal
a hMPV vaccine of the present disclosure comprises a RNA
( e.g.,mRNA ) polynucleotide encoding a F1 or F2 subunit of
a hMPV F protein . In some embodiments , a hMPV vaccine
of Virology, 81 (7):3051-57, 2007 , the contents of each of 55 of the present disclosure comprises a RNA ( e.g. , mRNA )
which is here incorporated by reference in their entirety ). polynucleotide encoding hMPV glycoprotein (G ). In some
The present disclosure also provides , in some embodiments, embodiments, a hMPV vaccine of the present disclosure
combination vaccines that comprise at least one RNA (e.g., comprises a RNA (e.g., mRNA ) polynucleotide encoding
mRNA ) polynucleotide encoding at least two antigenic hMPV matrix protein (M ). In some embodiments , a hMPV
polypeptides selected from hMPV antigenic polypeptides, 60 vaccine of the present disclosure comprises a RNA (e.g.,
PIV3 antigenic polypeptides, RSV antigenic polypeptides, mRNA ) polynucleotide encoding hMPV phosphoprotein
MeV antigenic polypeptides and BetaCoV antigenic poly
(P ). In some embodiments, a hMPV vaccine of the present
peptides . Also provided herein are methods of administering disclosure comprises a RNA (e.g. ,mRNA) polynucleotide
the RNA (e.g. , mRNA ) vaccines,methods of producing the encoding hMPV nucleoprotein (N ). In some embodiments,
RNA (e.g., mRNA ) vaccines, compositions (e.g., pharma- 65 a hMPV vaccine of the present disclosure comprises a RNA
ceutical compositions ) comprising the RNA ( e.g., mRNA ) ( e.g., mRNA) polynucleotide encoding hMPV SH protein
vaccines , and nucleic acids ( e.g., DNA ) encoding the RNA (SH ).
US 10,702,600 B1
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28
In some embodiments , a hMPV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein , G protein , M protein , P protein , N
protein and SH protein .
In some embodiments , a hMPV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein and G protein . In some embodiments , a
hMPV vaccine of the present disclosure comprises a RNA
( e.g., mRNA ) polynucleotide encoding F protein and M
protein . In some embodiments , a hMPV vaccine of the
present disclosure comprises a RNA (e.g., mRNA ) poly
nucleotide encoding F protein and P protein .
In some embodiments , a hMPV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein and N protein . In some embodiments , a
hMPV vaccine of the present disclosure comprises a RNA
(e.g., mRNA ) polynucleotide encoding F protein and SH
protein .
In some embodiments, a hMPV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding G protein and M protein . In some embodiments , a
hMPV vaccine of the present disclosure comprises a RNA
(e.g., mRNA) polynucleotide encoding G protein and P
protein . In some embodiments, a hMPV vaccine of the
present disclosure comprises a RNA ( e.g., mRNA ) polynucleotide encoding G protein and N protein . In some
embodiments, a hMPV vaccine of the present disclosure
comprises a RNA ( e.g. , mRNA ) polynucleotide encoding G
protein and SH protein .
In some embodiments, a hMPV vaccine of the present
disclosure comprises a RNA (e.g. , mRNA ) polynucleotide
encoding F protein , G protein and M protein . In some
embodiments, a hMPV vaccine of the present disclosure
comprises a RNA (e.g.,mRNA ) polynucleotide encoding F
protein , G protein and P protein . In some embodiments, a
hMPV vaccine of the present disclosure comprises a RNA
(e.g., mRNA ) polynucleotide encoding F protein , G protein
and N protein . In some embodiments , a hMPV vaccine of
the present disclosure comprises a RNA ( e.g., mRNA )
Virology 2008 ; 89: 975-83 ; Peret T C T et al . The Journal of
Infectious Disease 2002; 185 : 1660-63 , incorporated herein
by reference ). In some embodiments, at least one antigenic
polypeptide is obtained from the CAN98-75 (CAN75 )
5 hMPV strain . In some embodiments , at least one antigenic
polypeptide is obtained from the CAN97-83 (CAN83)
hMPV strain . In some embodiments, at least one antigenic
polypeptide is obtained from hMPV isolate TN /92-4 ( e.g.,
SEQ ID NO : 1 and 5 ). In some embodiments, at least one
10 antigenic polypeptide is obtained from hMPV isolate NL /1 /
99 (e.g., SEQ ID NO : 2 and 6 ). In some embodiments, at
least one antigenic polypeptide is obtained from hMPV
isolate PER /CF10497/2010 /B ( e.g. , SEQ ID NO : 3 and 7 ).
In some embodiments, hMPV vaccines comprise RNA
15 (e.g., mRNA ) polynucleotides encoding a hMPV antigenic
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with hMPV F protein
and having F protein activity .
A protein is considered to have F protein activity if, for
20 example , the protein acts to fuse the viral envelope and host
cell plasmamembrane , mediates viral entry into a host cell
via an interaction with arginine - glycine-aspartate RGD
binding integrins, or a combination thereof (see , e.g., Cox R
G et al. J Virol . 2012 ; 88( 22 ):12148-60 , incorporated herein
25 by reference ).
In some embodiments , hMPV vaccines comprise RNA
(e.g., mRNA ) polynucleotides encoding hMPV antigenic
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99% identity with hMPV G protein
30 and having G protein activity .
A protein is considered to have G protein activity if, for
example , the protein acts to modulate (e.g. , inhibit ) hMPV
induced cellular (immune ) responses (see, e.g., Bao X et al.
PLoS Pathog. 2008 ; 4 (5 ):e1000077 , incorporated herein by
35 reference ).
Human Parainfluenza Virus Type 3 (PIV3)
Parainfluenza viruses belong to the family Paramyxoviri
dae . These are enveloped viruses with a negative -sense
single -stranded RNA genome. Parainfluenza viruses belong
polynucleotide encoding F protein , G protein and SH pro- 40 to the subfamily Paramyxoviridae , which is subdivided into
tein .
three genera: Respirovirus (PIV - 1, PIV -3, and Sendai virus
A hMPV vaccine may comprise, for example , at least one (SeV )), Rubulavirus (PIV - 2, PIV -4 and mumps virus) and
RNA (e.g., mRNA ) polynucleotide having an open reading Morbillivirus (measles virus, rinderpest virus and canine
frame encoding at least one hMPV antigenic polypeptide distemper virus (CDV )). Their genome, a ~ 15 500 nucleo
identified by any one of SEQ ID NO : 5-8 ( Table 3 ; see also 45 tide-long negative -sense RNA molecule , encodes two enve
lope glycoproteins, the hemagglutinin -neuraminidase (HN ),
amino acid sequences of Table 4 ).
A hMPV vaccine may comprise , for example, at least one the fusion protein (F or FO ), which is cleaved into F1 and F2
RNA (e.g., mRNA ) polynucleotide encoded by a nucleic subunits, a matrix protein (M ), a nucleocapsid protein (N )
acid (e.g., DNA ) identified by any one of SEQ ID NO : 1-4 and several nonstructural proteins including the viral repli
50 case (L ).All parainfluenza viruses , except for PIV -1, express
( Table 2 ).
The present disclosure is not limited by a particular strain a non -structural V protein that blocks IFN signaling in the
of hMPV . The strain of hMPV used in a vaccine may be any
infected cell and acts therefore as a virulence factor (see ,
strain of hMPV. Non - limiting examples of strains of hMPV e.g., Nishio M et al. J Virol. 2008; 82 ( 13 ):6130-38 ).
for use as provide herein include the CAN98-75 (CAN75 )
M H et al. J Virol. 20014 ; 78 ( 13 )6927-37, incorporated
PIV3 hemagglutinin -neuraminidase (HN ), a structural
for attachment and cell entry . It recognizes and binds to
and the CAN97-83 (CAN83) hMPV strains ( Skiadopoulos 55 protein , is found on the viral envelope, where it is necessary
herein by reference ), a hMPV A1, A2, B1 or B2 strain (see ,
e.g., de Graaf M et al . The Journal of General Virology
2008; 89 :975-83 ; Peret TC T et al. The Journal of Infectious
Disease 2002 ; 185: 1660-63, incorporated herein by refer- 60
ence ), a hMPV isolate TN / 92-4 (e.g. , SEQ ID NO : 1 and 5 ),
a hMPV isolate NL /1 /99 (e.g., SEQ ID NO : 2 and 6 ), or a
sialic acid -containing receptors on the host cell's surface .As
a neuroaminidase , HN removes sialic acid from virus par
ticles , preventing self -aggregation of the virus, and promot
ing the efficient spread of the virus. Furthermore , HN
promotes the activity of the fusion ( F or F0 ) protein ,
contributing to the penetration of the host cell's surface .
hMPV isolate PER /CFI0497 /2010 /B ( e.g., SEQ ID NO : 3
PIV3 fusion protein (PIV3 F ) is located on the viral
and 7 ) .
envelope, where it facilitates the viral fusion and cell entry .
In some embodiments , at least one hMPV antigenic 65 The F protein is initially inactive, but proteolytic cleavage
polypeptide is obtained from a hMPV A1, A2, B1 or B2 leads to its active forms, F1 and F2, which are linked by
strain (see , e.g., de Graaf M et al. The Journal of General disulfide bonds. This occurs when the HN protein binds its
US 10,702,600 B1
29
30
receptor on the host cell's surface. During early phases of
infection , the F glycoprotein mediates penetration of the
host cell by fusion of the viral envelope to the plasma
membrane . In later stages of the infection , the F protein
In some embodiments , a PIV3 vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding HN protein and M protein . In some embodiments ,
a PIV3 vaccine of the present disclosure comprises a RNA
uninfected cells,which leads to the formation of a syncytium
protein . In some embodiments , a PIV3 vaccine of the
present disclosure comprises a RNA ( e.g., mRNA ) poly
nucleotide encoding HN protein and N protein .
facilitates the fusion of the infected cells with neighboring 5 (e.g., mRNA ) polynucleotide encoding HN protein and P
and spread of the infection .
PIV3 matrix protein (M ) is found within the viral enve
In some embodiments , a PIV3 vaccine of the present
lope and assists with viral assembly . It interacts with the
nucleocapsid
and envelope glycoproteins, where it facili- 10 encoding
disclosure Fcomprises
a RNA ( e.g. ,mRNA) polynucleotide
protein , HN protein and M protein . In some
tates the budding of progeny viruses through its interactions embodiments, a PIV3 vaccine of the present disclosure
with specific sites on the cytoplasmic tail of the viral comprises
RNA ( e.g., mRNA ) polynucleotide encoding F
glycoproteins and nucleocapsid . It also plays a role in protein , HNa protein
and P protein . In some embodiments, a
transporting viral components to the budding site .
15
PIV3
vaccine
of
the
present disclosure comprises a RNA
PIV3 phosphoprotein (P ) and PIV3 large polymerase (e.g. , mRNA ) polynucleotide
encoding F protein , HN pro
protein (L ) are found in the nucleocapsid where they form tein and N protein .
part of the RNA polymerase complex . The L protein , a viral
A PIV3 vaccine may comprise , for example , at least one
RNA -dependent RNA polymerase, facilitates genomic tran
RNA ( e.g., mRNA) polynucleotide having an open reading
scription , while the host cell's ribosomes translate the viral 20 frame encoding at least one PIV3 antigenic polypeptide
identified by any one of SEQ ID NO : 12-13 ( Table 6 ; see
mRNA into viral proteins .
PIV3 V is a non -structural protein that blocks IFN sig
naling in the infected cell, therefore acting as a virulence
also amino acid sequences of Table 7 ).
A PIV3 vaccine may comprise , for example , at least one
The encapsidated genomic RNA is termed the NC and
of PIV3. The strain of PIV3 used in a vaccine may be any
RNA (e.g., mRNA ) polynucleotide encoded by a nucleic
PIV3 nucleoprotein (N ) encapsidates the genome in a 25 acid (e.g., DNA ) identified by any one of SEQ ID NO : 9-12
ratio of 1 N per 6 ribonucleotides, protecting it from
( Table 5 ; see also nucleic acid sequences of Table 7 ).
nucleases. The nucleocapsid (NC ) has a helical structure.
The present disclosure is not limited by a particular strain
factor.
serves as template for transcription and replication . During
replication , encapsidation by PIV3 N is coupled to RNA 30
synthesis and all replicative products are resistant to nucle
ases. PIV3 N homo-multimerizes to form the nucleocapsid
and binds to viral genomic RNA . PIV3 N binds the P protein
and thereby positions the polymerase on the template.
In some embodiments, a PIV3 vaccine of the present 35
disclosure comprises a RNA (e.g. , mRNA ) polynucleotide
encoding PIV3 fusion protein (F ). In some embodiments, a
PIV3 vaccine of the present disclosure comprises a RNA
(e.g., mRNA ) polynucleotide encoding a F1 or F2 subunit of
a PIV3 F protein . In some embodiments , a PIV3 vaccine of 40
the present disclosure comprises RNA (e.g., mRNA )
polynucleotide encoding PIV3 hemagglutinin -neuramini
dase (HN ) (see, e.g., van Wyke Coelingh K L et al. J Virol.
1987 ; 61 (5 ) :1473-77 , incorporated herein by reference ). In
some embodiments, a PIV3 vaccine of the present disclosure 45
comprises a RNA (e.g., mRNA ) polynucleotide encoding
PIV3 matrix protein (M ). In some embodiments , a PIV3
vaccine of the present disclosure comprises a RNA (e.g.,
mRNA ) polynucleotide encoding PIV3 phosphoprotein (P ) .
In some embodiments , a PIV3 vaccine of the present dis- 50
closure comprises a RNA ( e.g., mRNA) polynucleotide
encoding PIV3 nucleoprotein ( N ).
In some embodiments, a PIV3 vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
strain of PIV3 . A non-limiting example of a strain of PIV3
for use as provide herein includes HPIV3 /Homo sapiens /
PER / FLA4815 /2008 .
In some embodiments, PIV3 vaccines comprise RNA
( e.g., mRNA) polynucleotides encoding a PIV3 antigenic
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with PIV3 F protein and
having F protein activity .
In some embodiments, PIV3 vaccines comprise RNA
(e.g., mRNA) polynucleotides encoding PIV3 antigenic
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with PIV3 hemagglu
tinin -neuraminidase (HN ) and having hemagglutinin
neuraminidase activity .
A protein is considered to have hemagglutinin -neuramini
dase activity if, for example , it is capable of both receptor
binding and receptor cleaving . Such proteins are major
surface glycoproteins that have functional sites for cell
attachment and for neuraminidase activity. They are able to
cause red blood cells to agglutinate and to cleave the
glycosidic linkages of neuraminic acids, so they have the
potential to both bind a potential host cell and then release
the cell if necessary, for example, to prevent self -aggrega
tion of the virus.
In some embodiments , PIV3 vaccines comprise RNA
(e.g., mRNA ) polynucleotides encoding PIV3 antigenic
encoding F protein , HN protein, M protein , P protein , and N 55 polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with PIV3 HN , F (e.g.,
In some embodiments , a PIV3 vaccine of the present F , F1 or F2 ), M , N , L or V and having HN , F (e.g., F, F1 or
disclosure comprises a RNA (e.g., mRNA polynucleotide F2), M , N , L or V activity, respectively.
encoding F protein and HN protein . In some embodiments, Respiratory Syncytial Virus (RSV )
a PIV3 vaccine of the present disclosure comprises a RNA 60 RSV is a negative -sense, single- stranded RNA virus of the
(e.g., mRNA ) polynucleotide encoding F protein and M
genus Pneumovirinae . The virus is present in at least two
protein . In some embodiments, a PIV3 vaccine of the antigenic subgroups, known as Group A and Group B ,
present disclosure comprises a RNA ( e.g., mRNA ) poly
primarily resulting from differences in the surface G glyco
nucleotide encoding F protein and P protein . In some proteins. Two RSV surface glycoproteins G and F -me
embodiments, a PIV3 vaccine of the present disclosure 65 diate attachment with and attachment to cells of the respi
comprises a RNA (e.g.,mRNA ) polynucleotide encoding F ratory epithelium . F surface glycoproteins mediate
protein and N protein .
coalescence of neighboring cells . This results in the forma
protein .
US 10,702,600 B1
31
32
encoding G protein and N protein . In some embodiments , a
RSV vaccine of the present disclosure comprises a RNA
( e.g., mRNA) polynucleotide encoding G protein and M2
protein . In some embodiments , a RSV vaccine of the present
disclosure comprises a RNA ( e.g., mRNA ) polynucleotide
encoding G protein and M protein .
In some embodiments, a RSV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein , G protein and L protein . In some
embodiments, a RSV vaccine of the present disclosure
comprises a RNA (e.g., mRNA ) polynucleotide encoding F
protein , G protein and P protein . In some embodiments , a
RSV vaccine of the present disclosure comprises a RNA
protein M is found in the inner layer of the lipid bilayer and 15 and
(e.g.,N mRNA
) polynucleotide encoding F protein , G protein
protein . In some embodiments, a RSV vaccine of the
assists virion formation . Nucleocapsid proteins L , P, N , and
M2 modulate replication and transcription of the RSV present disclosure comprises a RNA ( e.g., mRNA ) poly
genome. It is thought that glycoprotein G tethers and stabi nucleotide encoding F protein , G protein and M2 protein . In
lizes the virus particle at the surface of bronchial epithelial some embodiments, a RSV vaccine of the present disclosure
cells, while glycoprotein F interacts with cellular gly- 20 comprises a RNA ( e.g., mRNA ) polynucleotide encoding F
cosaminoglycans to mediate fusion and delivery of the RSV protein , G protein and M protein .
virion contents into the host cell (Krzyzaniak M A et al.
The present disclosure is not limited by a particular strain
tion of syncytial cells . RSV is the most common cause of
bronchiolitis. Most infected adults develop mild cold -like
symptoms such as congestion , low - grade fever, and wheez
ing . Infants and small children may suffer more severe
symptoms such as bronchiolitis and pneumonia. The disease 5
may be transmitted among humans via contact with respi
ratory secretions.
The genome of RSV encodes at least three surface gly
coproteins, including F, G , and SH , four nucleocapsid pro
teins, including L , P , N , and M2, and one matrix protein ,M. 10
Glycoprotein F directs viral penetration by fusion between
the virion and the host membrane. Glycoprotein G is a type
II transmembrane glycoprotein and is the major attachment
protein . SH is a short integral membrane protein . Matrix
PLoS Pathog 2013 ; 9 (4 )).
of RSV . The strain of RSV used in a vaccine may be any
strain of RSV .
disclosure comprises a RNA (e.g., mRNA ) polynucleotide 25 In some embodiments , RSV vaccines comprise RNA
encoding F protein . In some embodiments , a PIV3 vaccine (e.g., mRNA) polynucleotides encoding a RSV antigenic
In some embodiments, a RSV vaccine of the present
of the present disclosure comprises a RNA (e.g. , mRNA )
polynucleotide encoding G protein . In some embodiments , a
PIV3 vaccine of the present disclosure comprises a RNA
(e.g., mRNA ) polynucleotide encoding L protein . In some 30
embodiments , a PIV3 vaccine of the present disclosure
comprises a RNA (e.g., mRNA ) polynucleotide encoding P
protein . In some embod its , a PIV3 vaccine of the
present disclosure comprises a RNA ( e.g., mRNA) poly
nucleotide encoding N protein . In some embodiments , a 35
PIV3 vaccine of the present disclosure comprises a RNA
( e.g., mRNA ) polynucleotide encoding M2 protein . In some
embodiments , a PIV3 vaccine of the present disclosure
comprises a RNA (e.g., mRNA ) polynucleotide encoding M
protein .
40
In some embodiments, a RSV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein , G protein , L protein, P protein, N
protein , M2 protein and M protein .
In some embodiments, a RSV vaccine of the present 45
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding F protein and G protein . In some embodiments, a
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with RSV F protein and
having F protein activity .
In some embodiments , RSV vaccines comprise RNA
( e.g., mRNA ) polynucleotides encoding RSV antigenic
polypeptides having at least 95 % , at least 96 % , at least 97 % ,
at least 98 % or at least 99 % identity with RSV G protein and
having G protein activity.
A protein is considered to have G protein activity if, for
example , the protein acts to modulate (e.g. , inhibit) hMPV
induced cellular ( immune ) responses ( see, e.g., Bao X et al.
PLoS Pathog. 2008 ; 4 (5 ):e1000077, incorporated herein by
reference ).
Measles Virus (MeV ) Molecular epidemiologic investiga
tions and virologic surveillance contribute notably to the
control and prevention of measles. Nearly half ofmeasles
related deaths worldwide occur in India , yet virologic sur
veillance data are incomplete for many regions of the
country. Previous studies have documented the presence of
measles virus genotypes D4, D7, and D8 in India , and
genotypes D5 , D9, D11 , H1, and G3 have been detected in
RSV vaccine of the present disclosure comprises a RNA neighboring countries . Recently, MeV genotype B3 was
( e.g., mRNA ) polynucleotide encoding F protein and L detected in India (Kuttiatt V S et al. Emerg Infect Dis. 2014 ;
protein . In some embodiments , a RSV vaccine of the present 50 20 ( 10 ): 1764-66 ).
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
The glycoprotein complex of paramyxoviruses mediates
encoding F protein and P protein . In some embodiments, a receptor binding and membrane fusion . In particular, the
RSV vaccine of the present disclosure comprises a RNA MeV fusion (F ) protein executes membrane fusion , after
(e.g., mRNA ) polynucleotide encoding F protein and N receptor binding by the hemagglutinin (HA ) protein (Muhle
protein . In some embodiments , a RSV vaccine of the present 55 bach MD et al. Journal of Virology 2008 ; 82 (22 ):11437-45) .
disclosure comprises a RNA (e.g., mRNA ) polynucleotide The MeV P gene codes for three proteins : P , an essential
encoding F protein and M2 protein . In some embodiments , polymerase cofactor, and V and C , which have multiple
a RSV vaccine of the present disclosure comprises a RNA functions but are not strictly required for viral propagation
(e.g., mRNA ) polynucleotide encoding F protein and M
in cultured cells . V shares the amino -terminal domain with
protein .
60 P but has a zinc -binding carboxyl-terminal domain , whereas
In some embodiments, a RSV vaccine of the present
disclosure comprises a RNA (e.g. , mRNA ) polynucleotide
encoding G protein and L protein . In some embodiments, a
RSV vaccine of the present disclosure comprises a RNA
(e.g., mRNA ) polynucleotide encoding G protein and P 65
protein . In some embodiments , a RSV vaccine of the present
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
C is translated from an overlapping reading frame. The MeV
C protein is an infectivity factor. During replication , the P
protein binds incoming monomeric nucleocapsid (N ) pro
teins with its amino - terminal domain and positions them for
assembly into the nascent ribonucleocapsid . The P protein
amino -terminal domain is natively unfolded (Deveaux P et
al. Journal of Virology 2004 ; 78(21): 11632-40 ).
US 10,702,600 B1
33
34
In some embodiments, a MeV vaccine of the present
A MeV vaccine may comprise, for example, at least one
disclosure comprises a RNA (e.g., mRNA ) polynucleotide RNA ( e.g., mRNA ) polynucleotide encoded by a nucleic
encoding HA protein . In some embodiments, a MeV vaccine acid ( e.g., DNA ) identified by any one of SEQ ID NO : 35 ,
of the present disclosure comprises a RNA (e.g., mRNA ) 36 , 38 , 39 , 41, 42 , 44 and 45 ( Table 13 ) .
polynucleotide encoding F protein . In some embodiments, a 5 The present disclosure is not limited by a particular strain
MeV vaccine of the present disclosure comprises a RNA of MeV. The strain of MeV used in a vaccine may be any
( e.g., mRNA ) polynucleotide encoding P protein . In some strain of MeV . Non - limiting examples of strains ofMeV for
use as provide herein include B3/B3.1, C2, D4, D6 , D7, D8,
embodiments, a MeV vaccine of the present disclosure G3
, Moraten , Rubeovax , MVi/New Jersey.USA /45.05 ,
comprises a RNA ( e.g., mRNA ) polynucleotide encoding V 10 MVi, H1
/ Texas.USA /4.07 , AIK - C , MVi/New York.USA / 26.09 /
protein . In some embodiments, a MeV vaccine of the present 3 , MVi
/California USA / 16.03, MVi/ Virginia.USA / 15.09,
disclosure comprises a RNA (e.g., mRNA ) polynucleotide
encoding C protein .
MVi
/California.USA /8.04 , and MVi/Pennsylvania.USAI
20.09 .
In some embodiments , a MeV vaccine of the present
proteins may be from MeV genotype D4, D5, D7,
disclosure comprises a RNA ( e.g., mRNA ) polynucleotide 15 D8MeV
,
D9
,
D11, H1, G3 or B3. In some embodiments, a MeV
encoding HA protein , F protein , P protein , V protein and C
HA protein or a MeV F protein is from MeV genotype D8.
In some embodiments , a MeV HA protein or a MeV F
protein is from MeV genotype B3.
disclosure comprises a RNA (e.g., mRNA ) polynucleotide Betacoronaviruses (BetaCoV )
encoding HA protein and F protein . In some embodiments, 20 MERS -CoV. MERS - CoV is a positive -sense , single
a MeV vaccine of the present disclosure comprises a RNA stranded RNA virus of the genus Betacoronavirus. The
(e.g., mRNA ) polynucleotide encoding HA protein and P genomes are phylogenetically classified into two clades ,
protein . In some embodiments, a MeV vaccine of the present clade A and clade B. It has a strong tropism for non - ciliated
disclosure comprises a RNA (e.g. , mRNA ) polynucleotide bronchial epithelial cells, evades the innate immune
encoding HA protein and V protein . In some embodiments, 25 response and antagonizes interferon ( IFN ) production in
a MeV vaccine of the present disclosure comprises a RNA infected cells. Dipeptyl peptidase 4 (DDP4 , also known as
(e.g. , mRNA ) polynucleotide encoding HA protein and C CD26 ) has been identified as a functional cellular receptor
protein .
for MERS- CoV. Its enzymatic activity is not required for
some embodiments, a MeV vaccine of the present disclo infection , although its amino acid sequence is highly con
sure comprises a RNA (e.g., mRNA ) polynucleotide encod- 30 served across species and is expressed in the human bron
ing F protein and P protein . In some embodiments, a MeV chial epithelium and kidneys. Most infected individuals
vaccine of the present disclosure comprises a RNA (e.g., develop severe acute respiratory illnesses, including fever,
mRNA ) polynucleotide encoding F protein and V protein . In cough , and shortness of breath , and the virus can be fatal.
protein .
In some embodiments, a MeV vaccine of the present
some embodiments, a MeV vaccine of the present disclosure The disease may be transmitted among humans, generally
protein and C protein .
The genome ofMERS -CoV encodes at least four unique
In some embodiments, a MeV vaccine of the present accessory proteins, such as 3 , 4a, 4b and 5 , two replicase
disclosure comprises a RNA (e.g., mRNA ) polynucleotide proteins (open reading frame la and 1b ), and four major
encoding HA protein , F protein and P protein . In some structural proteins, including spike (S ), envelope ( E ),
embodiments, a MeV vaccine of the present disclosure 40 nucleocapsid (N ), and membrane (M ) proteins (Almazan F
comprises a RNA (e.g., mRNA ) polynucleotide encoding et al. MBio 2013 ; 4 (5 ):e00650-13 ). The accessory proteins
HA protein , F protein and V protein . In some embodiments , play nonessential roles in MERS- CoV replication , but they
a MeV vaccine of the present disclosure comprises a RNA are likely structural proteins or interferon antagonists,
( e.g., mRNA ) polynucleotide encoding HA protein , F pro modulating in vivo replication efficiency and /or pathogen
tein and C protein .
45 esis, as in the case of SARS -CoV (Almazan F et al. MBio
In some embodiments, MeV vaccines comprise RNA 2013 ; 4 (5 ):e00650-13 ; Totura A L et al . Curr Opin Virol
comprises a RNA (e.g., mRNA) polynucleotide encoding F 35 among those in close contact.
( e.g., mRNA) encoding a MeV antigenic polypeptide having 2012; 2 (3 ): 264-75 ; Scobey T et al. Proc Natl Acad Sci USA
at least 95 % , at least 96 % , at least 97 % , at least 98 % or at 2013 ; 110 (40 ): 16157-62). The other proteins ofMERS- CoV
least 99 % identity with MeV HA protein and having MeV maintain different functions in virus replication . The E
HA protein activity .
50 protein , for example , involves in virulence, and deleting the
In some embodiments, MeV vaccines comprise RNA E -coding gene results in replication - competent and propa
(e.g., mRNA ) encoding a MeV antigenic polypeptide having gation -defective viruses or attenuated viruses (Almazan F et
at least 95 % , at least 96 % , at least 97 % , at least 98 % or at al. MBio 2013 ; 4 (5 ):e00650-13 ). The S protein is particu
least 99 % identity with MeV F protein and having MeV F larly essential in mediating virus binding to cells expressing
protein activity.
55 receptor dipeptidyl peptidase- 4 (DPP4 ) through receptor
A protein is considered to have HA protein activity if the binding domain (RBD ) in the S1 subunit , whereas the S2
protein mediates receptor binding and/or membrane fusion. subunit subsequently mediates virus entry via fusion of the
MeV F protein executes membrane fusion , after receptor virus and target cell membranes (Li F. J Virol 2015 ; 89 ( 4 ):
binding by the MeV HA protein .
1954-64 ; Raj V S et al. Nature 2013 ; 495 ( 7440):251-4 ).
A MeV vaccine may comprise, for example, at least one 60 In some embodiments, a MERS -CoV vaccine of the
RNA (e.g., mRNA ) polynucleotide having an open reading
frame encoding at least one MeV antigenic polypeptide
identified by any one of SEQ ID NO : 47-50 ( Table 14 ; see
also amino acid sequences of Table 15 ) .
A MeV vaccine may comprise, for example, at least one 65
RNA (e.g., mRNA ) polynucleotide identified by any one of
SEQ ID NO : 37 , 40 , 43, 46 ( Table 13) .
present disclosure comprises a RNA ( e.g., mRNA ) poly
nucleotide encoding S protein . In some embodiments , a
MERS- CoV vaccine of the present disclosure comprises a
RNA ( e.g.,mRNA ) polynucleotide encoding the S1 subunit
of the S protein . In some embodiments , a MERS - CoV
vaccine of the present disclosure comprises a RNA ( e.g.,
mRNA) polynucleotide encoding the S2 subunit of the S
US 10,702,600 B1
36
protein . In some embodiments, a MERS- CoV vaccine of the
In some embodiments , a SARS - CoV vaccine of the
present disclosure comprises a RNA ( e.g., mRNA ) poly
present disclosure comprises a RNA ( e.g., mRNA ) poly
nucleotide encoding E protein . In some embodiments , a nucleotide encoding S protein (S , S1 and /or S2) and E
MERS- CoV vaccine of the present disclosure comprises a protein . In some embodiments, a SARS-CoV vaccine of the
RNA ( e.g., mRNA ) polynucleotide encoding N protein . In 5 present disclosure comprises a RNA (e.g., mRNA ) poly
some embodiments , a MERS- CoV vaccine of the present nucleotide encoding S protein (S , S1 and /or S2) and N
disclosure comprises a RNA (e.g., mRNA ) polynucleotide protein . In some embodiments , a SARS -CoV vaccine of the
encoding M protein .
present disclosure comprises a RNA (e.g., mRNA ) poly
In some embodiments , a MERS- CoV vaccine of the nucleotide encoding S protein (S , S1 and/or S2 ) and M
present disclosure comprises a RNA ( e.g., mRNA ) poly- 10 protein .
nucleotide encoding S protein (S , S1 and /or S2), E protein ,
In some embodiments, a SARS -CoV vaccine of the
N protein and M protein .
present disclosure comprises a RNA (e.g. , mRNA ) poly
In some embodiments, a MERS -CoV vaccine of the nucleotide encoding S protein (S , S1 and /or S2), E protein
35
present disclosure comprises a RNA (e.g. , mRNA ) poly
and M protein . In some embodiments , a SARS- CoV vaccine
nucleotide encoding S protein (S , S1 and /or S2 ) and E 15 of the present disclosure comprises a RNA (e.g., mRNA )
protein . In some embodiments , a MERS - CoV vaccine of the polynucleotide encoding S protein (S , S1 and /or S2 ), E
present disclosure comprises a RNA ( e.g., mRNA ) poly protein and N protein . In some embodiments, a SARS-COV
nucleotide encoding S protein (S , S1 and /or S2) and N vaccine of the present disclosure comprises a RNA ( e.g.,
protein . In some embodiments, a MERS -CoV vaccine of the mRNA ) polynucleotide encoding S protein (S , S1 and/or
present disclosure comprises a RNA (e.g., mRNA ) poly- 20 S2 ), M protein and N protein . In some embodiments , a
nucleotide encoding S protein (S , S1 and/or S2 ) and M
SARS - CoV vaccine of the present disclosure comprises a
protein .
RNA ( e.g., mRNA ) polynucleotide encoding E protein , M
In some embodiments , a MERS - CoV vaccine of the protein and N protein .
present disclosure comprises a RNA ( e.g., mRNA ) poly
A SARS- CoV vaccine may comprise , for example, at
nucleotide encoding S protein (S, S1 and/or S2 ), E protein 25 least one RNA (e.g.,mRNA) polynucleotide having an open
and M protein . In some embodiments , a MERS -CoV vac
reading frame encoding at least one SARS-CoV antigenic
cine of the present disclosure comprises a RNA ( e.g., polypeptide identified by any one of SEQ ID NO : 29 , 32 or
mRNA ) polynucleotide encoding S protein (S , S1 and /or 34 ( Table 11; see also amino acid sequences of Table 12 ).
S2 ), E protein and N protein . In some embodiments , a
The present disclosure is not limited by a particular strain
MERS- CoV vaccine of the present disclosure comprises a 30 of SARS- CoV . The strain of SARS- CoV used in a vaccine
RNA ( e.g., mRNA ) polynucleotide encoding S protein (S , may be any strain of SARS-CoV.
S1 and/or S2), M protein and N protein . In some embodi
HCOV -OC43 .
ments, a MERS -CoV vaccine of the present disclosure
Human coronavirus OC43 is an enveloped , positive
comprises a RNA (e.g., mRNA ) polynucleotide encoding E sense , single -stranded RNA virus in the species Betacoro
protein , M protein and N protein .
35 navirus -1 (genus Betacoronavirus, subfamily Coronaviri
A MERS - CoV vaccine may comprise , for example, at nae, family Coronaviridae, order Nidovirales). Four HCOV
least one RNA (e.g.,mRNA ) polynucleotide having an open OC43 genotypes (A to D ), have been identified with
reading frame encoding at least one MERS- CoV antigenic genotype D most likely arising from recombination . The
polypeptide identified by any one of SEQ ID NO : 24-38 or complete genome sequencing of two genotype C and D
33 (Table 11; see also amino acid sequences of Table 12 ). 40 strains and bootscan analysis shows recombination events
A MERS-CoV vaccine may comprise, for example , at between genotypes B and C in the generation of genotype D.
least one RNA (e.g., mRNA ) polynucleotide encoded by a Of 29 strains identified , none belong to the more ancient
nucleic acid (e.g., DNA) identified by any one of SEQ ID genotype A. Along with HCV-229E , a species in the
Alphacoronavirus genus, HCV-OC43 are among the
NO : 20-23 ( Table 10 ).
The present disclosure is not limited by a particular strain 45 known viruses that cause the common cold . Both viruses can
ofMERS-CoV. The strain ofMERS -CoV used in a vaccine cause severe lower respiratory tract infections , including
may be any strain ofMERS -CoV. Non -limiting examples of pneumonia in infants, the elderly , and immunocompromised
strains of MERS -CoV for use as provide herein include individuals such as those undergoing chemotherapy and
those with HIV -AIDS .
Riyadh_14_2013 , and 2cEMC /2012 , Hasa_1_2013 .
SARS - CoV. The genome of SARS- CoV includes of a 50 HCOV -HKU1.
single, positive-strand RNA that is approximately 29,700
Human coronavirus HKU1 (HCOV -H KU 1 ) is a positive
nucleotides long. The overall genome organization of sense, single -stranded RNA virus with the HE gene, which
SARS -CoV is similar to that of other coronaviruses. The distinguishes it as a group 2 , or betacoronavirus. It was
reference genome includes 13 genes, which encode at least discovered in January 2005 in two patients in Hong Kong.
14 proteins. Two large overlapping reading frames (ORFs) 55 The genome of HCOV-HKU1 is a 29,926 -nucleotide , poly
encompass 71% of the genome. The remainder has 12 adenylated RNA . The GC content is 32 % , the lowest among
potential ORFs, including genes for structural proteins S all known coronaviruses. The genome organization is the
(spike ), E ( small envelope ), M (membrane), and N (nucleo
same as that of other group II coronaviruses , with the
capsid ). Other potential ORFs code for unique putative characteristic gene order la, 1b , HE , S , E , M , and N.
SARS-CoV-specific polypeptides that lack obvious 60 Furthermore , accessory protein genes are present between
sequence similarity to known proteins. A detailed analysis of the S and E genes (ORF4 ) and at the position of the N gene
the SARS- CoV genome has been published in J Mol Biol (ORF8). The TRS is presumably located within the AAUC
2003 ; 331: 991-1004 .
UAAAC sequence , which precedes each ORF except E.As
In some embodiments , a SARS - CoV vaccine of the in sialodacryoadenitis virus and mouse hepatitis virus
present disclosure comprises a RNA ( e.g., mRNA ) poly- 65 (MHV ), translation of the E protein possibly occurs via an
nucleotide encoding S protein (S , S1 and/or S2 ) , E protein , internal ribosomal entry site . The 3 ' untranslated region
N protein and M protein .
contains a predicted stem - loop structure immediately down
US 10,702,600 B1
37
stream of the N ORF (nucleotide position 29647 to 29711).
Further downstream , a pseudoknot structure is present at
nucleotide position 29708 to 29760. Both RNA structures
are conserved in group II coronaviruses and are critical for
virus replication .
HCOV -NL63 .
The RNA genome of human coronavirus NL63 (HCOV
NL63 ) is 27,553 nucleotides, with a poly ( A ) tail ( FIG . 1).
With a GC content of 34 % , HCOV -NL63 has one of the
lowest GC contents of the coronaviruses, for which GC
content ranges from 32 to 42 % . Untranslated regions of 286
and 287 nucleotides are present at the 5' and 3' termini,
respectively. Genes predicted to encode the S , E , M , and N
proteins are found in the 3' part of the HCOV -NL63 genome.
The HE gene, which is present in some group II coronaviruses, is absent, and there is only a single , monocistronic
accessory protein ORF (ORF3 ) located between the S and E
genes. Subgenomic mRNAs are generated for all ORFs (S ,
ORF3 , E , M , and N ), and the core sequence of the TRS of
HCOV -NL63 is defined as AACUAAA. This sequence is
situated upstream of every ORF except for the E ORF, which
contains the suboptimal core sequence AACUAUA . Inter
estingly , a 13 -nucleotide sequence with perfect homology to
the leader sequence is situated upstream of the suboptimal E
TRS . Annealing of this 13 -nucleotide sequence to the leader
sequence may act as a compensatory mechanism for the
disturbed leader - TRS/body - TRS interaction .
HCOV -229E .
Human coronavirus 229E (HCOV -229E ) is a single
coronavirus genus of the subfamily Coronavirinae , in the
family Coronaviridae, of the order Nidovirales. Along with
38
antigenic polypeptides selected from hMPV antigenic poly
peptides, PIV3 antigenic polypeptides , RSV antigenic poly
peptides, MeV antigenic polypeptides, and BetaCoV anti
genic polypeptides (e.g. , selected from MERS-CoV, SARS
5 COV, HCV-OC43 , HCOV - 229E , HCOV -NL63 , HCOV -NL ,
HCOV-NH and HCOV -HKU1).
In some embodiments, a combination RNA (e.g.,mRNA )
vaccine comprises a RNA ( e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide , a PIV3 antigenic
10 polypeptide, a RSV antigenic polypeptide, a MeV antigenic
polypeptide, and a BetaCoV antigenic polypeptide (e.g.,
selected from MERS - CoV, SARS -CoV, HCOV -OC43 ,
HCOV- 229E , HCOV-NL63, HCOV -NL , HCOV -NH and
HCOV-HKU1).
15 In some embodiments , a combination RNA ( e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide and a PIV3 anti
genic polypeptide .
In some embodiments, a combination RNA (e.g., mRNA )
20 vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide and a RSV anti
genic polypeptide .
In some embodiments, a combination RNA (e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
25 encoding a hMPV antigenic polypeptide and a MeV anti
genic polypeptide .
In some embodiments , a combination RNA ( e.g.,mRNA )
vaccine comprises a RNA (e.g. , mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide and a BetaCoV
In some embodiments , a combination RNA (e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
stranded , positive-sense, RNA virus species in the Alpha- 30 antigenic polypeptide .
Human coronavirus OC it is responsible for the common encoding a PIV3 antigenic polypeptide and a RSV antigenic
cold . HCOV -NL63 and HCOV - 229E are two of the four polypeptide.
human coronaviruses that circulate worldwide . These two 35 In some embodiments, a combination RNA ( e.g., mRNA )
viruses are unique in their relationship towards each other. vaccine comprises a RNA ( e.g. , mRNA ) polynucleotide
Phylogenetically, the viruses are more closely related to each encoding a PIV3 antigenic polypeptide and a MeV antigenic
ronavirus, yet they only polypeptide.
other than to any other human coror
share 65 % sequence identity . Moreover , the viruses use
In some embodiments , a combination RNA (e.g. , mRNA )
different receptors to enter their target cell. HCOV-NL63 is 40
associated with croup in children , whereas all signs suggest
that the virus probably causes the common cold in healthy
adults . HCOV- 229E is a proven common cold virus in
healthy adults , so it is probable that both viruses induce
comparable symptoms in adults, even though their mode of 45
infection differs (HCOV-NL63 and HCOV- 229E are two of
vaccine comprises a RNA ( e.g., mRNA ) polynucleotide
encoding a PIV3 antigenic polypeptide and a BetaCoV
antigenic polypeptide (e.g., selected from MERS - CoV,
SARS -CoV, HCV-OC43 , HCOV - 229E , HCOV -NL63 ,
HCOV-NL , HCOV -NH and HCOV -HKU1).
In some embodiments, a combination RNA (e.g., mRNA )
vaccine comprises a RNA ( e.g. , mRNA ) polynucleotide
These two viruses are unique in their relationship towards
polypeptide .
the four human coronaviruses that circulate worldwide .
encoding a RSV antigenic polypeptide and a MeV antigenic
each other. Phylogenetically, the viruses are more closely
related to each other than to any other human coronavirus, 50
yet they only share 65 % sequence identity. Moreover, the
viruses use different receptors to enter their target cell .
HCOV-NL63 is associated with croup in children , whereas
all signs suggest that the virus probably causes the common
cold in healthy adults. HCOV - 229E is a proven common cold 55
virus in healthy adults, so it is probable that both viruses
induce comparable symptoms in adults, even though their
mode of infection differs (Dijkman R. et al. J Formos Med
In some embodiments, a combination RNA ( e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a RSV antigenic polypeptide and a BetaCoV
antigenic polypeptide ( e.g. , selected from MERS -CoV,
SARS - CoV , HCOV-OC43 , HCOV- 229E , HCOV-NL63,
HCOV-NL , HCOV -NH and HCOV -HKU1).
In some embodiments , a combination RNA (e.g. , mRNA )
vaccine comprises a RNA (e.g. , mRNA ) polynucleotide
encoding a MeV antigenic polypeptide and a BetaCoV
antigenic polypeptide ( e.g., selected from MERS-CoV,
Embodiments of the present disclosure also provide com
bination RNA (e.g., mRNA ) vaccines. A combination RNA
(e.g. , mRNA ) vaccine” of the present disclosure refers to a
vaccine comprising at least one (e.g., at least 2 , 3, 4 , or 5 ) 65
RNA (e.g., mRNA polynucleotide having an open reading
frame encoding a combination of any two or more (or all of)
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide, a PIV3 antigenic
polypeptide, a RSV antigenic polypeptide and a MeV anti
genic polypeptide.
In some embodiments, a combination RNA (e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
Assoc. 2009 April ; 108 (4 ):270-9 , the contents of which is SARS - CoV, HCV-OC43 , HCOV- 229E , HCOV -NL63,
incorporated herein by reference in their entirety ).
60 HCOV-NL , HCOV -NH and HCOV -HKU1).
Combination Vaccines
In some embodiments, a combination RNA ( e.g., mRNA )
US 10,702,600 B1
39
encoding a hMPV antigenic polypeptide, a PIV3 antigenic
polypeptide, a RSV antigenic polypeptide and a BetaCoV
40
selected from MERS-CoV, SARS -COV, HCV-OC43 ,
HCOV- 229E , HCOV -NL63, HCOV -NL , HCOV -NH and
antigenic polypeptide (e.g., selected from MERS- CoV , HCOV-HKU1).
SARS- CoV , HCV-OC43 , HCOV- 229E , HCOV - NL63 ,
In some embodiments, a combination RNA ( e.g.,mRNA )
5 vaccine comprises a RNA ( e.g., mRNA) polynucleotide
HCOV -NL , HCOV-NH and HCOV -HKU1).
In some embodiments, a combination RNA ( e.g.,mRNA ) encoding a RSV antigenic polypeptide, a MeV antigenic
vaccine comprises a RNA (e.g., mRNA ) polynucleotide polypeptide and a BetaCoV antigenic polypeptide (e.g.,
encoding a hMPV antigenic polypeptide, a PIV3 antigenic selected from MERS-CoV, SARS -CoV, HCV-OC43 ,
polypeptide, a MeV antigenic polypeptide and a BetaCoV HCOV- 229E, HCOV-NL63 , HCOV-NL , HCOV -NH and
antigenic polypeptide (e.g., selected from MERS -CoV, 10 HCOV -HKU1).
SARS -CoV, HCOV -OC43 , HCOV -229E , HCOV -NL63,
Other combination respiratory virus RNA ( e.g., mRNA)
HCOV -NL , HCOV -NH and HCOV -HKU1) .
vaccines are encompassed by the present disclosure.
In some embodiments, a combination RNA (e.g., mRNA )
It has been discovered that the mRNA vaccines described
vaccine comprises a RNA ( e.g., mRNA ) polynucleotide herein are superior to current vaccines in several ways. First,
encoding a hMPV antigenic polypeptide, a RSV antigenic 15 the lipid nanoparticle (LNP) delivery is superior to other
polypeptide, a MeV antigenic polypeptide and a BetaCoV
antigenic polypeptide (e.g., selected from MERS- CoV ,
SARS -COV, HCV-OC43 , HCOV -229E , HCOV-NL63,
HCOV-NL , HCOV -NH and HCOV -HKU1) .
In some embodiments, a combination RNA ( e.g., mRNA ) 20
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a PIV3 antigenic polypeptide, a RSV antigenic
polypeptide , a MeV antigenic polypeptide and a BetaCoV
antigenic polypeptide ( e.g. , selected from MERS -CoV ,
formulations including a protamine base approach described
in the literature and no additional adjuvants are to be
necessary. The use of LNPs enables the effective delivery of
chemically modified or unmodified mRNA vaccines. Addi
tionally it has been demonstrated herein that both modified
and unmodified LNP formulated mRNA vaccines were
superior to conventional vaccines by a significant degree . In
some embodiments the mRNA vaccines of the invention are
superior to conventional vaccines by a factor of at least 10
SARS -CoV, HCV-OC43 , HCOV -229E , HCOV -NL63, 25 fold , 20 fold , 40 fold, 50 fold , 100 fold , 500 fold or 1,000
fold .
HCOV-NL , HCOV -NH and HCOV -HKU1).
In some embodiments, a combination RNA (e.g.,mRNA )
Although attempts have been made to produce functional
vaccine comprises a RNA (e.g. , mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide, a PIV3 antigenic
RNA vaccines, includingmRNA vaccines and self-replicat
ing RNA vaccines, the therapeutic efficacy of these RNA
polypeptide and a RSV antigenic polypeptide.
30 vaccines have not yet been fully established . Quite surpris
In some embodiments , a combination RNA (e.g., mRNA ) ingly, the inventors have discovered , according to aspects of
vaccine comprises a RNA (e.g., mRNA ) polynucleotide the invention a class of formulations for delivering mRNA
encoding a hMPV antigenic polypeptide, a PIV3 antigenic
polypeptide and a MeV antigenic polypeptide .
vaccines in vivo that results in significantly enhanced , and in
many respects synergistic , immune responses including
In some embodiments, a combination RNA (e.g.,mRNA ) 35 enhanced antigen generation and functional antibody pro
vaccine comprises a RNA (e.g., mRNA ) polynucleotide duction with neutralization capability . These results can be
encoding a hMPV antigenic polypeptide , a PIV3 antigenic
polypeptide and a BetaCoV antigenic polypeptide ( e.g.,
selected from MERS-CoV, SARS -CoV, HCV-OC43 ,
HCOV - 229E , HCOV -NL63, HCOV -NL , HCOV -NH and 40
HCOV-HKU1).
In some embodiments, a combination RNA (e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide , a RSV antigenic
polypeptide and a MeV antigenic polypeptide .
45
In some embodiments, a combination RNA (e.g., mRNA )
vaccine comprises a RNA (e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide , a RSV antigenic
polypeptide and a BetaCoV antigenic polypeptide ( e.g.,
selected from MERS- CoV, SARS - COV , HCV-OC43 , 50
HCOV - 229E , HCOV-NL63, HCOV -NL , HCOV -NH and
HCOV -HKU1).
In some embodiments, a combination RNA (e.g., mRNA)
vaccine comprises a RNA ( e.g., mRNA ) polynucleotide
encoding a hMPV antigenic polypeptide, a MeV antigenic 55
polypeptide and a BetaCoV antigenic polypeptide (e.g.,
selected from MERS -CoV, SARS- CoV, HCV-OC43 ,
achieved even when significantly lower doses of the mRNA
are administered in comparison with mRNA doses used in
other classes of lipid based formulations. The formulations
of the invention have demonstrated significant unexpected in
vivo immune responses sufficient to establish the efficacy of
functionalmRNA vaccines as prophylactic and therapeutic
agents . Additionally, self -replicating RNA vaccines rely on
viral replication pathways to deliver enough RNA to a cell
to produce an immunogenic response. The formulations of
the invention do not require viral replication to produce
enough protein to result in a strong immune response . Thus,
the mRNA of the invention are not self-replicating RNA and
do not include components necessary for viral replication .
The invention involves, in some aspects, the surprising
finding that lipid nanoparticle (LNP ) formulations signifi
cantly enhance the effectiveness ofmRNA vaccines, includ
ing chemically modified and unmodified mRNA vaccines .
The efficacy of mRNA vaccines formulated in LNP was
examined in vivo using several distinct antigens. The results
presented herein demonstrate the unexpected superior effi
cacy of the mRNA vaccines formulated in LNP over other
HCOV- 229E , HCOV -NL63, HCOV -NL , HCOV -NH and commercially available vaccines.
HCOV -HKU1).
In addition to providing an enhanced immune response ,
In some embodiments , a combination RNA (e.g. ,mRNA ) 60 the formulations of the invention generate a more rapid
vaccine comprises a RNA (e.g., mRNA ) polynucleotide immune response with fewer doses of antigen than other
encoding a PIV3 antigenic polypeptide, a RSV antigenic vaccines tested . The mRNA-LNP formulations of the inven
polypeptide and a MeV antigenic polypeptide.
tion also produce quantitatively and qualitatively better
In some embodiments, a combination RNA ( e.g., mRNA ) immune responses than vaccines formulated in a different
vaccine comprises a RNA (e.g., mRNA ) polynucleotide 65 carriers.
encoding a PIV3 antigenic polypeptide , a RSV antigenic
The data described herein demonstrate that the formula
polypeptide and a BetaCoV antigenic polypeptide (e.g., tions of the invention produced significant unexpected
US 10,702,600 B1
41
42
improvements over existing antigen vaccines . Additionally ,
RNA (e.g., mRNA ) sequence encoded by the DNA, where
the mRNA -LNP formulations of the invention are superior
to other vaccines even when the dose ofmRNA is lower than
other vaccines. Mice immunized with either 10 ug or 2 ug
doses of an hMPV fusion protein mRNA LNP vaccine or a
PIV3 mRNALNP vaccine produced neutralizing antibodies
which for instance , successfully neutralized the hMPV B2
virus . A 10 ug dose of mRNA vaccine protected 100 % of
mice from lethal challenge and drastically reduced the viral
titer after challenge (~ 2 log reduction).
Two 20 ug doses of MERS - CoV mRNA LNP vaccine
significantly reduced viral load and induced significant
amount of neutralizing antibodies against MERS -CoV
each “ T” of the DNA sequence is substituted with “ U.”
The basic components of an mRNA molecule typically
include at least one coding region , a 5 ' untranslated region
5
10
( ECso between 500-1000 ). The MERS - CoVmRNA vaccine
(UTR ), a 3' UTR , a 5' cap and a poly - A tail. Polynucleotides
of the present disclosure may function asmRNA but can be
distinguished from wild -type mRNA in their functional
and /or structural design features , which serve to overcome
existing problems of effective polypeptide expression using
nucleic -acid based therapeutics .
In some embodiments , a RNA polynucleotide of an RNA
(e.g., mRNA ) vaccine encodes 2-10 , 2-9 , 2-8 , 2-7 , 2-6 , 2-5 ,
2-4 , 2-3 , 3-10 , 3-9, 3-8 , 3-7, 3-6 , 3-5 , 3-4 , 4-10,4-9, 4-8 , 4-7 ,
induced antibody titer was 3-5 fold better than any other 15 7-9
4-6,, 4-5
, 5-10, 5-9 , 5-8, 5-7, 5-6, 6-10 ,6-9, 6-8 ,6-7, 7-10 ,
7-8 , 8-10 , 8-9 or 9-10 antigenic polypeptides . In some
vaccines tested in the same model.
The LNP used in the studies described herein has been embodiments, a RNA (e.g., mRNA ) polynucleotide of a
used previously to deliver siRNA in various animal models respiratory virus vaccine encodes at least 10 , 20 , 30 , 40 , 50 ,
as well as in humans . In view of the observations made in 60 , 70 , 80 , 90 or 100 antigenic polypeptides. In some
association with the siRNA delivery of LNP formulations, 20 embodiments, a RNA (e.g., mRNA ) polynucleotide of a
the fact that LNP is useful in vaccines is quite surprising. It respiratory virus vaccine encodes at least 100 or at least 200
has been observed that therapeutic delivery of siRNA for antigenic polypeptides . In some embodiments , a RNA poly
mulated in LNP causes an undesirable inflammatory nucleotide of an respiratory virus vaccine encodes 1-10 ,
response associated with a transient IgM response , typically 5-15 , 10-20 , 15-25 , 20-30 , 25-35 , 30-40, 35-45 , 40-50 , 1-50 ,
leading to a reduction in antigen production and a compro- 25 1-100 , 2-50 or 2-100 antigenic polypeptides.
mised immune response. In contrast to the findings observed
Polynucleotides of the present disclosure, in some
with siRNA, the LNP -mRNA formulations of the invention
embodiments , are codon optimized . Codon optimization
are demonstrated herein to generate enhanced IgG levels , methods are known in the art and may be used as provided
sufficient for prophylactic and therapeutic methods rather herein . Codon optimization , in some embodiments, may be
30
than transient IgM responses .
used to match codon frequencies in target and host organ
Nucleic Acids/ Polynucleotides
isms to ensure proper folding; bias GC content to increase
Respiratory virus vaccines, as provided herein , comprise mRNA
or reduce secondary structures; minimize
at least one (one or more ) ribonucleic acid (RNA ) (e.g., tandem stability
repeat
codons
or base runs that may impair gene
mRNA ) polynucleotide having an open reading frame construction or expression
transcriptional and
encoding at least one antigenic polypeptide selected from 35 translational control regions; ; customize
insert or remove protein traf
hMPV, PIV3 , RSV , MeV and BetaCoV (e.g., selected from ficking
sequences ; remove/add post translation modification
MERS -CoV, SARS- COV, HCV-OC43 , HCOV - 229E ,
HCOV -NL63, HCOV -NL , HCOV -NH and HCOV -HKU1) sites in encoded protein (e.g. glycosylation sites); add ,
antigenic polypeptides . The term “ nucleic acid ” includes remove or shuffle protein domains ; insert or delete restric
any compound and /or substance that comprises a polymer of 40
nucleotides (nucleotide monomer ). These polymers are
referred to as polynucleotides. Thus , the terms “ nucleic
acid ” and “ polynucleotide” are used interchangeably.
Nucleic acids may be or may include , for example ,
ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), 45
threose nucleic acids ( TNAs), glycol nucleic acids (GNAs),
peptide nucleic acids (PNAs), locked nucleic acids (LNAS,
including LNA having a B - D - ribo configuration , a -LNA
having an a -L -ribo configuration (a diastereomer of LNA ),
2'-amino -LNA having a 2'-amino functionalization , and 50
2'- amino -a -LNA having a 2'-amino functionalization ), eth
tion sites; modify ribosome binding sites and mRNA deg
radation sites; adjust translational rates to allow the various
domains of the protein to fold properly ; or to reduce or
eliminate problem secondary structures within the poly
nucleotide . Codon optimization tools, algorithms and ser
vices are known in the art — non - limiting examples include
services from Gene Art (Life Technologies ), DNA2.0
(Menlo Park Calif.) and /or proprietary methods. In some
embodiments , the open reading frame ( ORF ) sequence is
optimized using optimization algorithms.
peptide in vitro , in vivo, in situ or ex vivo . The skilled artisan 60
will appreciate that, except where otherwise noted , poly
nucleotide sequences set forth in the instant application will
recite “ T” s in a representative DNA sequence but where the
sequence represents RNA ( e.g.,mRNA ), the “ T ” s would be
substituted for “ U ” s. Thus , any of the RNA polynucleotides 65
encoded by a DNA identified by a particular sequence
about 85 % , or between about67 % and about 80 % ) sequence
identity to a naturally occurring sequence or a wild-type
sequence ( e.g., a naturally -occurring or wild - type mRNA
sequence encoding a polypeptide or protein of interest ( e.g.,
an antigenic protein or polypeptide )). In someembodiments ,
a codon -optimized sequence shares between 65 % and 75 % ,
or about 80 % sequence identity to a naturally -occurring
In some embodiments, a codon optimized sequence
shares less than 95 % sequence identity , less than 90 %
ylene nucleic acids (ENA ), cyclohexenyl nucleic acids sequence identity, less than 85 % sequence identity, less than
80 % sequence identity , or less than 75 % sequence identity
(CENA ) or chimeras or combinations thereof.
In some embodiments, polynucleotides of the present to a naturally -occurring or wild -type sequence (e.g., a natu
disclosure function as messenger RNA (mRNA ). “Messen- 55 rally -occurring or wild -type mRNA sequence encoding a
ger RNA ” (mRNA ) refers to any polynucleotide that polypeptide or protein of interest (e.g., an antigenic protein
encodes a (at least one) polypeptide (a naturally-occurring , or antigenic polypeptide )).
non -naturally occurring, or modified polymer of amino
In some embodiments , a codon- optimized sequence
acids ) and can be translated to produce the encoded poly
shares between 65 % and 85 % (e.g., between about 67 % and
identification number may also comprise the corresponding
sequence or wild -type sequence (e.g., a naturally -occurring
US 10,702,600 B1
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43
or wild -type mRNA sequence encoding a polypeptide or
protein of interest ( e.g., an antigenic protein or polypep
tide)).
In some embodiments a codon -optimized RNA (e.g.,
ing variants and derivatives. These include, for example ,
substitutional, insertional , deletion and covalentvariants and
derivatives. The term " derivative” is synonymous with the
term “ variant” and generally refers to a molecule that has
been modified and /or changed in any way relative to a
mRNA ) may, for instance ,be one in which the levels of G C5
/
are enhanced . The G /C - content of nucleic acid molecules reference molecule or a starting molecule .
may influence the stability of the RNA . RNA having an
As such , polynucleotides encoding peptides or polypep
increased amount of guanine (G ) and /or cytosine (C ) resi tides containing substitutions, insertions and /or additions,
dues may be functionally more stable than nucleic acids deletions and covalentmodifications with respect to refer
containing a large amountofadenine (A ) and thymine ( T ) or 10 ence sequences, in particular the polypeptide sequences
uracil (U ) nucleotides. WO02/098443 discloses a pharma disclosed herein , are included within the scope of this
ceutical composition containing an mRNA stabilized by disclosure. For example, sequence tags or amino acids, such
sequence modifications in the translated region . Due to the as one or more lysines, can be added to peptide sequences
degeneracy of the genetic code , the modifications work by (e.g., at the N -terminal or C - terminal ends). Sequence tags
substituting existing codons for those that promote greater 15 can be used for peptide detection , purification or localiza
RNA stability without changing the resulting amino acid . tion . Lysines can be used to increase peptide solubility or to
The approach is limited to coding regions of the RNA .
allow for biotinylation . Alternatively, amino acid residues
Antigens/Antigenic Polypeptides
located at the carboxy and amino terminal regions of the
In some embodiments, an antigenic polypeptide ( e.g., a amino acid sequence of a peptide or protein may optionally
hMPV , PIV3, RSV , MeV or BetaCoV antigenic polypeptide ) 20 be deleted providing for truncated sequences . Certain amino
is longer than 25 amino acids and shorter than 50 amino acids (e.g., C - terminal residues or N -terminal residues )
acids. Polypeptides include gene products, naturally occur alternatively may be deleted depending on the use of the
ring polypeptides, synthetic polypeptides , homologs , sequence , as for example , expression of the sequence as part
orthologs, paralogs, fragments and other equivalents, vari of a larger sequence that is soluble, or linked to a solid
ants , and analogs of the foregoing . A polypeptide may be a 25 support.
single molecule or may be a multi-molecular complex such
“ Substitutional variants” when referring to polypeptides
as a dimer, trimer or tetramer. Polypeptides may also com
are those that have at least one amino acid residue in a native
prise single chain polypeptides or multichain polypeptides, or starting sequence removed and a different amino acid
such as antibodies or insulin , and may be associated or inserted in its place at the same position . Substitutions may
linked to each other.Most commonly , disulfide linkages are 30 be single, where only one amino acid in the molecule has
found in multichain polypeptides. The term “ polypeptide" been substituted , or they may be multiple, where two or
may also apply to amino acid polymers in which at least one more (e.g., 3 , 4 or 5 ) amino acids have been substituted in
amino acid residue is an artificial chemical analogue of a
corresponding naturally -occurring amino acid .
the same molecule .
As used herein the term " conservative amino acid sub
A “ polypeptide variant” is a molecule that differs in its 35 stitution ” refers to the substitution of an amino acid that is
amino acid sequence relative to a native sequence or a normally present in the sequence with a different amino acid
reference sequence . Amino acid sequence variants may of similar size, charge , or polarity . Examples of conservative
possess substitutions, deletions, insertions, or a combination substitutions include the substitution of a non -polar (hydro
of any two or three of the foregoing , at certain positions phobic ) residue such as isoleucine , valine and leucine for
within the amino acid sequence , as compared to a native 40 another non -polar residue. Likewise, examples of conserva
sequence or a reference sequence. Ordinarily , variants pos tive substitutions include the substitution of one polar (hy
sess at least 50 % identity to a native sequence or a reference drophilic ) residue for another such as between arginine and
sequence . In some embodiments , variants share at least 80 %
lysine , between glutamine and asparagine, and between
identity or at least 90 % identity with a native sequence or a glycine and serine . Additionally, the substitution of a basic
reference sequence.
45 residue such as lysine, arginine or histidine for another, or
In some embodiments “ variant mimics ” are provided . A the substitution ofone acidic residue such as aspartic acid or
" variantmimic ” contains at least one amino acid that would
glutamic acid for another acidic residue are additional
mimic an activated sequence . For example , glutamate may examples of conservative substitutions. Examples of non
serve as a mimic for phosphoro -threonine and /or phosphoro
conservative substitutions include the substitution of a non
serine. Alternatively, variant mimics may result in deacti- 50 polar (hydrophobic ) amino acid residue such as isoleucine ,
vation or in an inactivated product containing the mimic . For valine, leucine, alanine,methionine for a polar (hydrophilic )
example , phenylalanine may act as an inactivating substi residue such as cysteine, glutamine, glutamic acid or lysine
tution for tyrosine , or alanine may act as an inactivating and /or a polar residue for a non -polar residue.
substitution for serine.
“ Features” when referring to polypeptide or polynucle
“ Orthologs ” refers to genes in different species that 55 otide are defined as distinct amino acid sequence -based or
evolved from a common ancestral gene by speciation . Nor nucleotide -based components of a molecule respectively.
mally, orthologs retain the same function in the course of Features of the polypeptides encoded by the polynucleotides
evolution . Identification of orthologs is important for reli include surface manifestations, local conformational shape ,
able prediction of gene function in newly sequenced folds, loops, half- loops, domains, half -domains, sites, ter
genomes .
60 mini and any combination (s) thereof.
“ Analogs ” is meant to include polypeptide variants that
As used herein when referring to polypeptides the term
differ by one or more amino acid alterations, for example , " domain ” refers to a motif of a polypeptide having one or
substitutions, additions or deletions of amino acid residues more identifiable structural or functional characteristics or
that still maintain one ormore of the properties of the parent properties (e.g., binding capacity, serving as a site for
or starting polypeptide .
65 protein -protein interactions).
The present disclosure provides several types of compo
As used herein when referring to polypeptides the terms
sitions that are polynucleotide or polypeptide based , includ “ site ” as it pertains to amino acid based embodiments is used
US 10,702,600 B1
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46
synonymously with “ amino acid residue ” and “ amino acid
percentage of residues (amino acid residues or nucleic acid
side chain .” As used herein when referring to polynucle
residues) in the candidate amino acid or nucleic acid
otides the terms “ site” as it pertains to nucleotide based
sequence that are identical with the residues in the amino
embodiments is used synonymously with “ nucleotide .” A acid sequence or nucleic acid sequence of a second sequence
site represents a position within a peptide or polypeptide or 5 after aligning the sequences and introducing gaps, if neces
polynucleotide thatmay be modified , manipulated , altered , sary, to achieve the maximum percent identity.Methods and
derivatized or varied within the polypeptide-based or poly computer programs for the alignment are well known in the
nucleotide -based molecules .
art. Identity depends on a calculation of percent identity but
As used herein the terms “ termini” or “ terminus ” when
differ in value due to gaps and penalties introduced in
referring
refers to an. 10 may
the calculation
.Generally, variants of a particular polynucle
extremity toofpolypeptides
a polypeptideororpolynucleotides
polynucleotide respectively
Such extremity is not limited only to the first or final site of
the polypeptide or polynucleotide but may include addi
tional amino acids or nucleotides in the terminal regions .
Polypeptide
-based moleculesmay be characterized as hav- 15
ing both an N -terminus (terminated by an amino acid with
a free amino group (NH2 )) and a C - terminus (terminated by
an amino acid with a free carboxyl group (COOH )). Proteins
are in some cases made up of multiple polypeptide chains
forces (multimers , oligomers ). These proteins have multiple
N- and C -termini. Alternatively , the termini of the polypep
tides may be modified such that they begin or end , as the
case may be, with a non -polypeptide based moiety such as
an organic conjugate.
25
As recognized by those skilled in the art, protein frag
ments , functional protein domains, and homologous proteins
are also considered to be within the scope of polypeptides of
interest. For example, provided herein is any protein frag
otide or polypeptide have at least 40 % , 45 % , 50 % , 55 % ,
60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 91% , 92 % , 93 % ,
94 % , 95 % , 96 % , 97 % , 98 % , 99 % but less than 100 %
sequence identity to thatparticular reference polynucleotide
or polypeptide as determined by sequence alignment pro
grams and parameters described herein and known to those
skilled in the art. Such tools for alignment include those of
the BLAST suite (Stephen F. Altschul, et al. ( 1997 ).”
protein database search programs,” Nucleic Acids Res.
25 :3389-3402 ). Another popular local alignment technique
is based on the Smith -Waterman algorithm (Smith , T. F. &
Waterman , M.S. (1981) “ Identification of common molecu
lar subsequences." J. Mol . Biol. 147: 195-197). A general
global alignment technique based on dynamic programming
is the Needleman - Wunsch algorithm (Needleman , S. B. &
Wunsch , C. D. ( 1970 ) “ A general method applicable to the
search for similarities in the amino acid sequences of two
brought together by disulfide bonds or by non -covalent 20 Gapped BLAST and PSI-BLAST: a new generation of
ment (meaning a polypeptide sequence at least one amino 30 proteins.” J. Mol. Biol . 48 :443-453). More recently , a Fast
acid residue shorter than a reference polypeptide sequence OptimalGlobal Sequence Alignment Algorithm (FOGSAA )
but otherwise identical) of a reference protein having a was developed that purportedly produces global alignment
length of 10 , 20 , 30 , 40 , 50 ,60 ,70 , 80 , 90 , 100 or longer than ofnucleotide and protein sequences faster than other optimal
100 amino acids. In another example , any protein that global alignment methods, including the Needleman -Wun
includes a stretch of 20 , 30 , 40, 50 , or 100 (contiguous) 35 sch algorithm . Other tools are described herein , specifically
amino acids that are 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , in the definition of “ identity ” below .
or 100 % identical to any of the sequences described herein
As used herein , the term “ homology ” refers to the overall
can be utilized in accordance with the disclosure . In some relatedness between polymeric molecules, e.g. between
embodiments, a polypeptide includes 2 , 3 , 4 , 5 , 6 , 7, 8, 9 , 10 , nucleic acid molecules (e.g. DNA molecules and / or RNA
or more mutations as shown in any of the sequences pro- 40 molecules ) and /or between polypeptide molecules . Poly
vided herein or referenced herein . In another example, any meric molecules ( e.g. nucleic acid molecules (e.g. DNA
protein that includes a stretch of 20 , 30 , 40 , 50 , or 100 amino molecules and /or RNA molecules) and/or polypeptide mol
acids that are greater than 80 % , 90 % , 95 % , or 100 % ecules ) that share a threshold level of similarity or identity
identical to any of the sequences described herein , wherein determined by alignment of matching residues are termed
the protein has a stretch of 5 , 10 , 15 , 20 , 25 , or 30 amino 45 homologous . Homology is a qualitative term that describes
acids that are less than 80 % , 75 % , 70 % , 65 % to 60 %
a relationship between molecules and can be based upon the
identical to any of the sequences described herein can be quantitative similarity or identity . Similarity or identity is a
utilized in accordance with the disclosure .
quantitative term that defines the degree of sequence match
Polypeptide or polynucleotide molecules of the present between two compared sequences. In some embodiments,
disclosure may share a certain degree of sequence similarity 50 polymeric molecules are considered to be “homologous” to
or identity with the reference molecules (e.g., reference one another if their sequences are at least 25 % , 30 % , 35 % ,
polypeptides or reference polynucleotides ), for example, 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % ,
with art-described molecules (e.g., engineered or designed 90 % , 95 % , or 99 % identical or similar. The term “ homolo
molecules or wild -type molecules). The term “ identity,” as gous” necessarily refers to a comparison between at least
known in the art, refers to a relationship between the 55 two sequences (polynucleotide or polypeptide sequences).
sequences of two or more polypeptides or polynucleotides ,
as determined by comparing the sequences. In the art,
identity also means the degree of sequence relatedness
between two sequences as determined by the number of
matches between strings of two or more amino acid residues 60
or nucleic acid residues. Identity measures the percent of
identical matches between the smaller of two or more
sequences with gap alignments ( if any ) addressed by a
particular mathematical model or computer program ( e.g. ,
“ algorithms” ). Identity of related peptides can be readily 65
calculated by known methods. “ % identity ” as it applies to
polypeptide or polynucleotide sequences is defined as the
Two polynucleotide sequences are considered homologous
if the polypeptides they encode are at least 50 % , 60 % , 70 % ,
80 % , 90 % , 95 % , or even 99 % for at least one stretch of at
least 20 amino acids. In some embodiments , homologous
polynucleotide sequences are characterized by the ability to
encode a stretch of at least 4-5 uniquely specified amino
acids. For polynucleotide sequences less than 60 nucleotides
in length , homology is determined by the ability to encode
a stretch of at least 4-5 uniquely specified amino acids . Two
protein sequences are considered homologous if the proteins
are at least 50 % , 60 % , 70 % , 80 % , or 90 % identical for at
least one stretch of at least 20 amino acids.
US 10,702,600 B1
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48
Homology implies that the compared sequences diverged Techniques for determining identity are codified in publicly
in evolution from a common origin . The term “ homolog” available computer programs. Exemplary computer soft
refers to a first amino acid sequence or nucleic acid sequence ware to determine homology between two sequences
(e.g., gene (DNA or RNA ) or protein sequence ) that is include, but are not limited to , GCG program package ,
related to a second amino acid sequence or nucleic acid 5 Devereux , J., et al., Nucleic Acids Research , 12( 1), 387
sequence by descent from a common ancestral sequence . ( 1984 )), BLASTP, BLASTN , and FASTA Altschul, S. F. et
The term " homolog" may apply to the relationship between al., J. Molec . Biol., 215 , 403 ( 1990 )).
genes and /or proteins separated by the event of speciation or Multiprotein and Multicomponent Vaccines
to the relationship between genes and /or proteins separated
The present disclosure encompasses respiratory virus
by the event of genetic duplication . “Orthologs ” are genes 10 vaccines comprising multiple RNA (e.g., mRNA ) poly
( or proteins) in different species that evolved from a com
nucleotides, each encoding a single antigenic polypeptide, as
mon ancestral gene (or protein ) by speciation . Typically, well as respiratory virus vaccines comprising a single RNA
orthologs retain the same function in the course of evolution . polynucleotide encoding more than one antigenic polypep
“ Paralogs” are genes (or proteins) related by duplication tide (e.g., as a fusion polypeptide ). Thus , a vaccine compo
within a genome. Orthologs retain the same function in the 15 sition comprising a RNA ( e.g., mRNA ) polynucleotide
course of evolution , whereas paralogs evolve new functions, having an open reading frame encoding a first antigenic
polypeptide and a RNA ( e.g.,mRNA ) polynucleotide having
even if these are related to the original one .
The term “ identity” refers to the overall relatedness an open reading frame encoding a second antigenic poly
between polymeric molecules, for example, between poly peptide encompasses (a ) vaccines that comprise a first RNA
nucleotide molecules (e.g. DNA molecules and /or RNA 20 polynucleotide encoding a first antigenic polypeptide and a
molecules) and / or between polypeptide molecules . Calcula second RNA polynucleotide encoding a second antigenic
tion of the percent identity of two polynucleic acid polypeptide, and (b ) vaccines that comprise a single RNA
sequences , for example , can be performed by aligning the polynucleotide encoding a first and second antigenic poly
two sequences for optimal comparison purposes (e.g., gaps peptide (e.g., as a fusion polypeptide ). RNA (e.g., mRNA )
can be introduced in one or both of a first and a second 25 vaccines of the present disclosure , in some embodiments,
nucleic acid sequences for optimal alignment and non
comprise 2-10 (e.g., 2 , 3 , 4 , 5 , 6 , 7 , 8, 9 or 10 ), or more ,RNA
identical sequences can be disregarded for comparison pur
poses ). In certain embodiments , the length of a sequence
aligned for comparison purposes is at least 30 % , at least
40 % , at least 50 % , at least 60 % , at least 70 % , at least 80 % , 30
at least 90 % , at least 95 % , or 100 % of the length of the
reference sequence . The nucleotides at corresponding
nucleotide positions are then compared . When a position in
the first sequence is occupied by the same nucleotide as the
polynucleotides having an open reading frame, each of
which encodes a different antigenic polypeptide (or a single
RNA polynucleotide encoding 2-10 , or more, different anti
genic polypeptides). The antigenic polypeptides may be
selected from hMPV, PIV3, RSV, MEV and BetaCoV (e.g.,
selected from MERS-CoV, SARS -COV, HCOV -OC43 ,
HCOV -NH and
HCOV -229 HCOV -NL63 , HC
HCOV -HKU1) antigenic polypeptides.
corresponding position in the second sequence, then the 35 In some embodiments , a respiratory virus vaccine com
molecules are identical at that position. The percent identity prises a RNA (e.g. ,mRNA ) polynucleotide having an open
between the two sequences is a function of the number of reading frame encoding a viral capsid protein , a RNA ( e.g.,
identical positions shared by the sequences , taking into mRNA ) polynucleotide having an open reading frame
account the number of gaps , and the length of each gap , encoding a viral premembrane/membrane protein , and a
which needs to be introduced for optimal alignment of the 40 RNA ( e.g., mRNA ) polynucleotide having an open reading
two sequences. The comparison of sequences and determi frame encoding a viral envelope protein . In some embodi
nation of percent identity between two sequences can be ments , a respiratory virus vaccine comprises a RNA (e.g.,
accomplished using a mathematical algorithm . For example, mRNA polynucleotide having an open reading frame
the percent identity between two nucleic acid sequences can encoding a viral fusion ( F ) protein and a RNA polynucle
be determined using methods such as those described in 45 otide having an open reading frame encoding a viral major
Computational Molecular Biology , Lesk , A.M., ed ., Oxford surface glycoprotein (G protein ). In some embodiments, a
University Press, New York, 1988 ; Biocomputing : Informat
vaccine comprises a RNA ( e.g., mRNA ) polynucleotide
ics and Genome Projects, Smith , D. W., ed ., Academic Press, having an open reading frame encoding a viral F protein . In
New York , 1993 ; Sequence Analysis in Molecular Biology, some embodiments , a vaccine comprises a RNA (e.g. ,
von Heinje , G., Academic Press , 1987; Computer Analysis 50 mRNA ) polynucleotide having an open reading frame
of Sequence Data , Part I, Griffin , A. M., and Griffin , H. G., encoding a viral G protein . In some embodiments, a vaccine
eds., Humana Press, New Jersey, 1994 ; and Sequence comprises a RNA ( e.g., mRNA ) polynucleotide having an
Analysis Primer, Gribskov, M. and Devereux , J., eds., M
open reading frame encoding a HN protein .
Stockton Press, New York , 1991 ; each of which is incorpo
In some embodiments , a multicomponent vaccine com
rated herein by reference. For example, the percent identity 55 prises at least one RNA (e.g.,mRNA ) polynucleotide encod
between two nucleic acid sequences can be determined ing at least one antigenic polypeptide fused to a signal
using the algorithm of Meyers and Miller (CABIOS, 1989 , peptide (e.g. , any one of SEQ ID NO : 15-19). The signal
4 : 11-17 ), which has been incorporated into the ALIGN peptide may be fused at the N -terminus or the C -terminus of
program (version 2.0 ) using a PAM 120 weight residue an antigenic polypeptide . An antigenic polypeptide fused to
table , a gap length penalty of 12 and a gap penalty of 4. The 60 a signal peptide may be selected from hMPV , PIV3, RSV ,
percent identity between two nucleic acid sequences can , MEV and BetaCoV (e.g., selected from MERS - CoV, SARS
alternatively, be determined using the GAP program in the
GCG software package using an NWSgapdna.CMP matrix .
Methods commonly employed to determine percent identity
between sequences include, but are not limited to those 65
disclosed in Carillo , H., and Lipman , D., SIAM J Applied
Math ., 48: 1073 ( 1988 ); incorporated herein by reference .
COV, HCV-OC43 , HCOV - 229E , HCOV -NL63 , HCOV -NL ,
HCOV -NH and HCOV -HKU1) antigenic polypeptides .
Signal Peptides
In some embodiments, antigenic polypeptides encoded by
respiratory virus RNA (e.g., mRNA ) polynucleotides com
prise a signal peptide . Signal peptides, comprising the
US 10,702,600 B1
49
50
N - terminal 15-60 amino acids of proteins, are typically
targeting of a protein to the cell membrane may be used in
needed for the translocation across the membrane on the
accordance with the present disclosure .
secretory pathway and, thus, universally control the entry of
A signal peptide may have a length of 15-60 amino acids.
most proteins both in eukaryotes and prokaryotes to the For example, a signal peptide may have a length of 15 , 16 ,
secretory pathway. Signal peptides generally include three 5 17 , 18 , 19, 20 , 21, 22, 23 , 24 , 25 , 26 , 27 , 28 , 29, 30 , 31, 32,
regions: an N - terminal region of differing length , which 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40, 41, 42 , 43 , 44 , 45 , 46 , 47 , 48 ,
usually comprises positively charged amino acids, a hydro
49, 50 , 51, 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59, or 60 amino acids.
phobic region ; and a short carboxy -terminal peptide region . In some embodiments, a signal peptide has a length of
In eukaryotes , the signal peptide of a nascent precursor 20-60 , 25-60 , 30-60 , 35-60 , 40-60 , 45-60 , 50-60 , 55-60 ,
protein (pre -protein ) directs the ribosome to the rough 10 15-55 , 20-55 , 25-55 , 30-55 , 35-55 , 40-55 , 45-55 , 50-55 ,
endoplasmic reticulum (ER ) membrane and initiates the 15-50 , 20-50 , 25-50 , 30-50 , 35-50 , 40-50 , 45-50 , 15-45 ,
transport of the growing peptide chain across it for process 20-45 , 25-45 , 30-45 , 35-45 , 40-45 , 15-40 , 20-40 , 25-40,
ing . ER processing produces mature proteins, wherein the 30-40 , 35-40 , 15-35 , 20-35 , 25-35 , 30-35 , 15-30 , 20-30 ,
signal peptide is cleaved from precursor proteins , typically 25-30 , 15-25 , 20-25 , or 15-20 amino acids.
by a ER -resident signal peptidase of the host cell, or they 15 A signal peptide is typically cleaved from the nascent
remain uncleaved and function as a membrane anchor. A
polypeptide at the cleavage junction during ER processing .
signal peptide may also facilitate the targeting of the protein
The mature antigenic polypeptide produce by a respiratory
to the cell membrane. The signal peptide, however , is not virus RNA (e.g., mRNA ) vaccine of the present disclosure
responsible for the final destination of the mature protein . typically does not comprise a signal peptide .
Secretory proteins devoid of additional address tags in their 20 ChemicalModifications
sequence are by default secreted to the external environ
Respiratory virus vaccines of the present disclosure, in
ment. During recent years, a more advanced view of signal some embodiments, comprise at least RNA (e.g. mRNA )
peptides has evolved, showing that the functions and immu polynucleotide having an open reading frame encoding at
nodominance of certain signal peptides are much more least one antigenic polypeptide that comprises at least one
versatile than previously anticipated .
25 chemical modification .
Respiratory virus vaccines of the present disclosure may
The terms " chemical modification ” and “ chemically
comprise , for example , RNA (e.g., mRNA ) polynucleotides modified ” refer to modification with respect to adenosine
encoding an artificial signal peptide, wherein the signal ( A ), guanosine (G ), uridine (U ) , thymidine ( T ) or cytidine
peptide coding sequence is operably linked to and is in frame (C ) ribonucleosides or deoxyribnucleosides in at least one of
with the coding sequence of the antigenic polypeptide . Thus , 30 their position , pattern , percent or population . Generally ,
respiratory virus vaccines of the present disclosure, in some
embodiments , produce an antigenic polypeptide comprising
an antigenic polypeptide ( e.g., hMP V3, RSV, MeV or
BetaCoV ) fused to a signal peptide. In some embodiments,
these terms do not refer to the ribonucleotide modifications
in naturally occurring 5'-terminal mRNA cap moieties. With
respect to a polypeptide , the term “ modification ” refers to a
modification relative to the canonical set 20 amino acids .
a signal peptide is fused to the N -terminus of the antigenic 35 Polypeptides, as provided herein, are also considered “modi
polypeptide . In some embodiments , a signalpeptide is fused fied ” of they contain amino acid substitutions, insertions or
to the C - terminus of the antigenic polypeptide .
a combination of substitutions and insertions.
In some embodiments, the signal peptide fused to the
Polynucleotides ( e.g., RNA polynucleotides, such as
antigenic polypeptide is an artificial signal peptide. In some mRNA polynucleotides), in some embodiments, comprise
embodiments, an artificial signal peptide fused to the anti- 40 various (more than one) different modifications. In some
genic polypeptide encoded by the RNA ( e.g.,mRNA) vac
embodiments, a particular region of a polynucleotide con
cine is obtained from an immunoglobulin protein , e.g., an tains one, two or more (optionally different) nucleoside or
IgE signal peptide or an IgG signal peptide. In some nucleotide modifications. In some embodiments , a modified
embodiments , a signal peptide fused to the antigenic poly RNA polynucleotide (e.g., a modified mRNA polynucle
peptide encoded by a RNA ( e.g., mRNA ) vaccine is an Ig 45 otide), introduced to a cell or organism , exhibits reduced
heavy chain epsilon -1 signal peptide (IgE HC SP) having the degradation in the cell or organism , respectively, relative to
sequence of: MDWTWILFLVAAATRVHS (SEQ ID NO : an unmodified polynucleotide. In some embodiments , a
16 ). In some embodiments , a signal peptide fused to the modified RNA polynucleotide (e.g. , a modified mRNA
antigenic polypeptide encoded by the (e.g., mRNA ) RNA polynucleotide), introduced into a cell or organism , may
(e.g., mRNA ) vaccine is an IgGk chain V - III region HAH 50 exhibit reduced immunogenicity in the cell or organism ,
signal peptide ( IgGk SP ) having the sequence of MET respectively (e.g. , a reduced innate response ).
PAQLLFLLLLWLPDTTG (SEQ ID NO : 15 ). In some
Modifications of polynucleotides include, without limita
embodiments, the signal peptide is selected from : Japanese tion , those described herein . Polynucleotides (e.g., RNA
encephalitis PRM signal sequence (MLGSNSGQRV polynucleotides , such as mRNA polynucleotides) may com
VFTILLLLVAPAYS ; SEQ ID NO : 17), VSVg protein signal 55 prise modifications that are naturally -occurring , non -natu
sequence (MKCLLYLAFLFIGVNCA ; SEQ ID NO : 18 ) and rally -occurring or the polynucleotide may comprise a com
Japanese encephalitis JEV signal sequence (MWLVS bination of naturally occurring and non -naturally occurring
modifications. Polynucleotides may include any useful
LAIVTACAGA ; SEQ ID NO : 19 ) .
In some embodiments , the antigenic polypeptide encoded modification , for example , of a sugar, a nucleobase , or an
by a RNA (e.g., mRNA ) vaccine comprises an amino acid 60 internucleoside linkage (e.g., to a linking phosphate , to a
sequence identified by any one of SEQ ID NO : 5-8 , 12-13 , phosphodiester linkage or to the phosphodiester backbone ).
24-34 , 47-50 or 54-56 ( Tables 3 , 6 , 11 , 14 or 17 ; see also
Polynucleotides ( e.g., RNA polynucleotides, such as
amino acid sequences of Tables 4, 7 , 12 or 15 ) fused to a mRNA polynucleotides), in some embodiments, comprise
signal peptide identified by any one of SEQ ID NO : 15-19 non - naturalmodified nucleotides that are introduced during
( Table 8 ). The examples disclosed herein are notmeant to be 65 synthesis or post-synthesis of the polynucleotides to achieve
limiting and any signal peptide that is known in the art to desired functions or properties. The modifications may be
facilitate targeting of a protein to ER for processing and /or present on an internucleotide linkages , purine or pyrimidine
US 10,702,600 B1
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52
bases , or sugars . The modification may be introduced with adenine; N6 (methyl)adenine; N6-(isopentyl)adenine ;
chemical synthesis or with a polymerase enzyme at the 7 -deaza -8 - aza - adenosine ; 7 -methyladenine ; 1 -Deazaade
terminal of a chain or anywhere else in the chain . Any of the nosine TP; 2'Fluoro -N6 -Bz-deoxyadenosine TP ; 2 -OMe- 2
regions of a polynucleotide may be chemically modified . Amino -ATP ; 2'0 -methyl-N6 - Bz -deoxyadenosine TP ; 2'-a
The present disclosure provides for modified nucleosides 5 Ethynyladenosine TP ; 2 -aminoadenine ; 2 -Aminoadenosine
and nucleotides of a polynucleotide (e.g., RNA polynucle
TP ; 2- Amino - ATP ; 2'-a - Trifluoromethyladenosine TP ;
otides , such as mRNA polynucleotides ). A “nucleoside” 2 - Azidoadenosine TP ; 2 - b - Ethynyladenosine TP ; 2 -Bro
refers to a compound containing a sugar molecule (e.g., a moadenosine TP ; 2'-b - Trifluoromethyladenosine TP ;
pentose or ribose ) or a derivative thereof in combination 2 -Chloroadenosine TP ; 2 -Deoxy -2', 2 '-difluoroadenosine
with an organic base (e.g., a purine or pyrimidine) or a 10 TP ; 2 -Deoxy - 2'-a -mercaptoadenosine TP ; 2 - Deoxy- 2 -a
derivative thereof ( also referred to herein as “ nucleobase ” ). thiomethoxyadenosine TP ; 2 -Deoxy- 2'-b -aminoadenosine
A nucleotide ” refers to a nucleoside , including a phosphate TP ; 2'-Deoxy - 2'-b -azidoadenosine TP ; 2 -Deoxy - 2 - b -bro
group . Modified nucleotides may by synthesized by any moadenosine TP ; 2'-Deoxy -2'-b -chloroadenosine TP ; 2'-De
useful method , such as, for example , chemically, enzymati oxy - 2'-b -fluoroadenosine TP ; 2 -Deoxy- 2'-b - iodoadenosine
cally, or recombinantly , to include one or more modified or 15 TP ; 2'-Deoxy- 2'-b -mercaptoadenosine TP ; 2'-Deoxy -2'-b
non -natural nucleosides. Polynucleotides may comprise a thiomethoxyadenosine TP ; 2- Fluoroadenosine TP ; 2-lodo
region or regions of linked nucleosides. Such regions may adenosine TP ; 2 -Mercaptoadenosine TP ; 2-methoxy -ad
have variable backbone linkages. The linkages may be enine ; 2 -methylthio -adenine ; 2 - Trifluoromethyladenosine
standard phosphdioester linkages , in which case the poly
TP ; 3 -Deaza - 3 -bromoadenosine TP ; 3 -Deaza -3 - chloroad
nucleotides would comprise regions of nucleotides.
20 enosine TP ; 3 -Deaza - 3 - fluoroadenosine TP ; 3 - Deaza -3-10
Modified nucleotide base pairing encompasses not only doadenosine TP ; 3 -Deazaadenosine TP ; 4 '- Azidoadenosine
the standard adenosine -thymine , adenosine -uracil, or TP ; 4 '- Carbocyclic adenosine TP ; 4 '-Ethynyladenosine TP ;
guanosine-cytosine base pairs , but also base pairs formed 5'-Homo -adenosine TP ; 8 -Aza - ATP ; 8 -bromo -adenosine
between nucleotides and /or modified nucleotides compris
TP ; 8 - Trifluoromethyladenosine TP ; 9- Deazaadenosine TP ;
ing non -standard or modified bases, wherein the arrange- 25 2 -aminopurine; 7 -deaza -2,6 -diaminopurine ; 7 -deaza -8 -aza
mentof hydrogen bond donors and hydrogen bond acceptors 2,6 -diaminopurine; 7 - deaza -8 -aza -2 -aminopurine ; 2,6 -di
permits hydrogen bonding between a non -standard base and aminopurine ; 7 -deaza -8 - aza -adenine , 7 -deaza- 2 -aminopu
a standard base or between two complementary non - stan
rine; 2 -thiocytidine ; 3 -methylcytidine ; 5 -formylcytidine ;
dard base structures. One example of such non -standard base 5 - hydroxymethylcytidine; 5-methylcytidine ; N4 -acetylcyti
pairing is the base pairing between themodified nucleotide 30 dine; 2'-O -methylcytidine ; 2'-O -methylcytidine; 5,2 '- 0 -di
inosine and adenine, cytosine or uracil. Any combination of methylcytidine ; 5 - formyl- 2 - O -methylcytidine ; Lysidine ;
base/sugar or linker may be incorporated into polynucle N4,2 '-O -dimethylcytidine ; N4-acetyl-2'-O -methylcytidine;
N4-methylcytidine; N4,N4-Dimethyl-2'-OMe-Cytidine TP ;
otides of the present disclosure.
Modifications of polynucleotides (e.g., RNA polynucle
4 -methylcytidine ; 5 -aza -cytidine; Pseudo -iso -cytidine; pyr
otides, such as mRNA polynucleotides) that are useful in the 35 rolo -cytidine; a - thio -cytidine; 2-( thio )cytosine; 2'- Amino
vaccines of the present disclosure include, but are not 2 '-deoxy -CTP; 2'- Azido -2'-deoxy -CTP ; 2 -Deoxy - 2 -a
limited to the following: 2-methylthio- N6-( cis-hydroxyiso aminocytidine TP ; 2 -Deoxy - 2 -a -azidocytidine TP ; 3
pentenyl)adenosine ; 2 -methylthio - N6 -methyladenosine ; (deaza ) 5 ( aza )cytosine ; 3 (methyl)cytosine ; 3-(alkyl)cyto
2 -methylthio -N6 -threonyl carbamoyladenosine ; N6 - glyci
sine; 3-( deaza ) 5 ( aza )cytosine ; 3-(methyl) cytidine ; 4,2-0
nylcarbamoyladenosine;
N6-isopentenyladenosine; 40 dimethylcytidine ; 5 (halo )cytosine ; 5 (methyl)cytosine; 5
N6 -methyladenosine; N6 - threonylcarbamoyladenosine; (propynyl)cytosine ; 5 (trifluoromethyl)cytosine ; 5 - alkyl)
1,2-0 -dimethyladenosine ; 1-methyladenosine ; 2 - O -meth
cytosine; 5- alkynyl)cytosine ; 5 - halo )cytosine; 5-(propy
yladenosine ; 2 - O -ribosyladenosine (phosphate); 2-methyl nyl)cytosine; 5-( trifluoromethyl)cytosine ; 5-bromo-cyti
adenosine; 2-methylthio -N6 isopentenyladenosine; 2 -meth
dine; 5 -iodo -cytidine; 5 -propynyl cytosine; 6-(azo )cytosine ;
ylthio -N6 -hydroxynorvalyl carbamoyladenosine ; 2 '-0- 45 6 -aza -cytidine ; aza cytosine ; deaza cytosine ; N4 (acetyl)
methyladenosine ; 2 - O -ribosyladenosine (phosphate );
Isopentenyladenosine; N6- ( cis -hydroxyisopentenyl)adenos
ine; N6,2-0 -dimethyladenosine ; N6,2 -O - dimethyladenos
ine ; N6 ,N6,2 - O -trimethyladenosine; N6 ,N6 -dimethylad
N6 - acetyladenosine ; 50
enosine;
N6 -hydroxynorvalylcarbamoyladenosine; N6 -methyl-N6
threonylcarbamoyladenosine ; 2-methyladenosine ; 2-meth
ylthio -N6 -isopentenyladenosine;
7 -deaza -adenosine ;
N1-methyl-adenosine; N6 , N6 (dimethyl)adenine; N6 -cis
hydroxy -isopentenyl- adenosine; a -thio - adenosine; 2 55
(amino Jadenine ; 2 (aminopropyl)adenine ; 2 (methylthio ) N6
(isopentenyl) adenine ; 2- alkyl)adenine; 2 - aminoalkyl)ad
enine ; 2 - aminopropyl)adenine ; 2 - halo Jadenine ; 2 - Chalo )
adenine; 2- (propyl) adenine ; 2 '-Amino -2'-deoxy -ATP ;
2'-Azido - 2'-deoxy - ATP ; 2'-Deoxy - 2'- a -aminoadenosine TP ; 60
2 '-Deoxy - 2'-a -azidoadenosine TP ; 6 (alkyl)adenine ; 6
(methyl)adenine; 6-( alkyl)adenine ; 6-(methyl)adenine; 7
(deaza adenine ; 8 (alkenyl) adenine ; 8 (alkynyl)adenine ; 8
cytosine; 1 -methyl- 1 -deaza -pseudoisocytidine; l -methyl
2 -methoxy - 5 -methyl-cytidine ;
pseudoisocytidine ;
2 -methoxy -cytidine; 2 - thio -5 -methyl-cytidine; 4 -methoxy
1 -methyl-pseudoisocytidine; 4 -methoxy -pseudoisocytidine;
4 - thio - 1 -methyl- 1-deaza -pseudoisocytidine;
4 -thio - 1
methyl-pseudoisocytidine; 4 - thio -pseudoisocytidine ; 5 -aza
zebularine; 5 -methyl-zebularine ; pyrrolo -pseudoisocytidine ;
Zebularine ; (E )-5-(2 -Bromo -vinyl)cytidine TP ; 2,2 '-an
hydro -cytidine TP hydrochloride ; 2*Fluor-N4- BZ- cytidine
TP ; 2'Fluoro -N4- Acetyl-cytidine TP ; 2 - O -Methyl-N4
Acetyl-cytidine TP ; 2'O -methyl-N4 -Bz -cytidine TP ; 2'-a
Ethynylcytidine TP ; 2'-a - Trifluoromethylcytidine TP ; 2'-b
Ethynylcytidine TP ; 2'-b - Trifluoromethylcytidine TP ;
2'-Deoxy - 2', 2'-difluorocytidine TP ; 2 -Deoxy- 2'-a -mercap
tocytidine TP ; 2'-Deoxy -2'-a-thiomethoxycytidine TP ;
2 '-Deoxy -2'-b -aminocytidine TP; 2 -Deoxy - 2'-b -azidocyti
dine TP ; 2 -Deoxy - 2'- b -bromocytidine TP ; 2'- Deoxy - 2 '-b
chlorocytidine TP ; 2 -Deoxy - 2 -b - fluorocytidine TP ; 2 -De
8 -Chalo )adenine ; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine ;
8-(thiol) adenine; 8 -azido - adenosine ; aza adenine; deaza
(1 -propynyl cytidine TP ; 3'- Ethynylcytidine TP ; 4 '- Azido
cytidine TP ; 4'-Carbocyclic cytidine TP ; 4 '-Ethynylcytidine
(amino )adenine; 8 (thioalkyl)adenine; 8-(alkenyl) adenine ; oxy-2'-b -iodocytidine TP ; 2 - Deoxy-2 -b -mercaptocytidine
8 - alkyl)adenine; 8-(alkynyl)adenine; 8 - Camino )adenine; 65 TP ; 2 -Deoxy-2 -b -thiomethoxycytidine TP ; 2 -O -Methyl-5
US 10,702,600 B1
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TP ; 5-( 1-Propynyl) ara -cytidine TP ; 5-( 2 -Chloro -phenyl) -2 methyluridine; 5 -carboxymethylaminomethyl-2 - thiouri
thiocytidine TP ; 5-(4 -Amino -phenyl) -2 - thiocytidine TP ; dine ;
5 -carboxymethylaminomethyl-2 -thiouridine ;
5 -Aminoallyl-CTP ; 5 - Cyanocytidine TP ; 5 - Ethynylara -cy
5 -carboxymethylaminomethyluridine ; 5 -carboxymethylam
tidine TP ; 5- Ethynylcytidine TP ; 5'-Homo-cytidine TP ; inomethyluridine ; 5 - Carbamoylmethyluridine TP ;
5 -Methoxycytidine TP ; 5- Trifluoromethyl- Cytidine TP ; 5 5 -methoxycarbonylmethyl-2 -O -methyluridine ; 5-methoxy
N4 -Amino -cytidine TP ; N4 -Benzoyl-cytidine TP ; Pseudoi carbonylmethyl- 2-thiouridine ; 5 -methoxycarbonylmethylu
socytidine; 7-methylguanosine; N2,2-0 -dimethylguanos
ridine; 5 -methoxyuridine ; 5 -methyl- 2 -thiouridine ; 5 -meth
ine ; N2-methylguanosine; Wyosine ; 1,2-0 -dimethyl ylaminomethyl-2 -selenouridine; 5 -methylaminomethyl-2
5 -methylaminomethyluridine ;
guanosine ; l -methylguanosine ; 2 - O -methylguanosine ; thiouridine;
2 -O -ribosylguanosine (phosphate ); 2 -O -methylguanosine; 10 5 -Methyldihydrouridine; 5-Oxyacetic acid -Uridine TP ;
2'-O - ribosylguanosine (phosphate); 7-aminomethyl-7 5 - Oxyacetic acid -methyl ester-Uridine TP ; N1-methyl
deazaguanosine; 7 -cyano- 7 - deazaguanosine; Archaeosine; pseudo -uridine ; uridine 5 -oxyacetic acid ; uridine 5 -oxy
Methylwyosine ; N2,7 - dimethylguanosine; N2,N2,2-0
acetic acid methyl ester; 3-(3 -Amino -3 -carboxypropyl)-Uri
trimethylguanosine ; N2,N2,7 -trimethylguanosine ; N2,N2 dine TP ; 5 - iso -Pentenylaminomethyl)-2 -thiouridine TP ;
dimethylguanosine; N2,7,2 '-O - trimethylguanosine; 6 - thio- 15 5 - iso -Pentenylaminomethyl)-2'-O -methyluridine
TP ;
guanosine ;
7 - deaza - guanosine; 8 -oxo -guanosine ; 5 - iso -Pentenylaminomethyl)uridine TP ; 5 -propynyl uracil ;
N1-methyl-guanosine ; a -thio -guanosine ; 2 (propyl)guanine ; a -thio-uridine; 1 (aminoalkylamino -carbonylethylenyl )-2
2 - alkyl guanine ; 2' -Amino- 2'- deoxy -GTP ; 2 '-Azido - 2'- de
(thio )-pseudouracil; 1 ( aminoalkylaminocarbonylethyl
oxy -GTP ; 2'-Deoxy -2'-a -aminoguanosine TP ; 2'-Deoxy -2' enyl)-2,4-(dithio )pseudouracil ; 1 (aminoalkylaminocarbo
(thio )pseudouracil ;
a -azidoguanosine TP ; 6 (methyl )guanine ; 6-(alkyl)guanine; 20 nylethylenyl )-4
1
6 - methyl)guanine ; 6 -methyl- guanosine ; 7 (alkyl guanine ; 7 (aminoalkylaminocarbonylethylenyl) -pseudouracil ;
(deaza )guanine ; 7 (methyl)guanine; 7 - alkyl)guanine; (aminocarbonylethylenyl) -2 (thio ) -pseudouracil ; 1 (amin
7 - deaza guanine ; 7-(methyl) guanine ; 8 (alkyl)guanine ; 8 ocarbonylethylenyl)-2,4 - dithio )pseudouracil; 1 ( aminocar
( alkynyl)guanine ; 8 (halo ) guanine; 8 ( thioalkyl)guanine; bonylethylenyl) -4 (thio ) pseudouracil; 1 ( aminocarbonyleth
8-( alkenyl) guanine; 8 - alkyl)guanine; 8 - alkynyl)guanine; 25 ylenyl) -pseudouracil; 1 substituted 2 (thio ) -pseudouracil ; 1
8-(amino)guanine ; 8 - Chalo ) guanine; 8-(hydroxyl) guanine; substituted 2,4 - dithio )pseudouracil; 1 substituted 4 (thio )
8-(thioalkyl) guanine; 8-( thiol) guanine; aza guanine; deaza pseudouracil ; 1 substituted pseudouracil ; 1-( aminoalky
guanine; N (methyl) guanine; N-(methyl) guanine; 1 -methyl lamino -carbonylethylenyl)-2-(thio )-pseudouracil ;
6 -thio - guanosine; 6 -methoxy -guanosine; 6 - thio -7 - deaza -8
1-Methyl-3-(3 -amino -3-carboxypropyl) pseudouridine TP ;
aza -guanosine; 6 -thio -7-deaza -guanosine; 6 -thio -7 -methyl- 30 1-Methyl-3-(3 -amino -3-carboxypropyl)pseudo-UTP ;
guanosine ; 7 -deaza -8 -aza - guanosine ; 7 -methyl-8 - oxo
1 -Methyl-pseudo -UTP ; 2 (thio )pseudouracil; 2' deoxy uri
guanosine ; N2,N2-dimethyl-6 -thio - guanosine ; N2-methyl dine; 2' fluorouridine ; 2-(thio )uracil ; 2,4 - dithio )psuedoura
6 -thio -guanosine ; 1 -Me-GTP ; 2'Fluoro -N2-isobutyl cil; 2' methyl, 2'amino, 2' azido , 2'fluro -guanosine ;
guanosine TP ; 2'O -methyl-N2 -isobutyl- guanosine TP; 2 '-a
2 -Amino - 2'-deoxy-UTP;
2'-Azido - 2 '- deoxy -UTP ;
Ethynylguanosine TP ; 2 -a - Trifluoromethylguanosine TP ; 35 2'- Azido - deoxyuridine TP ; 2 - O -methylpseudouridine ; 2 '
2'-b -Ethynylguanosine TP ; 2'-b - Trifluoromethylguanosine deoxy uridine; 2' fluorouridine; 2'- Deoxy - 2'-a -aminouridine
TP ; 2 '-Deoxy - 2', 2 -difluoroguanosine TP ; 2 -Deoxy - 2 -a
TP ; 2 -Deoxy -2'-a -azidouridine TP ; 2 -methylpseudouridine ;
mercaptoguanosine TP ; 2'-Deoxy-2'- a- thiomethoxyguanos 3 (3 amino -3 carboxypropyl)uracil ; 4 (thio )pseudouracil ;
ine TP ; 2'-Deoxy -2 '-b -aminoguanosine TP ; 2 -Deoxy - 2 -b
4-(thio )pseudouracil ; 4-( thio Juracil ; 4 -thiouracil; 5 (1,3 -di
azidoguanosine TP ; 2'-Deoxy -2'-b -bromoguanosine TP ; 40 azole -1 - alkyl)uracil ; 5 ( 2-aminopropyluracil; 5 (amino
53
2'-Deoxy -2'-b -chloroguanosine TP ; 2'- Deoxy -2'-b - fluo alkyl)uracil; 5 (dimethylaminoalkyl) uracil; 5 (guanidini
roguanosine TP ; 2'-Deoxy -2'-b - iodoguanosine TP ; 2'-De umalkyl)uracil ; 5 (methoxycarbonylmethyl) -2-( thio Juracil ;
oxy - 2 -b -mercaptoguanosine TP ; 2 '-Deoxy - 2'-b -thio
5 (methoxycarbonyl-methyl )uracil ; 5 (methyl ) 2 ( thio )ura
methoxyguanosine TP ; 4'-Azidoguanosine TP ; cil ; 5 (methyl) 2,4 (dithio )uracil ; 5 (methyl) 4 ( thio ) uracil ; 5
4'-Carbocyclic guanosine TP ; 4 '-Ethynylguanosine TP ; 45 (methylaminomethyl) -2 (thio Juracil; 5 (methylaminom
5 -Homo-guanosine TP ; 8 -bromo-guanosine TP ; 9 -Deaza ethyl)-2,4 (dithio )uracil ; 5 (methylaminomethyl) -4 (thio )
guanosine TP ; N2-isobutyl-guanosine TP ; 1 -methylinosine ; uracil; 5 (propynyl)uracil ; 5 (trifluoromethyl)uracil ; 5-(2
Inosine ; 1,2'-O -dimethylinosine ; 2 -O -methylinosine; aminopropyl)uracil;
5 - alkyl) -2-(thio )pseudouracil;
7 -methylinosine; 2'-O -methylinosine; Epoxyqueuosine ; 5 - alkyl)-2,4 (dithio )pseudouracil ; 5- alkyl )-4 (thio
galactosyl -queuosine; Mannosylqueuosine ; Queuosine; 50 pseudouracil; 5 - alkyl)pseudouracil ; 5 - alkyl)uracil ;
allyamino -thymidine ; aza thymidine; deaza thymidine; 5-(alkynyl)uracil ; 5-( allylamino Juracil; 5-(cyanoalkyl)ura
deoxy -thymidine; 2 - O -methyluridine; 2 -thiouridine; cil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)
3 -methyluridine; 5 -carboxymethyluridine; 5 -hydroxyuri uracil; 5-(guanidiniumalkyl)uracil ; 5-(halo Juracil; 5-( 1,3 - di
dine; 5 -methyluridine; 5 -taurinomethyl- 2 - thiouridine ; 5 -tau
azole - 1 -alkyl) uracil ;
5-(methoxy ) uracil ;
rinomethyluridine ; Dihydrouridine; Pseudouridine; (3-(3- 55 5-(methoxycarbonylmethyl)-2-(thio Juracil ; 5- methoxycar
amino -3 - carboxypropyl)uridine; 1 -methyl- 3- (3 -amino -5
bonyl-methyl)uracil ; 5 - methyl) 2 (thio )uracil; 5-(methyl)
carboxypropyl)pseudouridine;
1-methylpseduouridine; 2,4 (dithio Juracil ; 5-(methyl) 4 ( thio )uracil ; 5-(methyl) -2
1 -methyl-pseudouridine ; 2 -O -methyluridine ; 2 - O -methylp
(thio pseudouracil ; 5-(methyl)-2,4 (dithio ) pseudouracil ;
seudouridine ; 2'-O -methyluridine ; 2 -thio - 2'- O -methyluri 5-(methyl)-4 (thio )pseudouracil ; 5-(methyl) pseudouracil ;
dine ; 3-(3 - amino -3 - carboxypropyl)uridine; 3,2 '- O -dimethy- 60 5 - methylaminomethyl)-2 ( thio Juracil ; 5- methylaminom
luridine ; 3 -Methyl- pseudo -Uridine TP ; 4 -thiouridine ; ethyl) -2,4 ( dithiouracil; 5-(methylaminomethyl) -4- (thio )
5-(carboxyhydroxymethyl )uridine; 5-(carboxyhydroxym
uracil; 5-(propynyl)uracil ; 5-( trifluoromethyl) uracil;
ethyl)uridine methyl ester; 5,2-0 -dimethyluridine ; 5,6 - di
hydro -uridine; 5 -aminomethyl-2- thiouridine; 5 -carbamoyl
methyl-2 - O -methyluridine ; 5 -carbamoylmethyluridine ; 65
5 -carboxyhydroxymethyluridine; 5 -carboxyhydroxymethy
luridine methyl ester; 5-carboxymethylaminomethyl-2'-O
5 -aminoallyl-uridine ; 5 -bromo-uridine; 5- iodo - uridine;
5 -uracil ; 6 (azo )uracil ; 6-( azo )uracil ; 6 - aza- uridine; ally
amino -uracil; aza uracil ; deaza uracil ; N3 (methyl) uracil ;
Pseudo -UTP - 1-2 -ethanoic acid ; Pseudouracil ; 4 - Thio
pseudo -UTP ; 1 -carboxymethyl-pseudouridine ; 1 -methyl- 1
US 10,702,600 B1
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56
deaza-pseudouridine; 1-propynyl-uridine ; 1 -taurinomethyl pholino )-pseudo -UTP ; 1 -Methyl-6-(substituted phenyl)
1 -methyl- uridine;
1 - taurinomethyl- 4 -thio -uridine; pseudo -UTP ; 1-Methyl-6 - amino -pseudo-UTP ; 1-Methyl-6
1-taurinomethyl-pseudouridine ; 2 -methoxy -4 - thio -pseudou
azido -pseudo -UTP ;
1-Methyl-6 -bromo -pseudo -UTP ;
ridine; 2 -thio - 1-methyl- 1 -deaza -pseudouridine; 2 -thio - 1
1-Methyl-6 - butyl-pseudo -UTP ; 1-Methyl-6 -chloro -pseudo
methyl-pseudouridine ; 2 -thio -5 -aza -uridine ; 2- thio -dihy- 5 UTP ; 1-Methyl-6 -cyano -pseudo -UTP ; 1 -Methyl-6 -dimeth
dropseudouridine ;
2 -thio -dihydrouridine ;
2 - thio
ylamino -pseudo -UTP ; 1 -Methyl-6 -ethoxy -pseudo-UTP ;
pseudouridine;
4 -methoxy - 2 -thio -pseudouridine; 1 -Methyl-6 - ethylcarboxylate -pseudo -UTP ; 1 -Methyl-6
4 -methoxy -pseudouridine ; 4 - thio - 1-methyl-pseudouridine ; ethyl- pseudo -UTP ;
1 -Methyl-6 - fluoro - pseudo -UTP ;
1 -Methyl-6 -hy
4 -thio -pseudouridine; 5 -aza -uridine; Dihydropseudouridine ; 1 -Methyl-6 - formyl-pseudo -UTP ;
(+ ) 1-( 2-Hydroxypropyl)pseudouridine TP ; (2R )-1-(2 -Hy- 10 droxyamino-pseudo -UTP ; 1 -Methyl-6 -hydroxy -pseudo
droxypropyl)pseudouridine TP ; (2S )-1-(2 -Hydroxypropyl) UTP ; 1-Methyl-6 - iodo -pseudo -UTP ; 1-Methyl -6 -iso -pro
pseudouridine TP ; ( E )-5- (2 -Bromo -vinyl) ara -uridine TP ; pyl -pseudo -UTP ;
1 -Methyl-6 -methoxy - pseudo -UTP ;
( E )-5-( 2- Bromo -vinyl)uridine TP ; (Z )-5-(2 - Bromo -vinyl) 1-Methyl-6 -methylamino - pseudo -UTP ; 1-Methyl-6 -phenyl
ara -uridine TP ; ( Z )-5- (2 -Bromo -vinyl)uridine TP ; 1-(2,2,2
pseudo-UTP; 1-Methyl-6 -propyl-pseudo-UTP; 1-Methyl-6
Trifluoroethyl) -pseudo -UTP ; 1- (2,2,3,3,3 -Pentafluoropro- 15 tert-butyl-pseudo -UTP ;
1 -Methyl-6 -trifluoromethoxy
pyl)pseudouridine TP ; 1-(2,2-Diethoxyethyl)pseudouridine pseudo -UTP ; 1-Methyl-6 - trifluoromethyl-pseudo-UTP ;
TP ; 1-( 2,4,6 - Trimethylbenzyl)pseudouridine TP ; 1-(2,4,6
1 -Morpholinomethylpseudouridine TP ; 1 -Pentyl-pseudo
Trimethyl- benzyl)pseudo -UTP ; 1-(2,4,6 - Trimethyl-phenyl) UTP ; 1 -Phenyl- pseudo -UTP ; 1- Pivaloylpseudouridine TP ;
pseudo -UTP ; 1-(2 -Amino -2 -carboxyethyl) pseudo -UTP ; 1 -Propargylpseudouridine TP ; 1 - Propyl-pseudo -UTP ;
1-(2 -Amino -ethyl) pseudo -UTP ;
1- (2 -Hydroxyethyl) 20 1- propynyl -pseudouridine ; 1 -p - tolyl-pseudo -UTP ; 1-tert
pseudouridine TP ; 1- ( 2 -Methoxyethyl)pseudouridine TP ; Butyl- pseudo-UTP ; 1-Thiomethoxymethylpseudouridine
1-( 3,4 -Bis - trifluoromethoxybenzyl)pseudouridine TP ; 1- (3 , TP ; 1 - Thiomorpholinomethylpseudouridine TP ; 1 - Trifluo
4 -Dimethoxybenzyl)pseudouridine TP ; 1-(3 -Amino - 3 -car roacetylpseudouridine TP ; 1 - Trifluoromethyl- pseudo -UTP ;
boxypropyl)pseudo -UTP ; 1-(3 - Amino -propyl)pseudo -UTP ; 1 - Vinylpseudouridine TP ; 2,2 -anhydro -uridine TP ;
1-(3 - Cyclopropyl-prop -2 - ynyl)pseudouridine TP ; 1-(4- 25 2'-bromo -deoxyuridine TP ; 2'- F -5 -Methyl- 2'-deoxy-UTP ;
Amino -4 - carboxybutyl) pseudo -UTP; 1- ( 4 -Amino -benzyl) 2 - Me- 5 -Me-UTP ; 2 -OMe-pseudo -UTP ; 2'-a - Ethynyluri
pseudo -UTP ; 1-(4 - Amino -butyl)pseudo -UTP ; 1-(4 - Amino
dine TP ; 2'-a - Trifluoromethyluridine TP ; 2'-b - Ethynyluri
phenyl)pseudo -UTP ; 1-( 4 -Azidobenzyl)pseudouridine TP ; dine TP; 2 - b - Trifluoromethyluridine TP ; 2 '-Deoxy - 2', 2'-di
1-(4 -Bromobenzyl)pseudouridine TP ; 1-(4 -Chlorobenzyl) fluorouridine TP ; 2 -Deoxy - 2'-a-mercaptouridine TP ;
pseudouridine TP ; 1- (4 -Fluorobenzyl)pseudouridine TP ; 30 2'-Deoxy -2'-a - thiomethoxyuridine TP ; 2 - Deoxy - 2 '-b -amin
1-(4 -Iodobenzyl)pseudouridine TP ; 1-(4 -Methanesulfonyl ouridine TP ; 2'-Deoxy -2'- b -azidouridine TP ; 2 - Deoxy - 2 '-b
benzyl )pseudouridine TP ; 1-(4 -Methoxybenzyl)pseudouri bromouridine TP ; 2'-Deoxy - 2 - b -chlorouridine TP; 2 -De
dine TP; 1-( 4 -Methoxy -benzyl)pseudo -UTP; 1-( 4 -Methoxy
oxy - 2'-b -fluorouridine TI 2 -Deoxy - 2'-b -iodouridine TP ;
phenyl)pseudo -UTP ; 1- (4 -Methylbenzyl)pseudouridine TP ; 2 -Deoxy -2 '-b -mercaptouridine TP ; 2'-Deoxy -2'-b -thio
1-(4 -Methyl-benzyl) pseudo -UTP;
1- (4 -Nitrobenzyl) 35 methoxyuridine
2 -methoxy- 4 - thio -uridine;
TP ;
pseudouridine TP ; 1-(4 -Nitro -benzyl)pseudo -UTP ; 1(4 -Ni 2 -methoxyuridine ; 2'-O -Methyl -5-(1-propynyl)uridine TP ;
tro -phenyl)pseudo -UTP ;
1- (4 - Thiomethoxybenzyl) 3 -Alkyl- pseudo -UTP ; 4 '-Azidouridine TP ; 4'-Carbocyclic
pseudouridine TP; 1-(4 - Trifluoromethoxybenzyl) uridine TP ; 4'-Ethynyluridine TP; 5- (1 -Propynyl)ara -uridine
pseudouridine
1- (4 - Trifluoromethylbenzyl) TP ; 5- (2 -Furanyl)uridine TP ; 5 - Cyanouridine TP ; 5 -Dim
TP ;
pseudouridine TP ; 1-(5 - Amino -pentyl) pseudo -UTP ; 1-(6- 40 ethylaminouridine TP ; 5 -Homo- uridine TP ; 5 - iodo - 2'
Amino -hexyl)pseudo -UTP ; 1,6 -Dimethyl-pseudo -UTP ; fluoro -deoxyuridine TP ; 5 -Phenylethynyluridine TP ; 5 - Tri
1-(3-(2- {2-[2-(2-Aminoethoxy )-ethoxy]-ethoxy} -ethoxy)
propionyl]pseudouridine TP ; 1- {3-[2-(2- Aminoethoxy )
ethoxyl-propionyl} pseudouridine TP ; 1- Acetylpseudouri
dine TP ; 1- Alkyl-6-( 1-propynyl)-pseudo-UTP ; 1- Alkyl-6- 45
( 2 -propynyl) -pseudo -UTP ; 1 -Alkyl-6 - allyl-pseudo -UTP ;
1 -Alkyl-6 -ethynyl-pseudo -UTP ;
1 -Alkyl-6 -homoallyl
deuteromethyl-6 -deuterouridine TP ; 5 - Trifluoromethyl
Uridine TP ; 5 - Vinylarauridine TP ; 6-(2,2,2 - Trifluoroethyl)
pseudo -UTP ; 6-(4 -Morpholino ) -pseudo -UTP ; 6-(4
Thiomorpholino) -pseudo -UTP ; 6- (Substituted - Phenyl)
pseudo -UTP ; 6 -Amino -pseudo -UTP ; 6 -Azido - pseudo-UTP ;
6 -Bromo -pseudo -UTP ; 6 -Butyl-pseudo-UTP ; 6 -Chloro
pseudo -UTP ; 1 -Alkyl-6 -vinyl-pseudo -UTP; 1 -Allylp pseudo -UTP ; 6 -Cyano -pseudo -UTP ; 6 -Dimethylamino
seudouridine TP ; 1- Aminomethyl-pseudo -UTP ; 1- Benzo pseudo -UTP ; 6 -Ethoxy -pseudo -UTP ; 6 -Ethylcarboxylate
ylpseudouridine TP ; 1- Benzyloxymethylpseudouridine TP ; 50 pseudo -UTP ; 6 -Ethyl-pseudo -UTP ; 6 -Fluoro -pseudo-UTP ;
1 -Benzyl-pseudo -UTP ; 1 -Biotinyl- PEG2-pseudouridine TP ; 6 -Formyl-pseudo -UTP ; 6 -Hydroxyamino -pseudo -UTP ;
1 -Biotinylpseudouridine TP ; 1-Butyl-pseudo -UTP ; 1 - Cya 6 -Hydroxy -pseudo-UTP ; 6 - Iodo -pseudo -UTP ; 6 - iso - Pro
nomethylpseudouridine TP ; 1- Cyclobutylmethyl-pseudo
pyl-pseudo-UTP ; 6 -Methoxy - pseudo -UTP; 6 -Methyl
UTP ; 1 -Cyclobutyl-pseudo -UTP ; 1- Cycloheptylmethyl
amino -pseudo -UTP ; 6 -Methyl-pseudo -UTP ; 6 - Phenyl
pseudo -UTP ;
1 -Cycloheptyl-pseudo-UTP ; 55 pseudo-UTP ; 6 -Phenyl-pseudo -UTP ; 6 -Propyl-pseudo
1 -Cyclohexylmethyl-pseudo -UTP ; 1- Cyclohexyl-pseudo UTP ; 6 -tert-Butyl-pseudo -UTP ; 6 - Trifluoromethoxy
UTP ; 1- Cyclooctylmethyl-pseudo -UTP ; 1- Cyclooctyl pseudo -UTP ; 6 - Trifluoromethyl-pseudo -UTP ; Alpha -thio
pseudo-UTP ; 1 - Cyclopentylmethyl-pseudo -UTP ; 1-Cyclo
pseudo -UTP; Pseudouridine 1- (4 -methylbenzenesulfonic
pentyl-pseudo -UTP ; 1 -Cyclopropylmethyl-pseudo-UTP ; acid ) TP ; Pseudouridine 1-(4 -methylbenzoic acid ) TP ;
1- Cyclopropyl-pseudo -UTP ;
1 -Ethyl-pseudo -UTP ; 60 Pseudouridine TP 1- [3-( 2 -ethoxylpropionic acid ; Pseudou
1 -Hexyl-pseudo -UTP ; 1 -Homoallylpseudouridine TP ; ridine TP 1- [3- { 2- (2-[2-( 2 - ethoxy )-ethoxy ]-ethoxy )
1-Hydroxymethylpseudouridine TP ; 1 - iso -propyl-pseudo ethoxy } ]propionic acid ; Pseudouridine TP 1-[3-{ 2- (2-[2- {2
UTP ; 1-Me-2 -thio -pseudo -UTP ; 1-Me-4 -thio - pseudo -UTP ; (2 - ethoxy )-ethoxy ) -ethoxy ] -ethoxy) -ethoxy } ]propionic
1-Me- alpha -thio -pseudo-UTP ; 1-Methanesulfonylmethylp
acid ; Pseudouridine TP 1- [3-2- (2- [2 - ethoxy]-ethoxy)
seudouridine TP ; 1-Methoxymethylpseudouridine TP ; 65 ethoxy }]propionic acid ; Pseudouridine TP 1-[3- {2-(2
1 -Methyl-6-(2,2,2 - Trifluoroethyl)pseudo -UTP; 1-Methyl-6
ethoxy)-ethoxy } ] propionic acid ; Pseudouridine TP 1-meth
(4 -morpholino ) -pseudo -UTP ;
1 -Methyl-6- (4 -thiomor
ylphosphonic acid ; Pseudouridine TP 1-methylphosphonic
US 10,702,600 B1
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58
acid diethyl ester ; Pseudo -UTP -N1-3 -propionic acid ;
Pseudo -UTP -N1-4 -butanoic acid ; Pseudo -UTP -N1-5 -pen
tanoic acid ; Pseudo-UTP -N1-6 -hexanoic acid ; Pseudo
UTP-N1-7 -heptanoic acid ; Pseudo -UTP -N1-methyl-p -ben
guanosine TP ; 5-2-carbomethoxyvinyl)uridine TP ; and N6
(19 - Amino -pentaoxanonadecyl)adenosine TP.
In some embodiments , polynucleotides (e.g., RNA poly
nucleotides, such asmRNA polynucleotides) include a com
zoic acid ; Pseudo -UTP -N1-p -benzoic acid ; Wybutosine ; 5 bination of at least two ( e.g., 2, 3 , 4 or more ) of the
Hydroxywybutosine; Isowyosine ; Peroxywybutosine; aforementioned modified nucleobases .
In some embodiments, modified nucleobases in poly
undermodified hydroxywybutosine ; 4 -demethylwyosine ;
(e.g., RNA polynucleotides, such as mRNA
2,6-(diamino )purine; 1-(aza )-2-( thio )-3-(aza )-phenoxazin nucleotides
) are selected from the group consisting of
1 -yl: 1,3-(diaza )-2- oxo )-phenthiazin - 1 -yl; 1,3-(diaza )-2 10 polynucleotides
pseudouridine
(
W
-methylpseudouridine (m'y ),N1- eth
(oxo)-phenoxazin - 1- yl; 1,3,5 - triaza -2,6 - dioxa)-naphtha ylpseudouridine , )2,-N1
thiouridine
-thiouridine , 5 -methylcyto
lene;2 (amino )purine; 2,4,5 - trimethyl)phenyl;2' methyl, sine, 2-thio -1 -methyl- 1-deaza, 4-'pseudouridine
2 -thio -1
2'amino, 2'azido, 2'fluro -cytidine ;2 ' methyl, 2' amino, methyl-pseudouridine, 2 -thio -5 -aza -uridine , , 2- thio
-dihy
2'azido, 2'fluro - adenine;2'methyl, 2'amino , 2' azido, 2'fluro dropseudouridine , 2 -thio -dihydrouridine, 2-thio -pseudouri
uridine ;2'-amino - 2'-deoxyribose; 2-amino-6 -Chloro -purine; 15 dine, 4-methoxy- 2-thio -pseudouridine, 4 -methoxy
2 -aza - inosinyl; 2' -azido - 2' -deoxyribose; 2'fluoro - 2'- deoxyri pseudouridine
, 4 -thio - 1 -methyl- pseudouridine , 4 -thio
bose; 2'- fluoro -modified bases; 2'-O -methyl- ribose ; 2 -oxo pseudouridine , 5 -aza -uridine, dihydropseudouridine,
7 -aminopyridopyrimidin -3 -yl; 2 -oxo -pyridopyrimidine-3 5 -methoxyuridine and 2 - O -methyl uridine. In some
yl; 2-pyridinone; 3 nitropyrrole ; 3-(methyl)-7-(propynyl) embodiments, polynucleotides (e.g., RNA polynucleotides ,
isocarbostyrilyl; 3- methyl)isocarbostyrilyl; 4-( fluoro ) -6- 20 such as mRNA polynucleotides ) include a combination of at
(methyl)benzimidazole ;
4-(methyl)benzimidazole ; least two ( e.g., 2, 3, 4 or more ) of the aforementioned
4-(methyl )indolyl; 4,6-(dimethyl) indolyl; 5 nitroindole; 5 modified nucleobases .
substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-ni
In some embodiments, modified nucleobases in poly
troindole; 6-(aza)pyrimidine; 6-(azo )thymine; 6-(methyl) -7 nucleotides (e.g. , RNA polynucleotides, such as mRNA
(aza) indolyl; 6 - chloro -purine ; 6 -phenyl -pyrrolo -pyrimidin- 25 polynucleotides ) are selected from the group consisting of
2 -on -3 -yl; 7-(aminoalkylhydroxy )-1 - aza )-2-(thio ) -3-(aza )
1 -methyl-pseudouridine (m'w ), 5 -methoxy -uridine (moU ),
phenthiazin - 1 - yl; 7-( aminoalkylhydroxy )-1-(aza )-2-(thio ) 5-methyl -cytidine (m?C ), pseudouridine (W ), a -thio - guanos
3 - aza )-phenoxazin - 1 - yl;
7- aminoalkylhydroxy )-1,3
ine and a -thio - adenosine . In some embodiments , polynucle
(diaza )-2-Coxo )-phenoxazin - 1-yl; 7-(aminoalkylhydroxy) -1 , otides includes a combination of at least two (e.g., 2 , 3 , 4 or
3- (diaza )-2 - oxo ) -phenthiazin - 1 - yl;
30 more ) of the aforementioned modified nucleobases.
7 - aminoalkylhydroxy )-1,3 - diaza )-2-( oxo )-phenoxazin - 1
In some embodiments, polynucleotides (e.g., RNA poly
yl; 7-(aza ) indolyl; 7-(guanidiniumalkylhydroxy )-1-(aza )-2 nucleotides , such as mRNA polynucleotides ) comprise
(thio )-3-(aza )-phenoxazinl-yl;
7-(guanidiniumalkylhy
pseudouridine (v ) and 5 -methyl-cytidine (m®C ). In some
embodiments, polynucleotides (e.g. , RNA polynucleotides,
droxy )-1- aza )-2- (thio )-3-(aza )-phenthiazin - 1 - yl;
7-(guanidiniumalkylhydroxy) -1 - aza )-2-( thio )-3-(aza )-phe- 35 such as mRNA polynucleotides) comprise 1-methyl
noxazin -1 -yl; 7-( guanidiniumalkylhydroxy )-1,3-(diaza) -2 pseudouridine (m + y ). In some embodiments, polynucle
(oxo )-phenoxazin - 1- yl; 7-(guanidiniumalkyl -hydroxy )-1,3
otides ( e.g., RNA polynucleotides, such as mRNA poly
nucleotides) comprise 1-methyl-pseudouridine my
( ) and
(diaza ) -2-(oxo ) -phenthiazin - 1 -yl;
7-( guanidiniumalkylhydroxy) -1,3-(diaza )-2-(oxo ) -phenox
5 -methyl-cytidine mC
( ). In some embodiments, polynucle
azin -1 -yl; 7-(propynyl) isocarbostyrilyl; 7-(propynyl )isocar- 40 otides ( e.g., RNA polynucleotides , such as mRNA poly
bostyrilyl, propynyl-7-(aza ) indolyl; 7 -deaza - inosinyl;
7 -substituted
1 - aza -2-(thio )-3 - aza )-phenoxazin - 1 -yl;
7 - substituted
1,3-(diaza )-2-(oxo)-phenoxazin - 1 -yl;
9 - methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl;
bis-ortho -Caminoalkylhydroxy) -6 -phenyl-pyrrolo -pyrimi
din -2 -on - 3 -yl; bis -ortho -substituted -6 -phenyl-pyrrolo -py
rimidin -2 -on- 3 -yl; Difluorotolyl; Hypoxanthine ; Imi
dizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine;
N2- substituted purines ; N6 -methyl-2 -amino -purine;
N6 -substituted purines; N -alkylated derivative ; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl;
Nitropyrazolyl; Nubularine ; 06 -substituted purines ; O - alky
lated derivative; ortho-(aminoalkylhydroxy ) -6 -phenyl-pyr
nucleotides) comprise 2-thiouridine (s’U ). In some embodi
ments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides ) comprise 2- thiouridine and
5 -methyl-cytidine (mC). In some embodiments , polynucle
45 otides (e.g., RNA polynucleotides, such as mRNA poly
nucleotides) comprise methoxy -uridine (mo®U ) . In some
embodiments, polynucleotides (e.g. , RNA polynucleotides,
such as mRNA polynucleotides ) comprise 5-methoxy -uri
dine (moU) and 5-methyl-cytidine (mC). In someembodi
50 ments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides ) comprise 2 -O -methyl uridine. In
some embodiments polynucleotides ( e.g., RNA polynucle
otides, such as mRNA polynucleotides) comprise 2-0
methyl uridine and 5 -methyl- cytidine (mC). In some
55 embodiments , polynucleotides (e.g., RNA polynucleotides,
such as mRNA polynucleotides) comprise N6 -methyl-ad
enosine (mºA ). In some embodiments, polynucleotides ( e.g.,
RNA polynucleotides, such as mRNA polynucleotides)
comprise N6 -methyl-adenosine (m?A ) and 5 -methyl-cyti
olo - pyrimidin - 2 -on -3 -yl; ortho -substituted -6 -phenyl-pyr
rolo -pyrimidin -2 -on -3 - yl; Oxoformycin TP ; para(aminoalkylhydroxy )-6 -phenyl-pyrrolo -pyrimidin -2 -on -3
yl; para-substituted -6 -phenyl-pyrrolo -pyrimidin - 2 -on -3 -yl;
Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7 - aza )in
dolyl; Pyrenyl; pyridopyrimidin -3 -yl; pyridopyrimidin - 3 -yl,
2 -oxo -7-amino-pyridopyrimidin -3- yl; pyrrolo-pyrimidin -2- 60 dine (m?C ).
on -3 -yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl;
In some embodiments , polynucleotides (e.g., RNA poly
substituted 1,2,4-triazoles; Tetracenyl ; Tubercidine; Xan nucleotides, such as mRNA polynucleotides ) are uniformly
thine; Xanthosine- 5 '- TP ; 2 - thio -zebularine ; 5 - aza - 2 -thio - ze
modified ( e.g., fully modified , modified throughout the
bularine ; 7 -deaza - 2-amino -purine ; pyridin -4 -one ribo
entire sequence ) for a particular modification . For example,
nucleoside ; 2 -Amino-riboside - TP ; Formycin A TP ; 65 a polynucleotide can be uniformly modified with 5 -methyl
Formycin B TP; Pyrrolosine TP ; 2 -OH -ara - adenosine TP ; cytidine (m®C ), meaning that all cytosine residues in the
2'-OH - ara -cytidine TP ; 2 -OH -ara - uridine TP ; 2 -OH -ara
mRNA sequence are replaced with 5-methyl-cytidine (m®C ).
US 10,702,600 B1
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60
Similarly , a polynucleotide can be uniformly modified for
pyrimidine such as a modified uracil or cytosine . In some
any type of nucleoside residue present in the sequence by embodiments, at least 5 % , at least 10 % , at least 25 % , at least
replacement with a modified residue such as those set forth 50 % , at least 80 % , at least 90 % or 100 % of the uracil in the
above .
polynucleotide is replaced with a modified uracil (e.g. , a
Exemplary nucleobases and nucleosides having a modi- 5 5 -substituted uracil ). The modified uracil can be replaced by
fied cytosine include N4-acetyl-cytidine (ac4C ), 5 -methyl a compound having a single unique structure , or can be
cytidine (m5C ), 5 - halo -cytidine ( e.g., 5 -iodo -cytidine ), replaced by a plurality of compounds having different struc
5 -hydroxymethyl-cytidine (hm5C ), 1 -methyl-pseudoisocy
tures (e.g., 2 , 3 , 4 or more unique structures). n some
tidine, 2 -thio -cytidine (s2C ), and 2 -thio -5 -methyl-cytidine . embodiments, at least 5 % , at least 10 % , at least 25 % , at least
In some embodiments , a modified nucleobase is a modi- 10 50 % , at least 80 % , at least 90 % or 100 % of the cytosine in
fied uridine. Exemplary nucleobases and In some embodi the polynucleotide is replaced with a modified cytosine (e.g.,
ments, a modified nucleobase is a modified cytosine. nucleo
a 5 -substituted cytosine ). The modified cytosine can be
sides having a modified uridine include 5 -cyano uridine , and replaced by a compound having a single unique structure, or
can be replaced by a plurality of compounds having different
4 '-thio uridine .
In some embodiments, a modified nucleobase is a modi- 15 structures (e.g., 2, 3 , 4 or more unique structures).
fied adenine. Exemplary nucleobases and nucleosides hav
Thus, in some embodiments , the RNA (e.g., mRNA)
ing a modified adenine include 7 -deaza -adenine, 1-methyl vaccines comprise a 5'UTR element, an optionally codon
adenosine (m1A ), 2-methyl-adenine (m2A ), and N6-methyl optimized open reading frame, and a 3'UTR element, a
poly ( A ) sequence and /or a polyadenylation signal wherein
adenosine (m6A ).
In some embodiments, a modified nucleobase is a modi- 20 the RNA is not chemically modified .
fied guanine. Exemplary nucleobases and nucleosides hav
In some embodiments, the modified nucleobase is a
ing a modified guanine include inosine (I), 1-methyl -inosine modified uracil . Exemplary nucleobases and nucleosides
(mll), wyosine ( img ) , methylwyosine (mim ), 7 -deaza having a modified uracil include pseudouridine ( ), pyridin
guanosine , 7 - cyano -7 -deaza-guanosine ( preQO ), 7-amin
4 -one ribonucleoside , 5 -aza -uridine , 6 - aza -uridine, 2 -thio
(m7G ), 1 -methyl- guanosine (ml ), 8 -oxo -guanosine,
7 -methyl-8 - oxo - guanosine.
The polynucleotides of the present disclosure may be
4 -thio - pseudouridine , 2 - thio -pseudouridine , 5-hydroxy-uri
25 % , from 1 % to 50 % , from 1 % to 60 % , from 1 % to 70 % ,
thymine ), 1-methyl-pseudouridine (m'y ), 5 -methyl-2- thio
omethyl-7-deaza -guanosine (preQ1), 7-methyl-guanosine 25 5-aza-uridine, 2-thio-uridine (s U ), 4- thio -uridine (stu ),
dine (ho®U ), 5 -aminoallyl-uridine , 5-halo -uridine (e.g. ,
5 -iodo -uridineor 5 -bromo-uridine), 3 -methyl- uridine mU
( ),
partially or fully modified along the entire length of the 5 -methoxy -uridine (mo?U ), uridine 5 -oxyacetic acid
molecule . For example , one or more or all or a given type of 30 (cmo U ), uridine 5 -oxyacetic acid methyl ester (mcmoSU ),
nucleotide (e.g., purine or pyrimidine, or any one ormore or 5 -carboxymethyl-uridine (cmu), 1 -carboxymethyl
all of A , G , U , C ) may be uniformly modified in a poly pseudouridine, 5- carboxyhydroxymethyl- uridine ( chm®U ),
nucleotide of the disclosure , or in a given predetermined 5 -carboxyhydroxymethyl-uridine methyl ester (mchmU),
sequence region thereof (e.g., in the mRNA including or 5 -methoxycarbonylmethyl-uridine (mcm®U ), 5 -methoxy
excluding the polyA tail). In some embodiments, all nucleo- 35 carbonylmethyl-2- thio -uridine (mcm s?U ), 5 -aminomethyl
tides X in a polynucleotide of the present disclosure (or in 2- thio -uridine (nm s?U ), 5 -methylaminomethyl-uridine
5 -methylaminomethyl- 2 - thio - uridine
a given sequence region thereof) are modified nucleotides , (mnm ),
wherein X may any one of nucleotides A , G , U , C , or any (mnm s- U ),
5 -methylaminomethyl- 2 - seleno -uridine
one of the combinations A + G , A + U , A + C , G + U , G + C , U + C , (mnm se’U ), 5 -carbamoylmethyl-uridine (ncmU), 5-car
40 boxymethylaminomethyl-uridine ( cmnmU), 5 - carboxym
A + G + U , A + G + C , G + U + C or A + G + C .
The polynucleotide may contain from about 1 % to about ethylaminomethyl-2 -thio -uridine ( cmnm'sU ), 5-propynyl
100 % modified nucleotides ( either in relation to overall uridine, 1 -propynyl-pseudouridine , 5 - taurinomethyl-uridine
nucleotide content, or in relation to one or more types of ( Tm U ), 1-taurinomethyl-pseudouridine , 5 -taurinomethyl-2
nucleotide, i.e., any one or more of A , G , U or C ) or any thio -uridine(m?sU ), 1 -taurinomethyl-4 -thio -pseudouridine,
intervening percentage (e.g., from 1 % to 20 % , from 1 % to 45 5 -methyl-uridine (m®U , i.e., having the nucleobase deoxy
from 1 % to 80 % , from 1 % to 90 % , from 1 % to 95 % , from
10 % to 20 % , from 10 % to 25 % , from 10 % to 50 % , from
10 %
10 %
20 %
20 %
20 %
to 60 % , from
to 25 % , from
to 70 % , from
to 95 % , from
10 %
20 %
20 %
20 %
to 90 % , from 10 %
50 % to 70 % , from 50 %
uridine (m5s2U ), 1-methyl-4 -thio -pseudouridine (m's w ),
4 -thio - 1 -methyl- pseudouridine , 3-methyl-pseudouridine
to 70 % , from 10 % to 80 % , from
(my), 2- thio - 1-methyl-pseudouridine , 1 -methyl- 1-deaza
to 95 % , from 10 % to 100 % , from 50 pseudouridine , 2 - thio - 1-methyl-1 -deaza -pseudouridine ,
to 50 % , from 20 % to 60 % , from
dihydrouridine (D ), dihydropseudouridine, 5,6 - dihydrouri
to 80 % , from 20 % to 90 % , from
dine , 5 -methyl-dihydrouridine (m®D ), 2 - thio -dihydrouri
to 100 % , from 50 % to 60 % , from
dine, 2 -thio -dihydropseudouridine, 2 -methoxy -uridine ,
to 80 % , from 50 % to 90 % , from
2 -methoxy - 4 - thio -uridine,
4 -methoxy -pseudouridine ,
50 % to 95 % , from 50 % to 100 % , from 70 % to 80 % , from 55 4 -methoxy-2 -thio -pseudouridine, N1-methyl-pseudouri
70 % to 90 % , from 70 % to 95 % , from 70 % to 100 % , from
80 % to 90 % , from 80 % to 95 % , from 80 % to 100 % , from
90 % to 95 % , from 90 % to 100 % , and from 95 % to 100 % ).
Any remaining percentage is accounted for by the presence
of unmodified A , G , U , or C.
The polynucleotides may contain at a minimum 1 % and
at maximum 100 % modified nucleotides , or any intervening
dine,
3- (3 -amino - 3 -carboxypropyl) uridine
(acpU ),
1-methyl-3-(3 -amino -3-carboxypropyl)pseudouridine
(acp ), 5-( isopentenylaminomethyl) uridine ( inmU),
5-( isopentenylaminomethyl)-2 - thio -uridine
(inm’s U ),
60 a - thio -uridine, 2'-O -methyl-uridine (Um ), 5,2 '-O -dimethyl
uridine (msUm ), 2 -O -methyl-pseudouridine (Wm ), 2 -thio
2'-O -methyl-uridine (s’Um ), 5 -methoxycarbonylmethyl-2
percentage , such as at least 5 % modified nucleotides , at least O -methyl-uridine (mcm Um ), 5 -carbamoylmethyl-2-0
10 % modified nucleotides , at least 25 % modified nucleo
methyl-uridine (ncm Um ), 5- carboxymethylaminomethyl
tides, at least 50 % modified nucleotides, at least 80 % 65 2'-O -methyl-uridine (cmnm Um ), 3,2'-O -dimethyl-uridine
modified nucleotides , or at least 90 % modified nucleotides. ( m Um ), and 5 - isopentenylaminomethyl) -2'-O -methyl-uri
For example , the polynucleotides may contain a modified
dine ( inm Um ), 1- thio -uridine, deoxythymidine, 2'-F -ara
US 10,702,600 B1
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62
uridine , 2'-F -uridine, 2'-OH -ara -uridine, 5-( 2 -car
bomethoxyvinyl) uridine, and 5-( 3-(1 - E -propenylamino ) ]
6 -thio -7 -deaza - 8 -aza - guanosine,
7 -methyl-guanosine
(m7G ), 6 -thio - 7 -methyl- guanosine , 7 -methyl- inosine ,
6 -methoxy - guanosine ,
1 -methyl- guanosine
(mg ),
N2-methyl-guanosine (m?G ), N2,N2-dimethyl-guanosine
uridine.
In some embodiments , the modified nucleobase is a
having a modified cytosine include 5 -aza -cytidine , 6 -aza
modified cytosine. Exemplary nucleobases and nucleosides 5 (mº2G ), N2,7 -dimethyl-guanosine (m2,7G ), N2, N2,7 -dim
ethyl-guanosine (m2,2,7G ), 8-oxo - guanosine, 7 -methyl-8
cytidine, pseudoisocytidine, 3 -methyl-cytidine (mºC ), oxo - guanosine, l-methyl-6 - thio -guanosine, N2-methyl-6
N4-acetyl-cytidine (ac+ C ), 5-formyl- cytidine ( f?C ), thio -guanosine, N2,N2-dimethyl-6 - thio -guanosine, a -thio
N4 -methyl- cytidine ( m ° c ) , 5 -methyl-cytidine (mC), guanosine , 2 -O -methyl- guanosine (Gm ), N2-methyl- 2 -O
5 -halo -cytidine ( e.g., 5-iodo -cytidine ), 5 -hydroxymethyl- 10 methyl-guanosine (m?Gm ), N2,N2-dimethyl-2 -O -methyl
cytidine (hmC), 1-methyl-pseudoisocytidine, pyrrolo -cyti guanosine (m²2Gm ), 1 -methyl- 2 - O -methyl- guanosine
dine, pyrrolo -pseudoisocytidine, 2-thio -cytidine (s- C ), (mGm ), N2,7 -dimethyl-2 '-O -methyl- guanosine (m ' 76m ) ,
2- thio -5 -methyl-cytidine , 4 -thio -pseudoisocytidine, 4- thio
2 -O -methyl- inosine ( Im ), 1,2 - O -dimethyl- inosine (m'Im ),
1 -methyl- pseudoisocytidine ,
4 -thio - 1 -methyl- 1 -deaza
2'- O -ribosylguanosine (phosphate ) (Gr(p ) ), 1- thio -guanos
pseudoisocytidine, 1-methyl- 1-deaza -pseudoisocytidine, 15 ine, 06 -methyl-guanosine, 2'-F -ara -guanosine , and 2'-F
zebularine, 5 -aza -zebularine, 5-methyl-zebularine , 5 -aza -2
guanosine .
thio -zebularine, 2 -thio -zebularine, 2-methoxy -cytidine , N -Linked Glycosylation Site Mutants
2 -methoxy - 5-methyl-cytidine , 4 -methoxy -pseudoisocyti
N - linked glycans of viral proteins play important roles in
dine , 4 -methoxy - 1-methyl-pseudoisocytidine , lysidine modulating the immune response .Glycans can be important
(dimethyl
k2C ), a --cytidine
thio -cytidine
, 2'-O -methyl-cytidine (Cm ), 5,2'-O- 20 for maintaining the appropriate antigenic conformations,
(
mCm
), N4-acetyl-2'-O -methyl-cytidine shielding potential neutralization epitopes, and may alter the
(ac+ Cm ), N4,2'-0 - dimethyl-cytidine (m * Cm ), 5 - formyl- 2' proteolytic susceptibility of proteins. Some viruses have
O -methyl-cytidine (fCm ), N4,N4,2'- O -trimethyl- cytidine putative N - linked glycosylation sites. Deletion or modifica
(m ^2Cm ), 1 -thio -cytidine , 2 -F -ara -cytidine, 2 -F -cytidine , tion of an N - linked glycosylation site may enhance the
and 2 -OH - ara -cytidine .
25 immune response . Thus , the present disclosure provides, in
In some embodiments , the modified nucleobase is a some embodiments , RNA ( e.g., mRNA ) vaccines compris
modified adenine . Exemplary nucleobases and nucleosides ing nucleic acids (e.g.,mRNA ) encoding antigenic polypep
having a modified adenine include 2 -amino -purine , 2 , 6 -di
tides that comprise a deletion or modification at one or more
aminopurine , 2 -amino -6 -halo -purine (e.g., 2-amino -6
N - linked glycosylation sites .
chloro -purine ), 6 -halo -purine (e.g., 6 -chloro -purine ), 30 In Vitro Transcription of RNA (e.g., mRNA )
2 - amino -6 -methyl-purine, 8 -azido -adenosine, 7 -deaza- ad
Respiratory virus vaccines of the present disclosure com
enine, 7 -deaza - 8 -aza -adenine, 7 - deaza - 2 -amino -purine, prise at least one RNA polynucleotide, such as a mRNA
7 -deaza - 8 -aza -2 -amino -purine , 7 -deaza - 2,6 -diaminopurine , ( e.g., modified mRNA ).mRN for example, is transcribed
7 -deaza -8 -aza -2,6 -diaminopurine ,
1 -methyl-adenosine in vitro from template DNA, referred to as an “ in vitro
(m'A ), 2-methyl-adenine (m²A ), N6 -methyl- adenosine 35 transcription template." In some embodiments, an in vitro
(m?A ), 2-methylthio -N6 -methyl-adenosine (ms?mºA ), transcription template encodes a 5 ' untranslated (UTR )
N6 - isopentenyl-adenosine ( i?A ), 2 -methylthio - N6 -isopente region , contains an open reading frame, and encodes a 3'
nyl-adenosine (ms? i?A ), N6-(cis -hydroxyisopentenyl) ad UTR and a polyA tail. The particular nucleic acid sequence
enosine (io A ), 2 -methylthio -N6-( cis -hydroxyisopentenyl) composition and length of an in vitro transcription template
adenosine (ms? io®A ), N6 - glycinylcarbamoyl-adenosine 40 will depend on the mRNA encoded by the template .
(g?A ), N6 -threonylcarbamoyl-adenosine ( tºA ), N6 -methyl
A “ 5' untranslated region ” (5'UTR ) refers to a region of an
N6- threonylcarbamoyl-adenosine (m?t6A ), 2 -methylthio mRNA that is directly upstream ( i.e., 5') from the start codon
N6 -threonylcarbamoyl-adenosine (ms> g®A ), N6 ,N6 -dim
(i.e., the first codon of an mRNA transcript translated by a
adenosine
ethyl-adenosine (m®2A ), N6- hydroxynorvalylcarbamoyl
(hn?A ),
ribosome) that does not encode a polypeptide.
methyl- adenosine
(Am ), N6,2 - O -dimethyl- adenosine
tide .
2 -methylthio -N6- 45 A “ 3' untranslated region ” (3'UTR ) refers to a region of an
hydroxynorvalylcarbamoyl- adenosine
(ms?hn A ), mRNA that is directly downstream ( i.e., 3') from the stop
N6 -acetyl-adenosine (acºA ), 7 -methyl-adenine , 2 -methyl codon ( i.e., the codon of an mRNA transcript that signals a
thio -adenine, 2 -methoxy - adenine, a -thio -adenosine , 2-0
termination of translation ) that does not encode a polypep
(m?Am ), N6,N6,2 - O -trimethyl-adenosine (m®2Am ), 1,2'- 50 An “ open reading frame” is a continuous stretch of DNA
O - dimethyl-adenosine (m'Am ), 2 - O -ribosyladenosine beginning with a start codon (e.g., methionine ( ATG )), and
(phosphate) ( Ar(p )), 2 - amino -N6-methyl-purine, 1-thio - ad
ending with a stop codon (e.g. , TAA , TAG or TGA) and
enosine , 8 -azido -adenosine , 2 '- F -ara -adenosine , 2 '- F -ad
enosine, 2 -OH -ara -adenosine, and N6-( 19 - amino -pentaox
anonadecyl) -adenosine.
55
In some embodiments , the modified nucleobase is a
modified guanine . Exemplary nucleobases and nucleosides
having a modified guanine include inosine (I), 1 -methyl
inosine (m?I), wyosine ( img ), methylwyosine (mimG ),
4 -demethyl-wyosine (img -14 ), isowyosine ( imG2), wybu- 60
tosine (yW ), peroxywybutosine (o yW ), hydroxywybuto
sine (OhyW ), undermodified hydroxywybutosine (OhyW * ),
7 -deaza- guanosine, queuosine (Q ), epoxyqueuosine ( Q ),
galactosyl- queuosine (galQ ) ,mannosyl-queuosine (man ) ,
encodes a polypeptide.
A “ polyA tail ” is a region ofmRNA that is downstream ,
e.g. , directly downstream (i.e., 3'), from the 3' UTR that
contains multiple , consecutive adenosine monophosphates .
A polyA tail may contain 10 to 300 adenosine monophos
phates . For example, a polyA tail may contain 10 , 20 , 30 , 40 ,
50 , 60 , 70 , 80 , 90 , 100, 110 , 120 , 130 , 140 , 150, 160, 170,
180, 190 , 200 , 210, 220 , 230 , 240, 250 , 260 , 270 , 280 , 290
or 300 adenosine monophosphates. In some embodiments, a
polyA tail contains 50 to 250 adenosinemonophosphates. In
a relevant biological setting (e.g., in cells, in vivo ) the
poly ( A ) tail functions to protect mRNA from enzymatic
7 -cyano -7 -deaza- guanosine (prelo), 7-aminomethyl -7- 65 degradation , e.g. , in the cytoplasm , and aids in transcription
deaza -guanosine (preQ .), archaeosine (G *), 7 -deaza -8 -aza
termination , export of the mRNA from the nucleus and
guanosine , 6 - thio - guanosine , 6 - thio -7 -deaza - guanosine, translation .
US 10,702,600 B1
63
64
In some embodiments, a polynucleotide includes 200 to
3,000 nucleotides. For example, a polynucleotide may
include 200 to 500 , 200 to 1000 , 200 to 1500 , 200 to 3000 ,
500 to 1000 , 500 to 1500 , 500 to 2000 , 500 to 3000 , 1000
in the 88-100 region , at least 6 substitutions are permitted
between Salmonella flagellin and other flagellins that still
preserve TLR5 activation . Thus, immunogenic fragments of
flagellin include flagellin like sequences that activate TLR5
to 1500 , 1000 to 2000, 1000 to 3000 , 1500 to 3000 , or 2000 5 and contain a 13 amino acid motif that is 53 % or more
identical to the Salmonella sequence in 88-100 of Flic
Flagellin Adjuvants
; SEQ ID NO : 84 ).
Flagellin is an approximately 500 amino acid monomeric (LQRVRELAVQSAN
In
some
embodiments
, the RNA (e.g., mRNA ) vaccine
protein that polymerizes to form the flagella associated with
to 3000 nucleotides .
includes an RNA that encodes a fusion protein of flagellin
bacterial
motion
.
Flagellin
is
expressed
by
a
variety
of
10
flagellated bacteria (Salmonella typhimurium for example ) and one or more antigenic polypeptides . A “ fusion protein ”
as well as non - flagellated bacteria (such as Escherichia coli ).
Sensing of flagellin by cells of the innate immune system
(dendritic cells ,macrophages , etc.) is mediated by the Toll
like receptor 5 ( TLR5) as well as by Nod - like receptors 15
(NLRs ) Ipaf and Naip5 . TLRs and NLRs have been identi
fied as playing a role in the activation of innate immune
response and adaptive immune response. As such , flagellin
provides an adjuvant effect in a vaccine.
The nucleotide and amino acid sequences encoding 20
known flagellin polypeptides are publicly available in the
NCBI GenBank database . The flagellin sequences from S.
Typhimurium , H. Pylori, V. Cholera, S. marcesens, S.
flexneri, T. Pallidum , L. pneumophila, B. burgdorferei, C.
difficile, R. meliloti, A. tumefaciens, R. lupini, B. clar- 25
ridgeiae, P. Mirabilis, B. subtilus, L. monocytogenes, P.
aeruginosa , and E. coli, among others are known .
A flagellin polypeptide, as used herein , refers to a full
length flagellin protein , immunogenic fragments thereof,
and peptides having at least 50 % sequence identify to a 30
flagellin protein or immunogenic fragments thereof. Exem
plary flagellin proteins include flagellin from Salmonella
typhi (UniPro Entry number: Q56086 ), Salmonella typhimu
rium
(A0A0C9DG09 ), Salmonella enteritidis
( AOAOC9BAB7), and Salmonella choleraesuis ( Q6V2X8), 35
and SEQ ID NO : 54-56 ( Table 17 ). In some embodiments,
the flagellin polypeptide has at least 60 % , 70 % , 75 % , 80 % ,
90 % , 95 % , 97 % , 98 % , or 99 % sequence identify to a
flagellin protein or immunogenic fragments thereof.
In some embodiments , the flagellin polypeptide is an
immunogenic fragment. An immunogenic fragment is a
portion of a flagellin protein that provokes an immune
response . In some embodiments, the immune response is a
TLR5 immune response . An example of an immunogenic
fragment is a flagellin protein in which all or a portion of a
hinge region has been deleted or replaced with other amino
acids. For example , an antigenic polypeptide may be
inserted in the hinge region . Hinge regions are the hyper
variable regions of a flagellin . Hinge regions of a flagellin
are also referred to as “D3 domain or region , “propeller
domain or region ,” “ hypervariable domain or region ” and
“ variable domain or region .” “ At least a portion of a hinge
region ,” as used herein , refers to any partof the hinge region
of the flagellin , or the entirety of the hinge region . In other
embodiments an immunogenic fragment of flagellin is a 20 ,
25 , 30 , 35 , or 40 amino acid C -terminal fragment of flagel
40
45
50
55
lin .
The flagellin monomer is formed by domains Do through
D3. DO and Di, which form the stem , are composed of
tandem
long alpha helices and are highly conserved among 60
different bacteria . The D1 domain includes several stretches
of amino acids that are useful for TLR5 activation . The
entire D1 domain or one or more of the active regions within
the domain are immunogenic fragments of flagellin .
Examples of immunogenic regions within the D1 domain 65
include residues 88-114 and residues 411-431 ( in Salmo
nella typhimurium Flic flagellin . Within the 13 amino acids
as used herein , refers to a linking of two components of the
construct. In some embodiments, a carboxy - terminus of the
antigenic polypeptide is fused or linked to an amino termi
nus of the flagellin polypeptide. In other embodiments , an
amino -terminus of the antigenic polypeptide is fused or
linked to a carboxy -terminus of the flagellin polypeptide.
The fusion protein may include, for example, one, two,
three , four, five , six or more flagellin polypeptides linked to
one, two, three, four , five , six or more antigenic polypep
tides. When two ormore flagellin polypeptides and /or two or
more antigenic polypeptides are linked such a constructmay
be referred to as a “multimer."
Each of the components of a fusion protein may be
directly linked to one another or they may be connected
through a linker. For instance , the linker may be an amino
acid linker. The amino acid linker encoded for by the RNA
(e.g., mRNA ) vaccine to link the components of the fusion
protein may include, for instance, at least one member
selected from the group consisting of a lysine residue, a
glutamic acid residue, a serine residue and an arginine
residue . In some embodiments the linker is 1-30 , 1-25 , 1-25 ,
5-10 , 5 , 15 , or 5-20 amino acids in length .
In other embodiments the RNA (e.g., mRNA ) vaccine
includes at least two separate RNA polynucleotides, one
encoding one or more antigenic polypeptides and the other
encoding the flagellin polypeptide. The at least two RNA
polynucleotides may be co -formulated in a carrier such as a
lipid nanoparticle .
Broad Spectrum RNA (e.g. , mRNA ) Vaccines
There may be situations where persons are at risk for
infection with more than one strain of hMPV , PIV3 , RSV,
MeV and /or BetaCoV ( including MERS -CoV, SARS- CoV,
HCOV -OC43, HCOV - 229E , HCOV-NL63 , HCOV-NL ,
HCOV-NH and /or HCOV-HKU1). RNA (e.g.,mRNA ) thera
peutic vaccines are particularly amenable to combination
vaccination approaches due to a number of factors includ
ing , but not limited to , speed of manufacture , ability to
rapidly tailor vaccines to accommodate perceived geo
graphical threat, and the like. Moreover, because the vac
cines utilize the human body to produce the antigenic
protein , the vaccines are amenable to the production of
larger,more complex antigenic proteins, allowing for proper
folding, surface expression , antigen presentation , etc. in the
human subject. To protect against more than one strain of
hMPV, PIV3, RSV, MeV and /or BetaCoV (including
MERS -CoV, SARS - CoV, HCV-OC43, HCOV -229E ,
HCOV -NL63, HCOV -NL , HCOV -NH and / or HCOV -HKU1),
a combination vaccine can be administered that includes
RNA
(e.g., mRNA ) encoding at least one antigenic poly
peptide protein (or antigenic portion thereof) of a first
respiratory virus and further includes RNA encoding at least
one antigenic polypeptide protein (or antigenic portion
thereof ) of a second respiratory virus . RNA ( e.g., mRNA )
can be co -formulated , for example , in a single lipid nano
particle ( LNP) or can be formulated in separate LNPs for
co - administration .
US 10,702,600 B1
65
Methods of Treatment
Provided herein are compositions ( e.g. pharmaceutical
66
for the vaccine . A traditional vaccine , as used herein , refers
to a vaccine other than the RNA ( e.g., mRNA ) vaccines of
compositions ), methods, kits and reagents for prevention
and/or treatment of respiratory diseases/ infections in
humans and other mammals. Respiratory virus RNA ( e.g.
mRNA) vaccines can be used as therapeutic or prophylactic
agents , alone or in combination with other vaccine(s ). They
may be used in medicine to prevent and/or treat respiratory
disease/ infection . In exemplary aspects , the RNA (e.g.,
mRNA) vaccines of the present disclosure are used to
provide prophylactic protection from hMPV, PIV3, RSV,
MeV and /or BetaCoV ( including MERS- CoV, SARS- COV ,
HCV-OC43 , HCV-229E , HCOV -NL63, HCOV -NL,
HCOV -NH and /or HCOV-HKU1). Prophylactic protection
from hMPV , PIV3, RSV, MeV and /or BetaCoV (including
MERS -CoV, SARS- CoV, HCV-OC43, HCV-229E ,
HCOV -NL63 , HCOV-NL , HCOV-NH and/ or HCOV-HKU1)
can be achieved following administration of a RNA ( e.g.,
mRNA ) vaccine of the present disclosure. Respiratory virus
RNA ( e.g., mRNA ) vaccines of the present disclosure may
be used to treat or prevent viral “ co - infections” containing
two or more respiratory infections. Vaccines can be admin
istered once , twice , three times, four times or more ,but it is
likely sufficient to administer the vaccine once (optionally
the present disclosure . For instance , a traditional vaccine
includes but is not limited to live/attenuated microorganism
5 vaccines, killed / inactivated microorganism vaccines, sub
HCOV-NL63 , HCOV -NL , HCOV -NH and /or HCOV -HKU1)
MERS -CoV, SARS -CoV, HCV-OC43 , HCOV- 229E ,
unit vaccines, protein antigen vaccines, DNA vaccines, VLP
vaccines, etc. In exemplary embodiments , a traditional vac
cine is a vaccine that has achieved regulatory approval
and /or is registered by a national drug regulatory body, for
10 example the Food and Drug Administration (FDA ) in the
United States or the European Medicines Agency (EMA).
In some embodiments the anti -antigenic polypeptide anti
body titer in the subject is increased 1 log to 10 log following
relative to anti -antigenic polypeptide antibody
15 vaccination
titer in a subject vaccinated with a prophylactically effective
dose of a traditional vaccine against hMPV, PIV3, RSV,
MeV and /or BetaCoV ( including MERS- CoV, SARS- COV ,
HCOV -OC43, HCOV -229E , HCOV-NL63 , HCOV-NL ,
20 HCOV-NH and / or HCOV-HKU1).
In some embodiments the anti-antigenic polypeptide anti
body titer in the subject is increased 1 log , 2 log, 3 log, 5 log
or 10 log following vaccination relative to anti -antigenic
polypeptide antibody titer in a subject vaccinated with a
followed by a single booster). It is possible, although less 25 prophylactically effective dose of a traditional vaccine
desirable, to administer the vaccine to an infected individual against hMPV , PIV3, RSV , MeV and /or BetaCoV (including
to achieve a therapeutic response . Dosing may need to be MERS-CoV , SARS -CoV, HCV-OC43, HCOV- 229E ,
adjusted accordingly .
-NL63, HCOV -NL , HCOV -NH and /or HCOV -HKU1).
A method of eliciting an immune response in a subject HCOV
A
method
eliciting an immune response in a subject
against hMPV , PIV3, RSV , MeV and /or BetaCoV ( including 30 against hMPVof, PIV3
,RSV , MeV and /or BetaCoV ( including
MERS-CoV, SARS-CoV, HCV-OC43 , HCV-229E ,
-NL63, HCOV -NL , HCOV -NH and /or HCOV -HKU1)
is provided in aspects of the present disclosure . The method HCOV
is
provided
in other aspects of the disclosure. The method
involves administering to the subject a respiratory virus
RNA (e.g., mRNA) vaccine comprising at least one RNA 35 RNA
involves(e.g.administering
to the subject a respiratory virus
) vaccine comprising at least one RNA
(e.g. , mRNA ) polynucleotide having an open reading frame ( e.g., mRNA, mRNA
) polynucleotide having an open reading frame
encoding at least one hMPV , PIV3, RSV , MeV and /or
BetaCoV (including MERS- CoV, SARS-CoV, HCV encoding at least one hMPV , PIV3, RSV , MeV and /or
OC43 , HCOV - 229E , HCOV -NL63, HCOV -NL , HCOV -NH BetaCoV ( including MERS-CoV, SARS - CoV, HCOV
and / or HCOV-HKU1) antigenic polypeptide thereof, thereby 40 OC43 , HCOV -229E , HCOV -NL63, HCOV- NL , HCOV-NH
inducing in the subject an immune response specific to and /or HCOV-HKU1) antigenic polypeptide or an immuno
hMPV, PIV3, RSV, MeV and/or BetaCoV ( including genic fragment thereof, thereby inducing in the subject an
MERS -CoV, SARS- CoV, HCV-OC43, HCOV -229E , immune response specific to hMPV, PIV3 , RSV , MeV
HCOV-NL63, HCOV-NL , HCOV -NH and /or HCOV -HKU1) and /or BetaCoV ( including MERS - CoV, SARS - CoV,
antigenic polypeptide or an immunogenic fragment thereof, 45 HCOV -OC43 , HCOV -229E , HCOV - NL63, HCOV -NL ,
wherein anti-antigenic polypeptide antibody titer in the HCOV -NH and/or HCOV -HKU1) antigenic polypeptide or
subject is increased following vaccination relative to anti an immunogenic fragment thereof, wherein the immune
antigenic polypeptide antibody titer in a subject vaccinated response in the subject is equivalent to an immune response
with a prophylactically effective dose of a traditional vac
in a subject vaccinated with a traditional vaccine against the
cine against hMPV, PIV3, RSV, MeV and / or BetaCoV 50 HMPV , PIV3, RSV, MeV and /or BetaCoV ( including
(including MERS- CoV , SARS- CoV, HCV-OC43, HCV MERS -CoV, SARS -CoV, HCV-OC43 , HCOV- 229E ,
229E , HCOV-NL63, HCOV -NL , HCOV-NH and/or HCOV
HCOV-NL63 , HCOV -NL , HCOV -NH and /or HCOV -HKU1)
HKU1). An “ anti -antigenic polypeptide antibody ” is a serum at 2 times to 100 times the dosage level relative to the RNA
antibody the binds specifically to the antigenic polypeptide. (e.g., mRNA ) vaccine.
In some embodiments , a RNA (e.g., mRNA ) vaccine 55 In some embodiments, the immune response in the sub
(e.g. , a hMPV, PIV3, RSV , MeV and/or BetaCoV ( including ject is equivalent to an immune response in a subject
MERS-CoV, SARS- COV, HCOV -OC43 , HCOV- 229E , vaccinated with a traditional vaccine at 2 , 3 , 4, 5, 10 , 50, 100
HCOV -NL63 , HCOV-NL , HCOV-NH and/or HCOV-HKU1 times the dosage level relative to the hMPV , PIV3, RSV,
RNA vaccine ) capable of eliciting an immune response is MeV and/or BetaCoV (including MERS - CoV, SARS - CoV,
administered intramuscularly via a composition including a 60 HCOV-OC43, HCOV-229E , HCOV-NL63, HCOV-NL,
compound according to Formula (I), (IA ), (II), (Ila ), (IIb ), HCOV -NH and/or HCOV -HKU1) RNA (e.g., mRNA ) vac
(IIC ), (IId ) or (Ile ) ( e.g., Compound 3 , 18 , 20 , 25 , 26 , 29, 30 , cine.
In some embodiments the immune response in the subject is
60 , 108-112, or 122 ).
A prophylactically effective dose is a therapeutically equivalent to an immune response in a subject vaccinated
effective dose that prevents infection with the virus at a 65 with a traditional vaccine at 10-100 times , or 100-1000
clinically acceptable level. In some embodiments the thera
times, the dosage level relative to the hMPV, PIV3, RSV,
peutically effective dose is a dose listed in a package insert MeV and /or BetaCoV ( including MERS -CoV, SARS -COV,
US 10,702,600 B1
67
HCOV -OC43, HCOV -229E , HCOV-NL63, HCOV -NL ,
HCOV -NH and /or HCOV -HKU1) RNA (e.g., mRNA ) vac
cine .
In some embodiments the immune response is assessed by
Some aspects of the present disclosure provide a method
of eliciting an immune response in a subject against a In
some embodiments the immune response in the subject is
determining (protein ) antibody titer in the subject .
equivalent to an immune response in a subject vaccinated
with a traditional vaccine at 2, 3 , 4, 5 , 10, 50 , 100 times the
68
blood mononuclear cells (PBMCs) ex vivo , which are then
infused ( re - infused ) into a subject.
In some embodiments, respiratory virus vaccine contain
ing
RNA (e.g., mRNA ) polynucleotides as described herein
5 can be administered to a subject (e.g. , a mammalian subject ,
such as a human subject ), and the RNA (e.g., mRNA )
polynucleotides are translated in vivo to produce an anti
genic polypeptide .
The respiratory virus RNA (e.g., mRNA ) vaccinesmay be
10 induced
for)translation
of a orpolypeptide
, antigen
immunogen
in a cell, tissue
organism . In(e.g.
some
embodior
ments, such translation occurs in vivo , although such trans
lation may occur ex vivo , in culture or in vitro . In some
embodiments, the cell, tissue or organism is contacted with
dosage level relative to the hMPV, PIV3, RSV ,MeV and /or
BetaCoV (including MERS-CoV, SARS - CoV, HCOV
OC43, HCV-229E , HCOV -NL63, HCOV-NL , HCOV -NH
and/or HCOV
(e.g.,mRNA
vaccine
admin-) 15 an effective amount of a composition containing a respira
istering
to the-HKU1
subject) aRNA
respiratory
virus )RNA
(e.g.by, mRNA
tory virus RNA (e.g., mRNA ) vaccine that contains a
vaccine comprising at least one RNA (e.g., mRNA ) poly polynucleotide that has at least one a translatable region
nucleotide having an open reading frame encoding at least encoding an antigenic polypeptide .
one hMPV, PIV3, RSV, MeV and /or BetaCoV ( including
An “ effective amount of an respiratory virus RNA (e.g.
MERS -CoV, SARS-CoV, HCOV -OC43 , HCOV- 229E , 20 mRNA ) vaccine is provided based , at least in part, on the
HCOV-NL63 , HCOV-NL, HCOV -NH and /or HCOV-HKU1) target tissue, target cell type , means of administration ,
antigenic polypeptide, thereby inducing in the subject an physical characteristics of the polynucleotide (e.g., size, and
immune response specific to the antigenic polypeptide or an extent ofmodified nucleosides ) and other components of the
immunogenic fragment thereof, wherein the immune vaccine , and other determinants . In general, an effective
response in the subject is induced 2 days to 10 weeks earlier 25 amount of the respiratory virus RNA ( e.g., mRNA ) vaccine
relative to an immune response induced in a subject vacci composition provides an induced or boosted immune
nated with a prophylactically effective dose of a traditional response as a function of antigen production in the cell ,
vaccine against the hMPV, PIV3, RSV, MeV and /or Beta
preferably more efficient than a composition containing a
CoV (including MERS-CoV, SARS- CoV, HCV-OC43 , corresponding unmodified polynucleotide encoding the
HCOV-229E , HCOV - NL63, HCOV-NL, HCOV -NH and /or 30 same antigen or a peptide antigen . Increased antigen pro
HCOV-HKU1). In some embodiments , the immune response duction may be demonstrated by increased cell transfection
in the subject is induced in a subject vaccinated with a (the percentage of cells transfected with the RNA , e.g.,
prophylactically effective dose of a traditional vaccine at 2 mRNA , vaccine ), increased protein translation from the
times to 100 times the dosage level relative to the RNA ( e.g., polynucleotide, decreased nucleic acid degradation (as dem
mRNA ) vaccine.
35 onstrated , for example , by increased duration of protein
In some embodiments, the immune response in the sub
translation from a modified polynucleotide ), or altered anti
ject is induced 2 days earlier, or 3 days earlier, relative to an gen specific immune response of the host cell.
immune response induced in a subject vaccinated with a
In some embodiments, RNA (e.g. mRNA ) vaccines ( in
prophylactically effective dose of a traditional vaccine.
cluding polynucleotides their encoded polypeptides) in
In some embodiments the immune response in the subject 40 accordance with the present disclosure may be used for
is induced 1 week , 2 weeks, 3 weeks , 5 weeks, or 10 weeks treatment of hMPV, PIV3, RSV , MeV and /or BetaCoV
earlier relative to an immune response induced in a subject ( including MERS- CoV , SARS- CoV , HCOV-OC43 , HCOV
vaccinated with a prophylactically effective dose of a tradi 229E , HCOV -NL63 , HCOV-NL , HCOV -NH and /or HCOV
tional vaccine .
HKU1).
Also provided herein is a method of eliciting an immune 45 Respiratory RNA ( e.g. mRNA ) vaccines may be admin
response in a subject against hMPV, PIV3, RSV,MeV and/or istered prophylactically or therapeutically as part of an
BetaCoV ( including MERS-CoV, SARS -CoV, HCOV active immunization scheme to healthy individuals or early
OC43, HCOV- 229E , HCOV -NL63 , HCOV -NL , HCOV -NH
in infection during the incubation phase or during active
and /or HCOV- HKUL) by administering to the subject a infection after onset of symptoms. In some embodiments,
respiratory virus RNA (e.g.,mRNA ) vaccine having an open 50 the amount of RNA (e.g. , mRNA ) vaccine of the present
reading frame encoding a first antigenic polypeptide , disclosure provided to a cell , a tissue or a subject may be an
wherein the RNA polynucleotide does not include a stabi amount effective for immune prophylaxis.
lization element, and wherein an adjuvant is not co -formu
Respiratory virus RNA (e.g. mRNA) vaccines may be
lated or co - administered with the vaccine .
administrated with other prophylactic or therapeutic com
Therapeutic and Prophylactic Compositions
55 pounds. As a non - limiting example, a prophylactic or thera
Provided herein are compositions (e.g., pharmaceutical peutic compound may be an adjuvant or a booster. As used
compositions), methods, kits and reagents for prevention , herein , when referring to a prophylactic composition , such
treatment or diagnosis of hMPV , PIV3, RSV, MeV and /or as a vaccine , the term “booster” refers to an extra adminis
BetaCoV (including MERS -CoV , SARS-CoV, HCOV
tration of the prophylactic (vaccine ) composition . A booster
OC43 , HCV-229E , HCOV-NL63, HCOV -NL , HCOV -NH60 (or booster vaccine )may be given after an earlier adminis
and / or HCOV-HKU1) in humans and other mammals , for tration of the prophylactic composition . The time of admin
example. Respiratory virus RNA (e.g. mRNA ) vaccines can istration between the initial administration of the prophy
be used as therapeutic or prophylactic agents . They may be lactic composition and the boostermay be, but is not limited
used in medicine to prevent and/or treat infectious disease . to , 1 minute , 2 minutes, 3 minutes, 4 minutes, 5 minutes , 6
In some embodiments , the respiratory RNA (e.g. , mRNA ) 65 minutes, 7 minutes, 8 minutes , 9 minutes, 10 minutes , 15
vaccines of the present disclosure are used fin the priming of
immune effector cells , for example , to activate peripheral
minutes , 20 minutes 35 minutes, 40 minutes, 45 minutes, 50
minutes , 55 minutes, 1 hour, 2 hours, 3 hours , 4 hours , 5
US 10,702,600 B1
69
70
hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours , 11 hours,
12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours ,
18 hours , 19 hours , 20 hours, 21 hours, 22 hours, 23 hours,
1 day , 36 hours, 2 days , 3 days , 4 days, 5 days, 6 days, 1
week , 10 days, 2 weeks, 3 weeks, 1 month , 2 months, 3
months, 4 months , 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years ,
3 years, years , 5 years, 6 years, 7 years , 8 years , 9 years,
general, such preparatory methods include the step of bring
ing the active ingredient (e.g., mRNA polynucleotide ) into
association with an excipient and/or one or more other
accessory ingredients, and then , if necessary and /or desir
able, dividing , shaping and /or packaging the product into a
desired single- or multi-dose unit.
Relative amounts of the active ingredient, the pharma
5
ceutically acceptable excipient, and/or any additional ingre
in a pharmaceutical composition in accordance with
years, 17 years , 18 years , 19 years, 20 years , 25 years , 30 10 dients
the
disclosure
vary, depending upon the identity, size ,
years, 35 years, 40 years , 45 years, 50 years , 55 years, 60 and /or conditionwillof the
treated and further depending
years, 65 years , 70 years, 75 years, 80 years, 85 years, 90 upon the route by whichsubject
the
composition
to be adminis
years, 95 years or more than 99 years . In some embodi tered . By way of example, the compositionis may
comprise
ments, the time of administration between the initial admin
istration of the prophylactic composition and the booster 15 between 1-30
0.1 %% , and
100 % , e.g., between 0.5 and 50 % ,
between 5-80 % , at least 80 % (w /w ) active
may be , but is not limited to , 1 week , 2 weeks, 3 weeks, 1 between
ingredient
.
month , 2 months , 3 months , 6 months or 1 year.
Respiratory virus RNA (e.g. mRNA ) vaccines can be
In some embodiments, respiratory virus RNA (e.g.
10 years, 11 years, 12 years, 13 years, 14 years, 15 years , 16
mRNA ) vaccines may be administered intramuscularly or
formulated using one or more excipients to : ( 1 ) increase
tained or delayed release (e.g., from a depot formulation );
intradermally
, similarly to the administration of inactivated 20 stability; (2) increase cell transfection ; (3 ) permit the sus
vaccines known in the art .
Respiratory virus RNA ( e.g. mRNA ) vaccines may be (4 ) alter the biodistribution (e.g., target to specific tissues or
utilized in various settings depending on the prevalence of cell types ); (5 ) increase the translation of encoded protein in
the infection or the degree or level of unmet medical need . vivo ; and /or (6 ) alter the release profile of encoded protein
As a non -limiting example , the RNA (e.g., mRNA ) vaccines 25 (antigen ) in vivo . In addition to traditional excipients such as
may be utilized to treat and /or prevent a variety of respira any and all solvents, dispersion media , diluents, or other
tory infections. RNA (e.g., mRNA ) vaccines have superior liquid vehicles, dispersion or suspension aids, surface active
properties in that they produce much larger antibody titers agents , isotonic agents , thickening or emulsifying agents ,
and produce responses early than commercially available preservatives, excipients can include, without limitation ,
30 lipidoids, liposomes, lipid nanoparticles, polymers , lipo
anti- viral agents/ compositions.
Provided herein are pharmaceutical compositions includ plexes , core-shell nanoparticles, peptides, proteins, cells
ing respiratory virus RNA ( e.g. mRNA ) vaccines and RNA transfected with respiratory virus RNA (e.g. mRNA )vac
( e.g. mRNA ) vaccine compositions and/or complexes cines ( e.g., for transplantation into a subject ), hyaluronidase ,
optionally in combination with one or more pharmaceuti nanoparticle mimics and combinations thereof.
cally acceptable excipients.
35 Stabilizing Elements
Respiratory virus RNA (e.g. mRNA ) vaccines may be
Naturally occurring eukaryotic mRNA molecules have
formulated or administered alone or in conjunction with one been found to contain stabilizing elements, including, but
or more other components. For instance, hMPV /PIV3/RSV not limited to untranslated regions (UTR ) at their 5 '-end
RNA ( e.g., mRNA ) vaccines ( vaccine compositions ) may (5'UTR ) and /or at their 3'- end (3'UTR ), in addition to other
comprise other components including, but not limited to , 40 structural features, such as a 5'-cap structure or a 3'-poly ( A )
adjuvants.
tail. Both the 5'UTR and the 3'UTR are typically transcribed
In some embodiments , respiratory virus (e.g. mRNA ) from the genomic DNA and are elements of the premature
vaccines do not include an adjuvant ( they are adjuvant free ). mRNA . Characteristic structural features ofmature mRNA ,
Respiratory virus RNA ( e.g. mRNA ) vaccines may be such as the 5'-cap and the 3'-poly (A ) tail are usually added
formulated or administered in combination with one ormore 45 to the transcribed (premature ) mRNA during mRNA pro
pharmaceutically -acceptable excipients. In some embodi cessing. The 3'-poly ( A ) tail is typically a stretch of adenine
ments, vaccine compositions comprise at least one addi
nucleotides added to the 3'- end of the transcribed mRNA . It
tional active substances, such as, for example, a therapeu can comprise up to about 400 adenine nucleotides . In some
tically -active substance , a prophylactically -active substance, embodiments the length of the 3'-poly ( A ) tail may be an
or a combination of both . Vaccine compositions may be 50 essential element with respect to the stability of the indi
sterile , pyrogen - free or both sterile and pyrogen -free . Gen
vidual mRNA .
eral considerations in the formulation and/ ormanufacture of
In some embodiments the RNA (e.g. , mRNA ) vaccine
pharmaceutical agents , such as vaccine compositions, may may include one or more stabilizing elements . Stabilizing
be found , for example, in Remington : The Science and elements may include for instance a histone stem - loop . A
Practice of Pharmacy 21st ed., Lippincott Williams & 55 stem - loop binding protein (SLBP ), a 32 kDa protein has
Wilkins, 2005 ( incorporated herein by reference in its been identified . It is associated with the histone stem -loop at
entirety ) .
the 3 '- end of the histone messages in both the nucleus and
In some embodiments , respiratory virus RNA (e.g. the cytoplasm . Its expression level is regulated by the cell
mRNA) vaccines are administered to humans, human cycle; it peaks during the S -phase , when histone mRNA
patients or subjects. For the purposes of the present disclo- 60 levels are also elevated . The protein has been shown to be
sure, the phrase " active ingredient” generally refers to the essential for efficient 3'-end processing of histone pre
RNA (e.g., mRNA ) vaccines or the polynucleotides con mRNA by the U7 snRNP. SLBP continues to be associated
tained therein , for example, RNA polynucleotides (e.g. , with the stem - loop after processing, and then stimulates the
mRNA polynucleotides) encoding antigenic polypeptides.
translation of mature histone mRNAs into histone proteins
Formulations of the respiratory virus vaccine composi- 65 in the cytoplasm . The RNA binding domain of SLBP is
tions described herein may be prepared by any method conserved through metazoa and protozoa ; its binding to the
known or hereafter developed in the art of pharmacology . In
histone stem -loop depends on the structure of the loop . The
US 10,702,600 B1
71
minimum binding site includes at least three nucleotides 5 '
and two nucleotides 3' relative to the stem -loop .
In some embodiments , the RNA ( e.g., mRNA ) vaccines
include a coding region , at least one histone stem -loop , and
optionally, a poly (A ) sequence or polyadenylation signal.
The poly (A ) sequence or polyadenylation signal generally
should enhance the expression level of the encoded protein .
The encoded protein , in some embodiments , is not a histone
protein , a reporter protein (e.g. Luciferase, GFP, EGFP,
B -Galactosidase , EGFP), or a marker or selection protein
(e.g. alpha -Globin , Galactokinase and Xanthine :guanine
phosphoribosyl transferase (GPT)) .
In some embodiments , the combination of a poly ( A )
sequence or polyadenylation signal and at least one histone
stem -loop , even though both represent alternative mechanisms in nature, acts synergistically to increase the protein
expression beyond the level observed with either of the
individual elements. It has been found that the synergistic
effect of the combination of poly ( A ) and at least one histone
stem - loop does not depend on the order of the elements or
the length of the poly ( A ) sequence .
In some embodiments, the RNA (e.g., mRNA ) vaccine
does not comprise a histone downstream element (HDE ) .
“ Histone downstream element" (HDE ) includes a purine
rich polynucleotide stretch of approximately 15 to 20
nucleotides 3' of naturally occurring stem -loops, represent
ing the binding site for the U7 snRNA , which is involved in
processing of histone pre-mRNA into mature histone
mRNA . Ideally , the inventive nucleic acid does not include
72
(e.g.,mRNA ) vaccines are formulated in a lipid nanoparticle
that includes a non -cationic lipid such as, but not limited to ,
cholesterol or dioleoyl phosphatidylethanolamine (DOPE ).
A lipid nanoparticle formulation may be influenced by,
5 but not limited to, the selection of the cationic lipid com
ponent, the degree of cationic lipid saturation , the nature of
the PEGylation , ratio of all components and biophysical
parameters such as size . In one example by Semple et al.
(Nature Biotech. 2010 28 : 172-176 ), the lipid nanoparticle
10 formulation is composed of 57.1 % cationic lipid, 7.1 %
dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and
1.4 % PEG -C -DMA . As another example, changing the com
position of the cationic lipid can more effectively deliver
siRNA to various antigen presenting cells (Basha et al.Mol
15 Ther. 2011 19 :2186-2200 ).
In some embodiments , lipid nanoparticle formulations
may comprise 35 to 45 % cationic lipid , 40 % to 50 % cationic
lipid , 50 % to 60 % cationic lipid and /or 55 % to 65 % cationic
lipid . In some embodiments , the ratio of lipid to RNA ( e.g.,
20 mRNA ) in lipid nanoparticles may be 5:1 to 20 :1, 10 :1 to
25: 1, 15 : 1 to 30 : 1 and/or at least 30 : 1 .
In some embodiments , the ratio of PEG in the lipid
nanoparticle formulations may be increased or decreased
and /or the carbon chain length of the PEG lipid may be
25 modified from C14 to C18 to alter the pharmacokinetics
and /or biodistribution of the lipid nanoparticle formulations.
As a non -limiting example , lipid nanoparticle formulations
may contain 0.5 % to 3.0 % , 1.0 % to 3.5 % , 1.5 % to 4.0 % ,
2.0 % to 4.5 % , 2.5 % to 5.0 % and /or 3.0 % to 6.0 % of the lipid
an intron .
30 molar ratio of PEG -C -DOMG (R -3 -[(w -methoxy -poly ( eth
In some embodiments , the RNA ( e.g., mRNA ) vaccine yleneglycol) 2000 )carbamoyl) ]- 1,2 - dimyristyloxypropyl-3
may or may not contain a enhancer and/or promoter amine ) (also referred to herein as PEG -DOMG ) as compared
sequence , which may be modified or unmodified or which to the cationic lipid , DSPC and cholesterol. In some embodi
may be activated or inactivated . In some embodiments , the ments, the PEG - C -DOMG may be replaced with a PEG lipid
histone stem - loop is generally derived from histone genes , 35 such as, but not limited to , PEG -DSG ( 1,2 - Distearoyl-sn
and includes an intramolecular base pairing of two neigh
glycerol, methoxypolyethylene glycol), PEG -DMG (1,2
bored partially or entirely reverse complementary sequences Dimyristoyl-sn - glycerol) and /or PEG -DPG ( 1,2 -Dipalmi
separated by a spacer, including (e.g., consisting of) a short toyl-sn - glycerol, methoxypolyethylene glycol). The cationic
sequence, which forms the loop of the structure. The lipid may be selected from any lipid known in the art such
unpaired loop region is typically unable to base pair with 40 as, but not limited to, DLin -MC3-DMA, DLin -DMA, C12
either of the stem loop elements . It occurs more often in
200 and DLin -KC2- DMA .
RNA , as is a key component of many RNA secondary
In some embodiments, an respiratory virus RNA (e.g.
structures, but may be present in single -stranded DNA as mRNA ) vaccine formulation is a nanoparticle that comprises
well. Stability of the stem - loop structure generally depends at least one lipid . The lipid may be selected from , but is not
on the length , number of mismatches or bulges , and base 45 limited to , DLin -DMA, DLin - K -DMA, 98N12-5 , C12-200,
composition of the paired region. In some embodiments , DLin -MC3 -DMA , DLin -KC2-DMA , DODMA , PLGA ,
wobble base pairing (non -Watson - Crick base pairing ) may PEG , PEG -DMG , PEGylated lipids and amino alcohol lip
result. In some embodiments , the at least one histone stem
ids. In some embodiments , the lipid may be a cationic lipid
loop sequence comprises a length of 15 to 45 nucleotides. such as, but not limited to , DLin -DMA , DLin - D -DMA,
In other embodiments the RNA (e.g., mRNA ) vaccine 50 DLin -MC3-DMA, DLin -KC2-DMA, DODMA and amino
may have one or more AU - rich sequences removed . These alcohol lipids.
sequences , sometimes referred to as AURES are destabiliz
The amino alcohol cationic lipid may be the lipids
ing sequences found in the 3'UTR . The AURES may be described in and/or made by the methods described in U.S.
removed from the RNA (e.g., mRNA ) vaccines . Alterna
Patent Publication No. US20130150625 , herein incorpo
tively the AURES may remain in the RNA (e.g., mRNA ) 55 rated by reference in its entirety. As a non - limiting example ,
vaccine .
the cationic lipid may be 2 -amino -3 -[(9Z ,12Z )-octadeca- 9,
Nanoparticle Formulations
12-dien - 1-yloxy ]-2 -{[(92,2Z )-octadeca -9,12 -dien - 1-yloxy ]
In some embodiments , respiratory virus RNA (e.g. methyl} propan -1 -ol (Compound 1 in US20130150625 );
mRNA ) vaccines are formulated in a nanoparticle. In some 2 -amino -3 - [(9Z )-octadec -9 - en - 1 -yloxyl-2 - {[ (9Z )-octadec
embodiments , respiratory virus RNA ( e.g. mRNA ) vaccines 60 9 - en -1 -yloxy ]methyl}propan -1 -ol (Compound 2 in
are formulated in a lipid nanoparticle . In some embodi US20130150625 ); 2 -amino - 3 -[ (97,12Z )-octadeca - 9,12
ments , respiratory virus RNA (e.g. mRNA ) vaccines are dien - 1-yloxy ]-2 -[(octyloxy)methyl ]propan -1 -ol (Compound
formulated in a lipid -polycation complex , referred to as a 3 in US20130150625); and 2-(dimethylamino )-3 -[(97,12Z )
cationic lipid nanoparticle. As a non - limiting example, the octadeca - 9,12 -dien - 1-yloxy ]-2 - {[( 92 , 12Z)-octadeca -9,12
polycation may include a cationic peptide or a polypeptide 65 dien - 1-yloxy ]methyl} propan -1 -ol (Compound 4 in
such as , but not limited to , polylysine , polyornithine and /or US20130150625 ); or any pharmaceutically acceptable salt
polyarginine . In some embodiments, respiratory virus RNA or stereoisomer thereof.
US 10,702,600 B1
74
73
Lipid nanoparticle formulations typically comprise a
In some embodiments, lipid nanoparticle formulations
include 45-65 % of a cationic lipid selected from 2,2 -dilino
2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ] -dioxolane
leyl-4 -dimethylaminoethyl- [ 1,3 ]-dioxolane (DLin -KC2
(DLin -KC2-DMA), dilinoleyl -methyl-4 - dimethylaminobu DMA), dilinoleyl-methyl-4 -dimethylaminobutyrate ( DLin
tyrate (DLin -MC3- DMA ), or di( (Z ) -non - 2 -en - 1 -yl) 9- (4- 5 MC3-DMA ), and di(( Z ) -non -2 -en -1 - yl) 9- ( 4
(dimethylamino )butanoyl)oxy )heptadecanedioate ( L319 ), (dimethylamino )butanoyl)oxylheptadecanedioate (L319 ),
and further comprise a neutral lipid , a sterol and a molecule 5-10 % of the neutral lipid , 25-40 % of the sterol, and
capable of reducing particle aggregation , for example a PEG 0.5-10 % of the PEG or PEG -modified lipid on a molar basis .
or PEG -modified lipid .
In some embodiments, lipid nanoparticle formulations
lipid , in particular, an ionizable cationic lipid , for example ,
In some embodiments , a lipid nanoparticle formulation 10 include 60 % of a cationic lipid selected from 2,2 - dilinoleyl
consists essentially of (i) at least one lipid selected from the 4 -dimethylaminoethyl-[ 1,3 ] -dioxolane (DLin -KC2-DMA),
group consisting of 2,2 -dilinoleyl- 4 -dimethylaminoethyl- [ 1, dilinoleyl-methyl-4 - dimethylaminobutyrate (DLin -MC3
3 ]-dioxolane (DLin -KC2-DMA), dilinoleyl-methyl-4 -dim
DMA), and di((Z ) -non - 2-en - 1-yl) 9- (4-(dimethylamino )bu
ethylaminobutyrate (DLin -MC3-DMA ), and di ((Z )-non -2
tanoyl) oxy) heptadecanedioate (L319 ), 7.5 % of the neutral
9-((4-(dimethylamino )butanoyl )oxy ) 15 lipid , 31 % of the sterol, and 1.5 % of the PEG or PEG
heptadecanedioate (L319); (ii) a neutral lipid selected from modified lipid on a molar basis.
DSPC , DPPC , POPC , DOPE and SM ; (iii ) a sterol, e.g.,
In some embodiments, lipid nanoparticle formulations
en - 1 -yl)
cholesterol; and (iv ) a PEG - lipid , e.g., PEG -DMG or PEG
include 50 % of a cationic lipid selected from 2,2 -dilinoleyl
CDMA , in a molar ratio of 20-60 % cationic lipid : 5-25 %
4 -dimethylaminoethyl- [ 1,3 ]-dioxolane (DLin -KC2-DMA ),
neutral lipid : 25-55 % sterol; 0.5-15 % PEG -lipid .
20 dilinoleyl-methyl-4 -dimethylaminobutyrate (DLin -MC3
In some embodiments, a lipid nanoparticle formulation DMA), and di((Z )-non -2 - en - 1 - yl) 9- (4- (dimethylamino )bu
includes 25 % to 75 % on a molar basis of a cationic lipid tanoyl) oxy )heptadecanedioate (L319 ), 10 % of the neutral
selected from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ]-di lipid , 38.5 % of the sterol, and 1.5 % of the PEG or PEG
oxolane (DLin -KC2- DMA), dilinoleyl -methyl-4 -dimethyl modified lipid on a molar basis.
aminobutyrate (DLin -MC3-DMA), and di( ( Z )-non - 2 -en - 1- 25 In some embodiments, lipid nanoparticle formulations
yl) 9- (4-(dimethylamino )butanoyl)oxy)heptadecanedioate include 50 % of a cationic lipid selected from 2,2 -dilinoleyl
(L319 ), e.g. , 35 to 65 % , 45 to 65 % , 60 % , 57.5 % , 50 % or 4 -dimethylaminoethyl- [ 1,3 ]-dioxolane (DLin -KC2-DMA ),
dilinoleyi-methyl-4 -dimethylaminobutyrate (DLin -MC3
40 % on a molar basis .
In some embodiments, a lipid nanoparticle formulation DMA ), and di((Z ) -non - 2 -en - 1 -yl) 9 -([4-(dimethylamino )bu
includes 0.5 % to 15 % on a molar basis of the neutral lipid , 30 tanoyl)oxy )heptadecanedioate (L319), 10 % of the neutral
e.g. , 3 to 12 % , 5 to 10 % or 15 % , 10 % , or 7.5 % on a molar
lipid , 35 % of the sterol, 4.5 % or 5 % of the PEG or
basis. Examples of neutral lipids include, without limitation ,
PEG -modified lipid, and 0.5 % of the targeting lipid on a
DSPC , POPC , DPPC , DOPE and SM . In some embodi
molar basis .
ments, the formulation includes 5 % to 50 % on a molar basis
In some embodiments, lipid nanoparticle formulations
of the sterol (e.g., 15 to 45 % , 20 to 40 % , 40 % , 38.5 % , 35 % , 35 include 40 % of a cationic lipid selected from 2,2 - dilinoleyl
or 31 % on a molar basis . A non -limiting example of a sterol 4 -dimethylaminoethyl- [1,3 ]-dioxolane (DLin -KC2-DMA),
is cholesterol. In some embodiments , a lipid nanoparticle dilinoleyl-methyl-4 -dimethylaminobutyrate (DLin -MC3
formulation includes 0.5 % to 20 % on a molar basis of the DMA), and di((Z ) -non -2 -en - 1-yl) 9 -([4-(dimethylamino )bu
PEG or PEG -modified lipid (e.g., 0.5 to 10 % , 0.5 to 5 % , tanoyl) oxy )heptadecanedioate (L319 ), 15 % of the neutral
1.5 % , 0.5 % , 1.5 % , 3.5 % , or 5 % on a molar basis . In some 40 lipid , 40 % of the sterol, and 5 % of the PEG or PEG -modified
embodiments , a PEG or PEG modified lipid comprises a lipid on a molar basis .
PEG molecule of an average molecular weight of 2,000 Da.
In some embodiments, lipid nanoparticle formulations
In some embodiments , a PEG or PEG modified lipid com
include 57.2 % of a cationic lipid selected from 2,2 -dilino
prises a PEG molecule of an average molecular weight of leyl-4 -dimethylaminoethyl-[ 1,3 ]-dioxolane (DLin -KC2
less than 2,000 , for example around 1,500 Da, around 1,000 45 DMA ), dilinoleyl-methyl -4- dimethylaminobutyrate (DLin
Da, or around 500 Da. Non -limiting examples of PEG
MC3-DMA ), and di( (Z ) -non - 2 - en - 1 -yl) 9 -((4
modified lipids include PEG -distearoyl glycerol (PEG
(dimethylamino)butanoyl)oxy )heptadecanedioate (L319 ),
DMG ) (also referred herein as PEG -C14 or C14 -PEG ),
PEG -CDMA ( further discussed in Reyes et al. J. Controlled
Release , 107 , 276-287 (2005 ) the contents of which are
herein incorporated by reference in their entirety ).
In some embodiments, lipid nanoparticle formulations
include 25-75 % of a cationic lipid selected from 2,2 -dilino
leyl-4 - dimethylaminoethyl- [ 1,3 ]-dioxolane (DLin -KC2
DMA), dilinoleyl-methyl-4 -dimethylaminobutyrate (DLinMC3-DMA), and di((Z ) -non - 2 -en - 1- yl) 9- (4
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319 ),
0.5-15 % of the neutral lipid , 5-50 % of the sterol, and
0.5-20 % of the PEG or PEG -modified lipid on a molar basis.
In some embodiments , lipid nanoparticle formulations
include 35-65 % of a cationic lipid selected from 2,2 -dilino
leyl-4 -dimethylaminoethyl- [1,3 ]-dioxolane (DLin -KC2
DMA ), dilinoleyl-methyl- 4 -dimethylaminobutyrate (DLin
MC3 -DMA ), and
di(( Z ) -non - 2 - en - 1 -yl)
9- ( 4
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
3-12 % of the neutral lipid , 15-45 % of the sterol, and
0.5-10 % of the PEG or PEG -modified lipid on a molar basis.
7.1 % of the neutral lipid , 34.3 % of the sterol, and 1.4 % of
the PEG or PEG -modified lipid on a molar basis .
50 In some embodiments, lipid nanoparticle formulations
include 57.5 % of a cationic lipid selected from the PEG lipid
is PEG -CDMA (PEG - CDMA is further discussed in Reyes et
al. (J. Controlled Release , 107 , 276-287 ( 2005), the contents
of which are herein incorporated by reference in their
55 entirety), 7.5 % of the neutral lipid , 31.5 % of the sterol, and
3.5 % of the PEG or PEG -modified lipid on a molar basis .
In some embodiments , lipid nanoparticle formulations
consists essentially of a lipid mixture in molar ratios of
20-70 % cationic lipid : 5-45 % neutral lipid : 20-55 % choles
60 terol: 0.5-15 % PEG -modified lipid . In some embodiments ,
lipid nanoparticle formulations consists essentially of a lipid
mixture in a molar ratio of 20-60 % cationic lipid : 5-25 %
neutral lipid : 25-55 % cholesterol: 0.5-15 % PEG -modified
65
lipid .
In some embodiments, the molar lipid ratio is 50 / 10 /38.5 /
1.5 (mol % cationic lipid /neutral lipid , e.g., DSPC /Chol/
PEG -modified lipid , e.g. , PEG -DMG , PEG -DSG or PEG
US 10,702,600 B1
75
76
DPG ), 57.2 /7.1134.3 / 1.4 (mol % cationic lipid /neutral lipid , example, the lipid nanoparticle comprise 55 % of the cationic
e.g., DPPC /Cho1/PEG -modified lipid , e.g., PEG -CDMA ), lipid L319 , 10 % of the non -cationic lipid DSPC , 2.5 % of the
40/15/40/5 (mol % cationic lipid/neutral lipid , e.g., DSPC/ PEG lipid PEG -DMG and 32.5 % of the structural lipid
Chol/PEG -modified lipid , e.g., PEG -DMG ), 50 /10 /35 /4.5 / cholesterol.
0.5 (mol % cationic lipid /neutral lipid , e.g., DSPC /Chol/ 5 Relative amounts of the active ingredient, the pharma
PEG -modified lipid , e.g., PEG -DSG ), 50/10/35/5 (cationic ceutically acceptable excipient, and /or any additional ingre
lipid /neutral lipid , e.g., DSPC /Cho1/PEG -modified lipid , dients in a vaccine composition may vary, depending upon
e.g., PEG -DMG ), 40/10/40/10 (mol % cationic lipid /neutral the identity , size, and /or condition of the subject being
lipid , e.g., DSPC /Chol/PEG -modified lipid , e.g. , PEG -DMG treated and further depending upon the route by which the
or PEG -CDMA), 35/15/40/10 (mol % cationic lipid /neutral 10 composition is to be administered . For example , the com
lipid , e.g., DSPC /Chol/PEG -modified lipid, e.g., PEG - DMG position may comprise between 0.1 % and 99 % (w /w ) of the
or PEG - CDMA ) or 52/13/30/5 (mol % cationic lipid /neutral active ingredient. By way of example , the composition may
lipid , e.g., DSPC /Chol/PEG -modified lipid , e.g. , PEG -DMG comprise between 0.1 % and 100 % , e.g., between 0.5 and
or PEG - CDMA ).
50 % , between 1-30 % , between 5-80 % , at least 80 % (w /w )
Non -limiting examples of lipid nanoparticle compositions 15 active ingredient.
and methods of making them are described , for example, in
In some embodiments , the respiratory virus RNA (e.g.
Semple et al. (2010 ) Nat. Biotechnol. 28 : 172-176 ; Jayarama mRNA ) vaccine composition may comprise the polynucle
et al. (2012), Angew . Chem . Int. Ed., 51 : 8529-8533 ; and otide described herein , formulated in a lipid nanoparticle
Maier et al . (2013 ) Molecular Therapy 21, 1570-1578 (the comprising MC3, Cholesterol, DSPC and PEG2000 -DMG ,
contents of each of which are incorporated herein by refer- 20 the buffer trisodium citrate, sucrose and water for injection .
As a non -limiting example, the composition comprises : 2.0
ence in their entirety ).
In some embodiments , lipid nanoparticle formulations mg/mL of drug substance (e.g., polynucleotides encoding
may comprise a cationic lipid , a PEG lipid and a structural H1ONS HMPV ), 21.8 mg/mL of MC3, 10.1 mg/mL of
lipid and optionally comprise a non -cationic lipid . As a cholesterol, 5.4 mg/mL of DSPC , 2.7 mg/mL of PEG2000
non - limiting example , a lipid nanoparticle may comprise 25 DMG , 5.16 mg/mL of trisodium citrate , 71 mg/mL of
40-60 % of cationic lipid , 5-15 % of a non -cationic lipid , sucrose and 1.0 mL of water for injection .
1-2 % of a PEG lipid and 30-50 % of a structural lipid . As
In some embodiments, a nanoparticle ( e.g., a lipid nano
another non-limiting example , the lipid nanoparticle may particle ) has a mean diameter of 10-500 nm , 20-400 nm ,
comprise 50 % cationic lipid, 10 % non -cationic lipid, 1.5 %
30-300 nm , 40-200 nm . In some embodiments, a nanopar
PEG lipid and 38.5 % structural lipid . As yet another non- 30 ticle (e.g., a lipid nanoparticle) has a mean diameter of
limiting example, a lipid nanoparticle may comprise 55 %
50-150 nm , 50-200 nm , 80-100 nm or 80-200 nm .
cationic lipid , 10 % non -cationic lipid , 2.5 % PEG lipid and Liposomes, Lipoplexes , and Lipid Nanoparticles
32.5 % structural lipid . In some embodiments, the cationic
The RNA (e.g., mRNA ) vaccines of the disclosure can be
lipid may be any cationic lipid described herein such as , but formulated using one ormore liposomes, lipoplexes, or lipid
not limited to , DLin -KC2 -DMA, DLin -MC3-DMA and 35 nanoparticles . In some embodiments , pharmaceutical com
L319 .
positions ofRNA (e.g. ,mRNA ) vaccines include liposomes .
In some embodiments , the lipid nanoparticle formulations Liposomes are artificially -prepared vesicles which may pri
described herein may be 4 component lipid nanoparticles . marily be composed of a lipid bilayer and may be used as a
The lipid nanoparticle may comprise a cationic lipid , a delivery vehicle for the administration of nutrients and
non -cationic lipid , a PEG lipid and a structural lipid . As a 40 pharmaceutical formulations. Liposomes can be of different
non - limiting example, the lipid nanoparticle may comprise sizes such as, but not limited to , a multilamellar vesicle
40-60 % of cationic lipid , 5-15 % of a non -cationic lipid , (MLV ) which may be hundreds of nanometers in diameter
1-2 % of a PEG lipid and 30-50 % of a structural lipid . As and may contain a series of concentric bilayers separated by
another non -limiting example, the lipid nanoparticle may narrow aqueous compartments, a small unicellular vesicle
comprise 50 % cationic lipid , 10 % non - cationic lipid , 1.5 % 45 (SUV ) which may be smaller than 50 nm in diameter , and a
PEG lipid and 38.5 % structural lipid. As yet another non
large unilamellar vesicle (LUV ) which may be between 50
limiting example , the lipid nanoparticle may comprise 55 %
and 500 nm in diameter. Liposome design may include, but
cationic lipid , 10 % non -cationic lipid , 2.5 % PEG lipid and is not limited to , opsonins or ligands in order to improve the
32.5 % structural lipid . In some embodiments , the cationic attachment of liposomes to unhealthy tissue or to activate
lipid may be any cationic lipid described herein such as, but 50 events such as, but not limited to , endocytosis. Liposomes
may contain a low or a high pH in order to improve the
L319 .
delivery of the pharmaceutical formulations.
In some embodiments , the lipid nanoparticle formulations
The formation of liposomes may depend on the physico
described herein may comprise a cationic lipid, a non
chemical characteristics such as, but not limited to , the
cationic lipid , a PEG lipid and a structural lipid . As a 55 pharmaceutical formulation entrapped and the liposomal
non -limiting example , the lipid nanoparticle comprise 50 % ingredients, the nature of the medium in which the lipid
of the cationic lipid DLin -KC2-DMA, 10 % of the non vesicles are dispersed , the effective concentration of the
cationic lipid DSPC , 1.5 % of the PEG lipid PEG -DOMG
entrapped substance and its potential toxicity , any additional
and 38.5 % of the structural lipid cholesterol. As a non
processes involved during the application and /or delivery of
limiting example , the lipid nanoparticle comprise 50 % of the 60 the vesicles, the optimization size , polydispersity and the
cationic lipid DLin -MC3-DMA, 10 % of the non -cationic shelf-life of the vesicles for the intended application , and the
lipid DSPC , 1.5 % of the PEG lipid PEG -DOMG and 38.5 % batch - to -batch reproducibility and possibility of large -scale
of the structural lipid cholesterol. As a non - limiting production of safe and efficient liposomal products .
example, the lipid nanoparticle comprise 50 % of the cationic
In some embodiments , pharmaceutical compositions
lipid DLin -MC3- DMA, 10 % of the non -cationic lipid 65 described herein may include,without limitation , liposomes
DSPC , 1.5 % of the PEG lipid PEG -DMG and 38.5 % of the such as those formed from 1,2 - dioleyloxy - N , N -dimethylam
structural lipid cholesterol. As yet another non -limiting inopropane (DODMA) liposomes, DiLa2 liposomes from
not limited to , DLin -KC2- DMA, DLin -MC3 -DMA and
US 10,702,600 B1
78
Marina Biotech (Bothell, Wash .), 1,2 -dilinoleyloxy -3 - dim
In some embodiments , the RNA ( e.g., mRNA ) vaccines
ethylaminopropane (DLin -DMA), 2,2 -dilinoleyl-4-( 2-dim
may be formulated in a lipid -polycation complex . The
ethylaminoethyl)-[ 1,3 ]-dioxolane (DLin -KC2-DMA ), and formation of the lipid -polycation complex may be accom
MC3 (US20100324120 ;herein incorporated by reference in plished by methods known in the art and/or as described in
its entirety ) and liposomes which may deliver small mol- 5 U.S. Pub . No. 20120178702, herein incorporated by refer
ecule drugs such as, but not limited to , DOXIL® from
ence in its entirety. As a non - limiting example, the polyca
tion may include a cationic peptide or a polypeptide such as,
Janssen Biotech , Inc. (Horsham , Pa .).
but
not limited to, polylysine , polyornithine and /or polyargi
In some embodiments, pharmaceutical compositions
77
nine. In some embodiments, the RNA (e.g., mRNA ) vac
described herein may include, without limitation , liposomes cines may be formulated in a lipid -polycation complex ,
such as those formed from the synthesis of stabilized plas 10 which
may further include a non -cationic lipid such as, but
mid -lipid particles (SPLP ) or stabilized nucleic acid lipid not limited
to , cholesterol or dioleoyl phosphatidyletha
particle (SNALP ) that have been previously described and nolamine (DOPE
).
shown to be suitable for oligonucleotide delivery in vitro
In some embodiments , the ratio of PEG in the lipid
and in vivo (see Wheeler et al. Gene Therapy. 1999 6 :271 15 nanoparticle (LNP) formulations may be increased or
281 ; Zhang et al. Gene Therapy. 1999 6 : 1438-1447; Jeffs et decreased and/or the carbon chain length of the PEG lipid
al. Pharm Res. 2005 22:362-372 ; Morrissey et al., Nat may be modified from C14 to C18 to alter the pharmacoki
Biotechnol. 2005 2 : 1002-1007 ; Zimmermann et al., Nature . netics and /or biodistribution of the LNP formulations . As a
2006 441: 111-114 ; Heyes et al . J Contr Rel. 2005 107 :276
non - limiting example, LNP formulations may contain from
287; Semple et al. Nature Biotech . 2010 28: 172-176 ; Judge 20 about 0.5 % to about 3.0 % , from about 1.0 % to about 3.5 % ,
et al. J Clin Invest. 2009 119 :661-673; deFougerolles Hum
Gene Ther. 2008 19 : 125-132 ; U.S. Patent Publication No
US20130122104 ; all of which are incorporated herein in
their entireties ). The original manufacture method by
from about 1.5 % to about 4.0 % , from about 2.0 % to about
4.5 % , from about 2.5 % to about 5.0 % and /or from about
3.0 % to about 6.0 % of the lipid molar ratio of PEG -C
DOMG (R -3 -[(0 -methoxy -poly (ethyleneglycol)2000 ) car
Wheeler et al. was a detergent dialysis method , which was 25 bamoyl) ]- 1,2 -dimyristyloxypropyl- 3 -amine ) (also referred
later improved by Jeffs et al. and is referred to as the to herein as PEG -DOMG ) as compared to the cationic lipid ,
spontaneous vesicle formation method. The liposome for DSPC and cholesterol. In some embodiments, the PEG - C
mulations are composed of 3 to 4 lipid components in DOMG may be replaced with a PEG lipid such as , but not
addition to the polynucleotide . As an example a liposome limited to , PEG -DSG ( 1,2 -Distearoyl-sn - glycerol, methoxy
can contain , but is not limited to , 55 % cholesterol, 20 % 30 polyethylene glycol), PEG - DMG ( 1,2 -Dimyristoyl-sn -glyc
disteroylphosphatidyl choline (DSPC ), 10 % PEG -S -DSG , erol) and/ or PEG -DPG ( 1,2 -Dipalmitoyl-sn - glycerol,
and 15 % 1,2 -dioleyloxy -N ,N -dimethylaminopropane methoxypolyethylene glycol). The cationic lipid may be
(DODMA), as described by Jeffs et al. As another example ,
certain liposome formulations may contain , but are not
limited to , 48 % cholesterol, 20 % DSPC , 2 % PEG -C -DMA, 35
and 30 % cationic lipid , where the cationic lipid can be
1,2 -distearloxy -N ,N -dimethylaminopropane (DSDMA ),
DODMA , DLin -DMA, or 1,2 - dilinolenyloxy - 3 -dimethyl
selected from any lipid known in the art such as , but not
limited to , DLin -MC3 -DMA , DLin -DMA , C12-200 and
DLin -KC2-DMA .
In some embodiments , the RNA (e.g., mRNA ) vaccines
may be formulated in a lipid nanoparticle .
In some embodiments , the RNA (e.g., mRNA ) vaccine
aminopropane (DLenDMA ), as described by Heyes et al .
formulation comprising the polynucleotide is a nanoparticle
In some embodiments , liposome formulations may com- 40 which may comprise at least one lipid . The lipid may be
prise from about 25.0 % cholesterol to about 40.0 % choles selected from , but is not limited to , DLin -DMA , DLin - K
terol, from about 30.0 % cholesterol to about 45.0 % choles DMA, 98N12-5 , C12-200 , DLin -MC3-DMA, DLin -KC2
terol, from about 35.0 % cholesterol to about 50.0 %
DMA, DODMA, PLGA, PEG , PEG -DMG , PEGylated lip
cholesterol and /or from about 48.5 % cholesterol to about ids and amino alcohol lipids. In another aspect, the lipid may
60 % cholesterol. In some embodiments , formulations may 45 be a cationic lipid such as, but not limited to , DLin -DMA,
comprise a percentage of cholesterol selected from the group DLin - D -DMA, DLin -MC3 -DMA, DLin -KC2-DMA,
consisting of 28.5 % , 31.5 % , 33.5 % , 36.5 % , 37.0 % , 38.5 % , DODMA and amino alcohol lipids. The amino alcohol
39.0 % and 43.5 % . In some embodiments, formulations may cationic lipid may be the lipids described in and/or made by
comprise from about 5.0 % to about 10.0 % DSPC and/or the methods described
U.S. Patent Publication No.
from about 7.0 % to about 15.0 % DSPC .
50 US20130150625, herein incorporated by reference in its
In some embodiments , the RNA (e.g. , mRNA ) vaccine entirety . As a non - limiting example , the cationic lipid may
pharmaceutical compositions may be formulated in lipo be 2-amino - 3-[(9Z , 12Z ) -octadeca -9,12- dien - 1-yloxyl-2- {
somes such as, but not limited to , DiLa2 liposomes (Marina [( 9Z , 2Z )-octadeca - 9,12 -dien -1 -yloxy ]methyl} propan - 1 -ol
Biotech , Bothell, Wash .), SMARTICLES® (Marina Bio
(Compound 1 in US20130150625 ) ; 2 - amino- 3 -[( 9Z )-octa
tech
,
Bothell
,
Wash
.
)
,
neutral
DOPC
(
1,2
dioleoyl
sn55
dec
-9 -en -1-yloxy )-2 -{[(92 ) -octadec-9 -en - 1-yloxy]
glycero -3 -phosphocholine ) based liposomes ( e.g., siRNA methyl }propan -1 -ol (Compound 2 in US20130150625);
delivery for ovarian cancer (Landen et al. Cancer Biology & 2 -amino -3-[( 9Z , 12Z )-octadeca - 9,12 -dien -1 -yloxy ]-2 -[(oc
Therapy 2006 5 (12 ) 1708-1713 ); herein incorporated by ref tyloxy )methyl]propan - 1 -ol
(Compound
3
in
erence in its entirety ) and hyaluronan -coated liposomes US20130150625 ); and 2-(dimethylamino )-3 -[( 92 , 12Z )-oc
(Quiet Therapeutics , Israel).
60 tadeca-9,12- dien - 1 - yloxyl- 2 - {{ ( 9Z, 12Z )-octadeca - 9,12
In some embodiments, the cationic lipid may be a low dien - 1 -yloxy]methyl} propan - 1 -ol (Compound 4 in
molecular weight cationic lipid such as those described in US20130150625 ); or any pharmaceutically acceptable salt
U.S. Patent Application No. 20130090372 , the contents of or stereoisomer thereof.
which are herein incorporated by reference in their entirety.
Lipid nanoparticle formulations typically comprise a
In some embodiments , the RNA (e.g., mRNA ) vaccines 65 lipid , in particular , an ionizable cationic lipid , for example,
may be formulated in a lipid vesicle, which may have 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ]-dioxolane
(DLin -KC2-DMA ), dilinoleyl-methyl-4 -dimethylaminobu
crosslinks between functionalized lipid bilayers.
US 10,702,600 B1
79
80
tyrate (DLin -MC3- DMA ), or di( (Z ) -non - 2 -en - 1 -yl) 9- (4
In some embodiments, the formulations of the present
( dimethylamino )butanoyl)oxy )heptadecanedioate (L319), disclosure include 45-65 % of a cationic lipid selected from
and further comprise a neutral lipid , a sterol and a molecule 2,2 -dilinoleyl-4 -dimethylaminoethyl-[ 1,3 ] -dioxolane
capable of reducing particle aggregation , for example a PEG (DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu
or PEG -modified lipid .
5 tyrate (DLin -MC3-DMA), and di((Z ) -non -2 - en -1 -yl) 9- (4
In some embodiments , the lipid nanoparticle formulation (dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
consists essentially of (i) at least one lipid selected from the 5-10 % of the neutral lipid , 25-40 % of the sterol, and
group consisting of 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1, 0.5-10 % of the PEG or PEG -modified lipid on a molar basis.
31-dioxolane (DLin -KC2 -DMA), dilinoleyl-methyl-4 - dim
In some embodiments, the formulations of the present
ethylaminobutyrate (DLin -MC3-DMA ), and di((Z ) -non - 2- 10 disclosure include about 60 % of a cationic lipid selected
en - 1 -yl)
9 -((4-(dimethylamino )butanoyl) oxy ) from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ]-dioxolane
heptadecanedioate (L319); ( ii ) a neutral lipid selected from
(DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu
DSPC , DPPC , POPC , DOPE and SM ; ( iii) a sterol, e.g. , tyrate (DLin -MC3-DMA) , and di((Z )-non - 2 - en - 1- yl) 9-( 4
cholesterol; and (iv ) a PEG - lipid , e.g., PEG -DMG or PEG
(dimethylamino)butanoyl)oxy )heptadecanedioate (L319) ,
CDMA , in a molar ratio of about 20-60 % cationic lipid : 15 about 7.5 % of the neutral lipid , about 31 % of the sterol, and
5-25 % neutral lipid : 25-55 % sterol; 0.5-15 % PEG -lipid .
about 1.5 % of the PEG or PEG -modified lipid on a molar
In some embodiments , the formulation includes from
basis .
about 25 % to about 75 % on a molar basis of a cationic lipid
In some embodiments, the formulations of the present
selected from 2,2- dilinoleyl-4 - dimethylaminoethyl-[ 1,3 ]-di disclosure include about 50 % of a cationic lipid selected
oxolane (DLin -KC2 -DMA), dilinoleyl -methyl-4 -dimethyl- 20 from 2,2 - dilinoleyl-4 -dimethylaminoethyl-[1,3 ]-dioxolane
aminobutyrate (DLin -MC3 -DMA ), and di((Z )-non - 2- en -1
(DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu
yl) 9 -([4-(dimethylamino )butanoyl)oxy )heptadecanedioate
(L319), e.g., from about 35 to about 65 % , from about 45 to
tyrate (DLin -MC3-DMA ), and di( (Z )-non -2 - en - 1 -yl) 9- (4
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
about 65 % , about 60 % , about 57.5 % , about 50 % or about about 10 % of the neutral lipid , about 38.5 % of the sterol, and
40 % on a molar basis .
25 about 1.5 % of the PEG or PEG -modified lipid on a molar
In some embodiments, the formulation includes from
basis .
about 0.5 % to about 15 % on a molar basis of the neutral lipid
In some embodiments, the formulations of the present
e.g., from about 3 to about 12 % , from about 5 to about 10 %
disclosure include about 50 % of a cationic lipid selected
or about 15 % , about 10 % , or about 7.5 % on a molar basis . from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,31-dioxolane
Examples of neutral lipids include , but are not limited to , 30 (DLin -KC2 -DMA), dilinoleyl-methyl- 4 -dimethylaminobu
DSPC , POPC , DPPC , DOPE and SM . In some embodi tyrate (DLin -MC3-DMA), and di((Z )-non -2 -en - 1 -yl) 9 -((4
ments , the formulation includes from about 5 % to about
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
50 % on a molar basis of the sterol ( e.g., about 15 to about
45 % , about 20 to about 40 % , about 40 % , about 38.5 % , about
35 % , or about 31 % on a molar basis . An exemplary sterol is 35
cholesterol. In some embodiments , the formulation includes
from about 0.5 % to about 20 % on a molar basis of the PEG
or PEG -modified lipid ( e.g., about 0.5 to about 10 % , about
0.5 to about 5 % , about 1.5 % , about 0.5 % , about 1.5 % , about
3.5 % , or about 5 % on a molar basis . In some embodiments, 40
the PEG or PEG modified lipid comprises a PEG molecule
of an average molecular weight of 2,000 Da. In other
embodiments, the PEG or PEG modified lipid comprises a
PEG molecule of an average molecular weight of less than
2,000 , for example around 1,500 Da, around 1,000 Da, or 45
around 500 Da. Examples of PEG -modified lipids include ,
but are not limited to , PEG - distearoyl glycerol (PEG -DMG )
about 10 % of the neutral lipid , about 35 % of the sterol, about
4.5 % or about 5 % of the PEG or PEG -modified lipid , and
about 0.5 % of the targeting lipid on a molar basis .
In some embodiments, the formulations of the present
disclosure include about 40 % of a cationic lipid selected
from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [1,3 ]-dioxolane
(DLin -KC2-DMA ), dilinoleyl-methyl-4 -dimethylaminobu
tyrate (DLin -MC3-DMA), and di((Z )-non -2 -en - 1 -yl) 9 -((4
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
about 15 % of the neutral lipid , about 40 % of the sterol, and
about 5 % of the PEG or PEG -modified lipid on a molar
basis .
In some embodiments, the formulations of the present
disclosure include about 57.2 % of a cationic lipid selected
from 2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 ]-dioxolane
herein incorporated by reference in their entirety )
In some embodiments , the formulations of the present
disclosure include 25-75 % of a cationic lipid selected from
2,2 -dilinoleyl-4 -dimethylaminoethyl- [ 1,3 )-dioxolane
(DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu- 55
tyrate (DLin -MC3-DMA) , and di((Z )-non - 2 -en - 1- yl) 9-( 4
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319 ),
0.5-15 % of the neutral lipid , 5-50 % of the sterol, and
0.5-20 % of the PEG or PEG -modified lipid on a molar basis .
In some embodiments, the formulations of the present 60
disclosure include 35-65 % of a cationic lipid selected from
about 7.1 % of the neutral lipid , about 34.3 % of the sterol,
and about 1.4 % of the PEG or PEG -modified lipid on a
molar basis .
In some embodiments, the formulations of the present
disclosure include about 57.5 % of a cationic lipid selected
from the PEG lipid is PEG -CDMA (PEG -CDMA is further
discussed in Reyes et al. (J. Controlled Release, 107, 276
287 ( 2005), the contents ofwhich are herein incorporated by
reference in their entirety ), about 7.5 % of the neutral lipid ,
about 31.5 % of the sterol, and about 3.5 % of the PEG or
PEG -modified lipid on a molar basis .
(also referred herein as PEG -C14 or C14 -PEG ), PEG
(DLin -KC2-DMA), dilinoleyl-methyl-4 -dimethylaminobu
CDMA (further discussed in Reyes et al. J. Controlled tyrate (DLin -MC3-DMA ), and di((Z ) -non -2 -en - 1-yl) 9-((4
Release, 107, 276-287 (2005 ) the contents of which are 50 (dimethylamino )butanoyl)oxy )heptadecanedioate (L319),
2,2 -dilinoleyl-4 -dimethylaminoethyl-[1,3 ] -dioxolane
In some embodiments, lipid nanoparticle formulation
( DLin -KC2 -DMA), dilinoleyl-methyl-4 -dimethylaminobu consists essentially of a lipid mixture in molar ratios ofabout
tyrate (DLin -MC3-DMA ), and di((Z ) -non - 2 -en - 1 -yl) 9 -((4
20-70 % cationic lipid : 5-45 % neutral lipid : 20-55 % choles
(dimethylamino )butanoyl)oxy )heptadecanedioate (L319 ), 65 terol: 0.5-15 % PEG -modified lipid ; more preferably in a
3-12 % of the neutral lipid , 15-45 % of the sterol, and molar ratio of about 20-60 % cationic lipid : 5-25 % neutral
0.5-10 % of the PEG or PEG -modified lipid on a molar basis . lipid : 25-55 % cholesterol: 0.5-15 % PEG -modified lipid .
US 10,702,600 B1
82
81
In some embodiments , the molar lipid ratio is approxi
mately 50 /10 /38.5/1.5 (mol % cationic lipid/neutral lipid ,
e.g., DSPC /Chol/PEG -modified lipid , e.g., PEG -DMG ,
PEG -DSG or PEG -DPG ), 57.2 /7.1134.3/ 1.4 (mol % cationic
lipid /neutral lipid , e.g. , DPPC /Chol/PEG -modified lipid ,
e.g., PEG -CDMA), 40/15/40/5 (mol % cationic lipid /neutral
lipid , e.g., DSPC /Chol/PEG -modified lipid , e.g., PEG
DMG ), 50 /10 /35/4.5/0.5 (mol % cationic lipid /neutral lipid ,
e.g. , DSPC /Chol/PEG -modified lipid , e.g., PEG -DSG ),
50/10/35/5 (cationic lipid /neutral lipid , e.g., DSPC / Chol/
PEG -modified lipid , e.g., PEG -DMG ), 40/10/40/10 (mol %
cationic lipid /neutral lipid , e.g., DSPC /Chol/PEG -modified
lipid , e.g., PEG -DMG or PEG -CDMA), 35/15/40/10 (mol %
cationic lipid /neutral lipid , e.g., DSPC /Chol/PEG -modified
lipid , e.g., PEG -DMG or PEG -CDMA) or 52/13/30/5 (mol %
cationic lipid /neutral lipid , e.g., DSPC /Chol/PEG -modified
lipid , e.g., PEG -DMG or PEG -CDMA).
Examples of lipid nanoparticle compositions and methods
of making same are described , for example , in Semple et al.
(2010 ) Nat. Biotechnol. 28 :172-176 ; Jayarama et al. ( 2012 ),
Angew . Chem . Int. Ed ., 51 : 8529-8533 ; and Maier et al.
the PEG lipid PEG -DOMG and about 38.5 % of the struc
tural lipid cholesterol. As a non - limiting example , the lipid
nanoparticle comprise about 50 % of the cationic lipid DLin
MC3 -DMA, about 10 % of the non -cationic lipid DSPC ,
5 about 1.5 % of the PEG lipid PEG -DMG and about 38.5 % of
the structural lipid cholesterol. As yet another non- limiting
example, the lipid nanoparticle comprise about 55 % of the
cationic lipid L319 , about 10 % of the non -cationic lipid
DSPC , about 2.5 % of the PEG lipid PEG -DMG and about
10 32.5 % of the structural lipid cholesterol.
As a non - limiting example , the cationic lipid may be
selected from (202,23Z )-N ,N - dimethylnonacosa - 20,23
dien-10-amine, ( 177,20Z )-N ,N -dimemylhexacosa - 17,20
dien- 9 -amine,
( 17,19Z )-N5N - dimethylpentacosa - 16 ,
15 19 -dien - 8 - amine, (132,16Z )-N , N -dimethyldocosa - 13,16
dien - 5 -amine, ( 12Z, 15Z )-N , N -dimethylhenicosa - 12,15
dien- 4 -amine, ( 14Z, 17Z)-N , N - dimethyltricosa -14,17 -dien
6 -amine, (15Z , 18Z )-N ,N -dimethyltetracosa -15,18 -dien -7
amine, ( 187,21Z ) -N ,N -dimethylheptacosa - 18,21 -dien - 10
20 amine, (152 , 182 )-N ,N -dimethyltetracosa -15,18 -dien - 5
amine,
(147 , 172 )-N , N - dimethyltricosa -14,17 -dien -4
( 2013 ) Molecular Therapy 21 , 1570-1578 (the contents of amine , ( 197,22Z )-N , N -dimeihyloctacosa - 19,22 -dien - 9
each of which are incorporated herein by reference in their amine, (187,21 Z )-N ,N -dimethylheptacosa -18,21 -dien -8
entirety ) .
amine, (172,20Z )-N ,N - dimethylhexacosa - 17,20 -dien -7
In some embodiments, the lipid nanoparticle formulations 25 amine, (167 , 19Z )-N ,N -dimethylpentacosa - 16,19 -dien -6
described herein may comprise a cationic lipid , a PEG lipid amine, ( 222,25Z ) -N ,N -dimethylhentriaconta - 22,25- dien
and a structural lipid and optionally comprise a non - cationic 10 -amine, (21 Z ,24Z ) -N ,N -dimethyltriaconta -21,24 -dien - 9
lipid . As a non - limiting example, the lipid nanoparticle may amine, ( 18Z )-N ,N -dimetylheptacos - 18 -en - 10 -amine , ( 172 )
comprise about 40-60 % of cationic lipid , about 5-15 % of a N , N - dimethylhexacos- 17 - en - 9 -amine, ( 197,22Z ) -N , N
N ,N
non-cationic lipid , about 1-2 % of a PEG lipid and about 30 dimethyloctacosa - 19,22 -dien - 7-amine ,
30-50 % of a structural lipid . As another non -limiting dimethylheptacosan - 10 -amine,
( 207,23Z ) -N - ethyl- N
example , the lipid nanoparticle may comprise about 50 % methylnonacosa - 20,23 -dien -10 -amine , 1 -[ ( 11Z , 14Z ) -1
cationic lipid , about 10 % non - cationic lipid , about 1.5 % nonylicosa - 11,14 -dien - 1 -yl] pyrrolidine, (202 )-N , N
PEG lipid and about 38.5 % structural lipid . As yet another dimethylheptacos -20 -en - 10 - amine , ( 15Z ) -N ,N -dimethyl
non - limiting example, the lipid nanoparticle may comprise 35 eptacos-15 - en - 10 - amine, (142 )-N ,N -dimethylnonacos- 14
about 55 % cationic lipid , about 10 % non -cationic lipid , en - 10 - amine , ( 172 )-N , N -dimethylnonacos - 17 -en - 10 -amine,
about 2.5 % PEG lipid and about 32.5 % structural lipid . In (24Z )-N ,N -dimethyltritriacont- 24 -en - 10 -amine, (20Z )-N ,N
some embodiments, the cationic lipid may be any cationic dimethylnonacos -20 - en - 10 -amine , (22Z )-N ,N -dimethylhen
lipid described herein such as, but not limited to , DLin
triacont-22 -en - 10 - amine , (16Z ) -N , N -dimethylpentacos - 16
40 en -8 -amine , ( 122 , 15Z )-N ,N -dimethyl- 2 -nonylhenicosa - 12 ,
KC2-DMA, DLin -MC3-DMA and L319 .
In some embodiments , the lipid nanoparticle formulations 15 -dien - 1-amine , ( 13Z , 16Z )-N ,N -dimethyl-3 -nonyldocosa
N ,N - dimethyl- 1 - [ ( 15,2R )-2
described herein may be 4 component lipid nanoparticles . 13,16- dien - 1 -amine,
The lipid nanoparticle may comprise a cationic lipid , a octylcyclopropyl ] eptadecan- 8 -amine, 1-[( 15,2R )-2
non -cationic lipid , a PEG lipid and a structural lipid . As a hexylcyclopropyl ]-N ,N -dimethylnonadecan -10 -amine,
non -limiting example , the lipid nanoparticle may comprise 45 N ,N -dimethyl -1 -[(18,2R )-2-octylcyclopropyl]nonadecan
about 40-60 % of cationic lipid , about 5-15 % of a non
10 -amine, N ,N -dimethyl-21-[( 18,2R ) -2 -octylcyclopropyl]
cationic lipid , about 1-2 % of a PEG lipid and about 30-50 % henicosan - 10 - amine ,N , N -dimethyl -1 -[(18,2S ) -2 - {[( 1R ,
of a structural lipid . As another non -limiting example , the 2R )-2 -pentylcyclopropyl]methyl} cyclopropyl]nonadecan
lipid nanoparticle may comprise about 50 % cationic lipid , 10 -amine, N ,N -dimethyl- 1 -[( 18,2R )-2- octylcyclopropyl]
N , N -dimethyl-[( 1R ,2S )-2
about 10 % non -cationic lipid , about 1.5 % PEG lipid and 50 hexadecan - 8 -amine ,
about 38.5 % structural lipid . As yet another non -limiting undecylcyclopropyl] tetradecan -5 -amine, N ,N -dimethyl-3
example , the lipid nanoparticle may comprise about 55 %
{7 -[(15,2R ) -2 -octylcyclopropyl]heptyl} dodecan -1-amine,
cationic lipid , about 10 % non -cationic lipid , about 2.5 %
1 -[(1R ,2S) -2 -heptylcyclopropyl]-N ,N -dimethyloctadecan
PEG lipid and about 32.5 % structural lipid . In some embodi
9 - amine, 1 -[( 15,2R )-2 -decylcyclopropyl]-N ,N - dimethyl
ments , the cationic lipid may be any cationic lipid described 55 pentadecan -6 -amine, N ,N -dimethyl-1 -[(18,2R )-2 -octylcy
herein such as, but not limited to , DLin -KC2-DMA , DLin
clopropyl]pentadecan -8 -amine , R - N ,N - dimethyl- 1-[ (9Z ,
MC3 - DMA and L319.
12Z ) -octadeca- 9,12 -dien -1 -yloxy ]-3-(octyloxy) propan -2
In some embodiments, the lipid nanoparticle formulations amine, S -N ,N -dimethyl- 1 -[ (9Z , 12Z )-octadeca - 9,12 - dien -1
described herein may comprise a cationic lipid, a non yloxy ] -3-(octyloxy)propan -2 -amine, 1- { 2 -[(97,12Z )-octa
cationic lipid, a PEG lipid and a structural lipid . As a 60 deca- 9,12 -dien - 1-yloxy ]-1-[(octyloxy )methyl]
non -limiting example, the lipid nanoparticle comprise about ethyl} pyrrolidine, (2S)-N ,N - dimethyl- 1- [(9Z , 12Z )
50 % of the cationic lipid DLin -KC2-DMA , about 10 % of octadeca -9,12 -dien - 1-yloxy ) -3 -[(5Z )-oct-5 -en - 1-yloxy]
the non -cationic lipid DSPC , about 1.5 % of the PEG lipid propan -2 -amine , 1- {2 -[(9Z , 12Z )-octadeca - 9,12 -dien - 1
PEG -DOMG and about 38.5 % of the structural lipid cho yloxy ] -1- [(octyloxy )methyllethyl} azetidine ,
( 2S ) -1
lesterol. As a non - limiting example , the lipid nanoparticle 65 (hexyloxy )-N ,N -dimethyl-3 -[ (9Z , 12Z ) -octadeca -9,12 -dien
comprise about 50 % of the cationic lipid DLin -MC3-DMA , 1 - yloxy ]propan - 2 -amine,
(2S )-1- (heptyloxy) -N ,N
about 10 % of the non -cationic lipid DSPC , about 1.5 % of dimethyl-3 -[( 9Z , 12Z )-octadeca - 9,12 -dien - 1 -yloxylpropan
US 10,702,600 B1
83
2 -amine , N ,N -dimethyl- 1- (nonyloxy )-3 -[(9Z ,
octadeca - 9,12 - dien - 1- yloxylpropan - 2 -amine,
84
12Z )
The nanoparticle formulationsmay comprise a phosphate
N ,N
conjugate . The phosphate conjugate may increase in vivo
dimethyl- 1 - [(9Z) -octadec -9 -en - 1 -yloxy ]-3-(octyloxy )
circulation times and /or increase the targeted delivery of the
propan - 2 -amine ; (2S )-N ,N -dimethyl- 1-[ (62,9Z , 12Z ) nanoparticle. As a non -limiting example , the phosphate
5 conjugates may include a compound of any one of the
octadeca -6,9,12 -trien -1 - yloxy ]-3-(octyloxy )propan - 2
amine, (2S ) -1 -[ (117,14Z ) -icosa - 11,14 -dien - 1 -yloxy ]-N ,N formulas described in International Application No.
dimethyl- 3-(pentyloxy )propan - 2-amine , (2S)-1- (hexyloxy ) WO2013033438 , the contents of which are herein incorpo
rated by reference in its entirety .
3 -[( 112,14Z )-icosa - 11,14 -dien - 1 -yloxy ]-N , N
The nanoparticle formulation may comprise a polymer
dimethylpropan -2 - amine, 1-[(112,14Z ) -icosa -11,14 -dien - 1 10 conjugate
. The polymer conjugate may be a water soluble
yloxy ] -N ,N -dimethyl-3-(octyloxy )propan - 2 -amine ,
conjugate
.
polymer conjugate may have a structure as
1 -[( 132 , 16Z )-docosa - 13,16 -dien - 1 -yloxy ]-N ,N -dimethyl described inThe
U.S.
Application No. 20130059360 , the
3-(octyloxy )propan -2 -amine , (2S )-1-[( 132,16Z )-docosa - 13, contents ofwhich Patent
are
herein
incorporated by reference in its
16 -dien - 1 -yloxy ]-3-(hexyloxy ) -N ,N -dimethylpropan -2
entirety . In some embodiments , polymer conjugates with the
amine , (2S )-1-[( 132 )-docos- 13 -en - 1-yloxy ]-3-(hexyloxy) 15 polynucleotides of the present disclosure may be made using
N ,N -dimethylpropan -2 - amine, 1 - [( 13Z )-docos - 13 - en - 1
methods and/or segmented polymeric reagents described
yloxy ] -N ,N -dimethyl-3-(octyloxypropan - 2 -amine , 1 - [(9Z) intheU.S.
Patent Application No. 20130072709 , the contents of
hexadec - 9 - en -1 -yloxy ]-N , N -dimethyl-3-( octyloxy ) propan
which are herein incorporated by reference in its entirety. In
2 -amine, (2R )-N ,N - dimethyl-H (1 -metoylo ctyl)oxy ] -3 some embodiments, the polymer conjugate may have pen
[( 9Z , 12Z ) -octadeca - 9,12 -dien - 1- yloxy ]propan - 2- amine, 20 dant side groups comprising ring moieties such as, but not
( 2R )-1-[ (3,7 -dimethyloctyl)oxy ]-N , N -dimethyl- 3 -[( 9Z ,
limited to , the polymer conjugates described in U.S. Patent
12Z )-octadeca - 9,12 - dien -1 - yloxy ]propan - 2- amine, N ,N - di Publication No. US20130196948 , the contents which are
methyl- 1-(octyloxy) -3 -({ 8- [( 18,2S )-2 - {[(1R 2R )-2- pentyl
herein incorporated by reference in its entirety .
cyclopropyl]methyl} cyclopropyl]octyl}oxy)propan -2
The nanoparticle formulationsmay comprise a conjugate
amine , N ,N -dimethyl-1 - {[8-(2 -oclylcyclopropyl)octyl] 25 to enhance the delivery of nanoparticles of the present
oxy } -3-(octyloxy )propan -2 -amine and (11E ,207,23Z)-N ,N
disclosure in a subject. Further , the conjugate may inhibit
a
or
dimethylnonacosa - 11,20,2 -trien - 10 - amine
phagocytic clearance of the nanoparticles in a subject. In one
pharmaceutically acceptable salt or stereoisomer thereof.
aspect, the conjugate may be a " self" peptide designed from
In some embodiments , the LNP formulations of the RNA the human membrane protein CD47 (e.g.,the “ self particles
(e.g.,mRNA ) vaccines may contain PEG -C -DOMG at 3 % 30 described by Rodriguez et al . (Science 2013 339 , 971-975 ),
lipid molar ratio . In some embodiments , the LNP formula
herein incorporated by reference in its entirety ). As shown
tions of the RNA (e.g., mRNA ) vaccines may contain by Rodriguez et al., the self peptides delayed macrophage
PEG -C -DOMG at 1.5 % lipid molar ratio .
mediated clearance of nanoparticles which enhanced deliv
In some embodiments , the pharmaceutical compositions ery of the nanoparticles . In another aspect , the conjugate
of the RNA (e.g., mRNA) vaccines may include at least one 35 may be the membrane protein CD47 ( e.g., see Rodriguez et
of the PEGylated lipids described in International Publica
al. Science 2013 339 , 971-975 , herein incorporated by
tion No. WO2012099755, the contents of which are herein reference in its entirety ). Rodriguez et al. showed that,
incorporated by reference in their entirety.
similarly to “ self” peptides, CD47 can increase the circu
In some embodiments, the LNP formulation may contain lating particle ratio in a subject as compared to scrambled
PEG -DMG 2000 (1,2 -dimyristoyl-sn -glycero -3 -phophoe- 40 peptides and PEG coated nanoparticles .
thanolamine -N-[methoxy (polyethylene glycol)-2000 ). In
In some embodiments, the RNA (e.g. ,mRNA) vaccines of
some embodiments, the LNP formulation may contain PEG
the present disclosure are formulated in nanoparticles which
DMG 2000 , a cationic lipid known in the art and at least one comprise a conjugate to enhance the delivery of the nano
other component. In some embodiments, the LNP formula particles of the present disclosure in a subject . The conjugate
tion may contain PEG -DMG 2000 , a cationic lipid known in 45 may be the CD47 membrane or the conjugate may be
the art, DSPC and cholesterol. As a non - limiting example , derived from the CD47 membrane protein , such as the " self"
the LNP formulation may contain PEG -DMG 2000 , DLin
peptide described previously . In some embodiments, the
DMA, DSPC and cholesterol. As another non -limiting nanoparticle may comprise PEG and a conjugate of CD47 or
example the LNP formulation may contain PEG - DMG a derivative thereof. In some embodiments, the nanoparticle
2000, DLin -DMA , DSPC and cholesterol in a molar ratio of 50 may comprise both the " self" peptide described above and
2: 40 :10 : 48 (see e.g., Geall et al., Nonviral delivery of the membrane protein CD47.
self -amplifying RNA (e.g., mRNA ) vaccines, PNAS 2012 ;
In some embodiments , a “ self" peptide and /or CD47
PMID : 22908294 , the contents of each of which are herein protein may be conjugated to a virus- like particle or
incorporated by reference in their entirety ).
pseudovirion , as described herein for delivery of the RNA
The lipid nanoparticles described herein may be made in 55 ( e.g. , mRNA) vaccines of the present disclosure .
a sterile environment.
In some embodiments , RNA (e.g., mRNA ) vaccine phar
In some embodiments , the LNP formulation may be maceutical compositions comprising the polynucleotides of
formulated in a nanoparticle such as a nucleic acid - lipid the present disclosure and a conjugate that may have a
particle . As a non -limiting example , the lipid particle may degradable linkage . Non - limiting examples of conjugates
comprise one or more active agents or therapeutic agents ; 60 include an aromatic moiety comprising an ionizable hydro
one or more cationic lipids comprising from about 50 mol %
gen atom , a spacer moiety , and a water -soluble polymer. As
to about 85 mol % of the total lipid present in the particle ; a non - limiting example , pharmaceutical compositions com
one or more non -cationic lipids comprising from about 13 prising a conjugate with a degradable linkage and methods
mol % to about 49.5 mol % of the total lipid present in the for delivering such pharmaceutical compositions are
particle; and one or more conjugated lipids that inhibit 65 described in U.S. Patent Publication No. US20130184443 ,
aggregation of particles comprising from about 0.5 mol % to the contents ofwhich are herein incorporated by reference in
about 2 mol % of the total lipid present in the particle .
their entirety.
US 10,702,600 B1
85
The nanoparticle formulations may be a carbohydrate
86
tissue such as , but not limited to , oral ( e.g., the buccal and
nanoparticle comprising a carbohydrate carrier and a RNA
( e.g., mRNA ) vaccine. As a non -limiting example, the
carbohydrate carrier may include , but is not limited to , an
esophageal membranes and tonsil tissue), ophthalmic , gas
trointestinal ( e.g., stomach , small intestine , large intestine ,
colon , rectum ), nasal, respiratory ( e.g., nasal , pharyngeal,
methods of making hydrophilic polymer particles are
made as described in U.S. Pat. No. 8,241,670 or Interna
anhydride -modified phytoglycogen or glycogen - type mate 5 tracheal and bronchial membranes ), genital (e.g., vaginal,
rial, phtoglycogen octenyl succinate , phytoglycogen beta
cervical and urethral membranes ). Nanoparticles larger than
dextrin , anhydride -modified phytoglycogen beta-dextrin . 10-200 nm which are preferred for higher drug encapsula
(See e.g., International Publication No.WO2012109121; the tion efficiency and the ability to provide the sustained
contents of which are herein incorporated by reference in delivery of a wide array ofdrugs have been thought to be too
their entirety).
10 large to rapidly diffuse through mucosal barriers. Mucus is
Nanoparticle formulations of the present disclosure may continuously secreted , shed , discarded or digested and
be coated with a surfactant or polymer in order to improve recycled so most of the trapped particles may be removed
the delivery of the particle . In some embodiments , the from the mucosa tissue within seconds or within few
nanoparticle may be coated with a hydrophilic coating such hours. Large polymeric nanoparticles (200 nm -500 nm in
as, butnot limited to, PEG coatings and /or coatings that have 15 diameter) which have been coated densely with a low
a neutral surface charge . The hydrophilic coatings may help molecular weight polyethylene glycol (PEG ) diffused
to deliver nanoparticles with larger payloads such as, but not through mucus only 4 to 6 -fold lower than the sameparticles
limited to , RNA (e.g., mRNA ) vaccines within the central diffusing in water (Lai et al. PNAS 2007 104 (5 ):1482-487 ;
nervous system . As a non - limiting example nanoparticles Lai et al. Adv Drug Deliv Rev. 2009 61 (2 ): 158-171; each of
comprising a hydrophilic coating and methods of making 20 which is herein incorporated by reference in their entirety ).
such nanoparticles are described in U.S. Patent Publication The transport of nanoparticles may be determined using
No. US20130183244 , the contents of which are herein rates of permeation and /or fluorescent microscopy tech
incorporated by reference in their entirety .
niques including , but not limited to , fluorescence recovery
In some embodiments, the lipid nanoparticles of the after photobleaching (FRAP ) and high resolution multiple
present disclosure may be hydrophilic polymer particles . 25 particle tracking (MPT). As a non -limiting example, com
Non - limiting examples ofhydrophilic polymer particles and positions which can penetrate a mucosal barrier may be
described in U.S. Patent Publication No. US20130210991 ,
tional Patent Publication No. WO2013110028 , the contents
the contents of which are herein incorporated by reference in of each of which are herein incorporated by reference in its
30 entirety.
their entirety.
In some embodiments , the lipid nanoparticles of the
The lipid nanoparticle engineered to penetrate mucusmay
present disclosure may be hydrophobic polymer particles. comprise a polymeric material (i.e. a polymeric core ) and /or
Lipid nanoparticle formulations may be improved by a polymer- vitamin conjugate and / or a tri-block co -polymer.
replacing the cationic lipid with a biodegradable cationic The polymeric material may include , but is not limited to ,
lipid which is known as a rapidly eliminated lipid nanopar- 35 polyamines, polyethers, polyamides, polyesters, polycar
ticle ( reLNP). Ionizable cationic lipids, such as, but not bamates , polyureas , polycarbonates, poly (styrenes), polyim
limited to , DLinDMA , DLin -KC2-DMA , and DLin -MC3
ides, polysulfones , polyurethanes , polyacetylenes, polyeth
DMA, have been shown to accumulate in plasma and tissues ylenes, polyethyeneimines, polyisocyanates, polyacrylates ,
over time and may be a potential source of toxicity . The polymethacrylates, polyacrylonitriles , and polyarylates. The
rapid metabolism of the rapidly eliminated lipids can 40 polymeric material may be biodegradable and /or biocom
improve the tolerability and therapeutic index of the lipid patible . Non -limiting examples of biocompatible polymers
nanoparticles by an order ofmagnitude from a 1 mg/kg dose are described in International Patent Publication No.
to a 10 mg/ kg dose in rat. Inclusion of an enzymatically WO2013116804 , the contents of which are herein incorpo
degraded ester linkage can improve the degradation and rated by reference in their entirety . The polymeric material
metabolism profile of the cationic component, while still 45 may additionally be irradiated. As a non - limiting example,
maintaining the activity of the reLNP formulation . The ester the polymeric material may be gamma irradiated (see e.g.,
linkage can be internally located within the lipid chain or it International App . No. WO201282165 , herein incorporated
may be terminally located at the terminal end of the lipid by reference in its entirety ). Non -limiting examples of
chain . The internal ester linkage may replace any carbon in specific polymers include poly (caprolactone) (PCL ), ethyl
the lipid chain .
50 ene vinyl acetate polymer (EVA ), poly (lactic acid ) (PLA ),
In some embodiments, the internal ester linkage may be poly (L - lactic acid ) (PLLA ), poly ( glycolic acid ) (PGA ), poly
located on either side of the saturated carbon .
( lactic acid -co - glycolic acid ) (PLGA), poly (L -lactic acid
In some embodiments, an immune response may be co - glycolic acid ) (PLLGA ), poly (D , L -lactide ) (PDLA ), poly
elicited by delivering a lipid nanoparticle which may include (L - lactide ) (PLLA ), poly (D ,L -lactide -co -caprolactone ),
a nanospecies , a polymer and an immunogen . (U.S. Publi- 55 poly (D ,L - lactide -co -caprolactone -co - glycolide), poly (D ,L
cation No. 20120189700 and International Publication No. lactide -co -PEO -CO - D ,L -lactide ), poly ( D ,L -lactide -co - PPO
WO2012099805 ; each of which is herein incorporated by Co - D ,L - lactide), polyalkyl cyanoacralate , polyurethane ,
reference in their entirety ). The polymer may encapsulate poly -L -lysine (PLL ),hydroxypropyl methacrylate (HPMA),
the nanospecies or partially encapsulate the nanospecies . polyethyleneglycol, poly -L - glutamic acid , poly (hydroxy
The immunogen may be a recombinant protein , a modified 60 acids ), polyanhydrides, polyorthoesters, poly (ester amides),
RNA and /or a polynucleotide described herein . In some polyamides, polyester ethers ), polycarbonates, polyalky
embodiments, the lipid nanoparticle may be formulated for lenes such as polyethylene and polypropylene, polyalkylene
use in a vaccine such as, but not limited to , against a glycols such as poly (ethylene glycol) (PEG ), polyalkylene
pathogen .
oxides (PEO ), polyalkylene terephthalates such as poly
Lipid nanoparticles may be engineered to alter the surface 65 (ethylene terephthalate ), polyvinyl alcohols (PVA ), polyvi
properties of particles so the lipid nanoparticles may pen nyl ethers, polyvinyl esters such as poly (vinyl acetate),
etrate the mucosal barrier. Mucus is located on mucosal
polyvinyl halides such as poly (vinyl chloride ) (PVC ), poly
US 10,702,600 B1
87
88
vinylpyrrolidone, polysiloxanes, polystyrene ( PS ), polyure
thanes, derivatized celluloses such as alkyl celluloses ,
hydroxyalkyl celluloses, cellulose ethers , cellulose esters ,
nitro celluloses, hydroxypropylcellulose , carboxymethylcel
herein . The polynucleotide may be encapsulated in the lipid
nanoparticle may be coated or associated with a co -polymer
WO2013110028, the contents of which are herein incorpo
glycol))-(poly (propylene oxide ))-(poly( ethylene glycol)) tri
Hypotonic solutions were found to increase the rate at
nanoparticle and /or disposed on the surface of the particle .
The polynucleotide may be covalently coupled to the lipid
nanoparticle. Formulations of mucus penetrating lipid nano
lulose ,polymers of acrylic acids, such as poly (methyl(meth ) 5 particles may comprise a plurality of nanoparticles. Further,
acrylate ) (PMMA ), poly ( ethyl(meth )acrylate ), poly (butyl the formulations may contain particles which may interact
(methacrylate ), polyisobutylmethacrylate ), poly (hexyl with the mucus and alter the structural and/or adhesive
(meth )acrylate ), poly ( isodecyl(meth )acrylate ), poly (lauryl properties of the surrounding mucus to decrease mucoad
(methacrylate), poly (phenylmethacrylate), poly (methyl hesion , which may increase the delivery of the mucus
acrylate ), polyisopropyl acrylate ), polyisobutyl acrylate ), 10 penetrating lipid nanoparticles to the mucosal tissue .
poly (octadecyl acrylate ) and copolymers and mixtures
In some embodiments , the mucus penetrating lipid nano
thereof, polydioxanone and its copolymers, polyhydroxyal particles may be a hypotonic formulation comprising a
kanoates, polypropylene fumarate , polyoxymethylene, mucosal penetration enhancing coating . The formulation
poloxamers , poly (ortho )esters, poly (butyric acid ), poly ( val may be hypotonice for the epithelium to which it is being
eric acid ), poly (lactide -co -caprolactone), PEG -PLGA- PEG 15 delivered . Non - limiting examples of hypotonic formulations
and trimethylene carbonate , polyvinylpyrrolidone. The lipid may be found in International Patent Publication No.
such as, but not limited to , a block co -polymer (such as a rated by reference in their entirety.
branched polyether -polyamide block copolymer described
In some embodiments, in order to enhance the delivery
in International Publication No. WO2013012476 , herein 20 through the mucosal barrier the RNA (e.g., mRNA ) vaccine
incorporated by reference in its entirety ) , and (polyethylene formulation may comprise or be a hypotonic solution .
block copolymer (see e.g., U.S. Publication 20120121718 which mucoinert particles such as, but not limited to ,
and U.S. Publication 20100003337 and U.S. Pat. No. 8,263 , mucus -penetrating particles , were able to reach the vaginal
665, the contents of each of which is herein incorporated by 25 epithelial surface (see e.g., Ensign et al. Biomaterials 2013
reference in their entirety ). The co -polymer may be a 34 ( 28 ):6922-9, the contents of which are herein incorpo
polymer that is generally regarded as safe (GRAS ) and the rated by reference in their entirety ).
formation of the lipid nanoparticle may be in such a way that
In some embodiments , the RNA ( e.g., mRNA ) vaccine is
no new chemical entities are created . For example , the lipid formulated as a lipoplex , such as , without limitation , the
nanoparticle may comprise poloxamers coating PLGA nano- 30 ATUPLEXTM system , the DACC system , the DBTC system
particles without forming new chemical entities which are and other siRNA - lipoplex technology from Silence Thera
still able to rapidly penetrate human mucus (Yang et al. peutics (London , United Kingdom ), STEMFECTTM from
Angew . Chem . Int. Ed . 2011 50 :2597-2600 ; the contents of STEMGENT® (Cambridge , Mass .), and polyethylenimine
which are herein incorporated by reference in their entirety ). (PEI) or protamine -based targeted and non - targeted delivery
A non -limiting scalable method to produce nanoparticles 35 of nucleic acids acids (Aleku et al. Cancer Res . 2008
which can penetrate human mucus is described by Xu et al.
68:9788-9798 ; Strumberg et al. Int J Clin Pharmacol Ther
The lipid nanoparticle engineered to penetrate mucusmay
include surface altering agents such as, but not limited to ,
polynucleotides, anionic proteins (e.g., bovine serum albu
min ), surfactants (e.g., cationic surfactants such as for 50
example dimethyldioctadecyl-ammonium bromide), sugars
or sugar derivatives (e.g., cyclodextrin ), nucleic acids, poly
mers ( e.g., heparin , polyethylene glycol and poloxamer),
mucolytic agents ( e.g., N -acetylcysteine, mugwort, brome
lain , papain , clerodendrum , acetylcysteine , bromhexine, car- 55
bocisteine , eprazinone, mesna , ambroxol, sobrerol, domi
odol, letosteine , stepronin , tiopronin , gelsolin , thymosin 34
dornase alfa, neltenexine, erdosteine) and various DNases
including rhDNase. The surface altering agent may be
embedded or enmeshed in the particle's surface or disposed 60
( e.g., by coating , adsorption , covalent linkage , or other
process ) on the surface of the lipid nanoparticle. (see e.g.,
are incorporated herein by reference in their entirety ).
In some embodiments, such formulations may also be
constructed or compositions altered such that they passively
or actively are directed to different cell types in vivo ,
including but not limited to hepatocytes, immune cells ,
tumor cells , endothelial cells, antigen presenting cells, and
leukocytes ( Akinc et al. Mol Ther. 2010 18 : 1357-1364 ;
Song et al., Nat Biotechnol. 2005 23 :709-717 ; Judge et al.,
J Clin Invest. 2009 119 :661-673 ; Kaufmann et al., Micro
vasc Res 2010 80 :286-293 ; Santel et al., Gene Ther 2006
13 : 1222-1234; Santel et al., Gene Ther 2006 13 : 1360-1370 ;
Gutbier et al., Pulm Pharmacol. Ther. 2010 23 :334-344 ;
Basha et al., Mol. Ther. 2011 19 :2186-2200 ; Fenske and
Cullis, Expert Opin Drug Deliv. 2008 5 :25-44 ; Peer et al .,
Science . 2008 319 :627-630 ; Peer and Lieberman , Gene
Ther. 2011 18 : 1127-1133, the contents of each of which are
( see, e.g., J ControlRelease 2013 , 170 (2 ):279-86 ; the con
2012 50 : 76-78 ; Santel et al., Gene Ther 2006 13 : 1222-1234 ;
tents of which are herein incorporated by reference in their Santel et al., Gene Ther 2006 13:1360-1370 ; Gutbier et al.,
entirety ) .
Pulm Pharmacol. Ther. 2010 23: 334-344 ; Kaufmann et al.
The vitamin of the polymer -vitamin conjugate may be 40 Microvasc Res 2010 80 :286-293 Weide et al. J Immunother.
vitamin E. The vitamin portion of the conjugate may be 2009 32 : 498-507 ; Weide et al. J Immunother. 2008 31: 180
substituted with other suitable components such as , but not 188 ; Pascolo Expert Opin . Biol. Ther. 4 : 1285-1294 ; Fotin
limited to , vitamin A , vitamin E , other vitamins , cholesterol, Mleczek et al., 2011 J. Immunother. 34: 1-15 ; Song et al.,
a hydrophobic moiety , or a hydrophobic component ofother Nature Biotechnol. 2005 , 23 : 709-717; Peer et al., Proc Natl
surfactants (e.g. , sterol chains , fatty acids, hydrocarbon 45 Acad Sci USA . 2007 6 ; 104 :4095-4100 ; deFougerolles Hum
Gene Ther. 2008 19 : 125-132, the contents of each of which
chains and alkylene oxide chains).
U.S. Publication 20100215580 and U.S. Publication
20080166414 and US20130164343 ; the contents of each of
incorporated herein by reference in their entirety ). One
example of passive targeting of formulations to liver cells
which are herein incorporated by reference in their entirety ). 65 includes the DLin -DMA , DLin -KC2-DMA and DLin -MC3
In some embodiments , the mucus penetrating lipid nano
DMA- based lipid nanoparticle formulations, which have
particles may comprise at least one polynucleotide described been shown to bind to apolipoprotein E and promote binding
US 10,702,600 B1
89
90
and uptake of these formulations into hepatocytes in vivo pharmaceutical composition or compound of the disclosure
(Akinc et al. Mol Ther. 2010 18 : 1357-1364, the contents of may be enclosed , surrounded or encased within the delivery
which are incorporated herein by reference in their entirety ). agent. “ Partially encapsulation ” means that less than 10 , 10 ,
Formulations can also be selectively targeted through 20, 30 , 40 50 or less of the pharmaceutical composition or
expression of different ligands on their surface as exempli- 5 compound of the disclosure may be enclosed, surrounded or
fied by , but not limited by, folate, transferrin , N -acetylga encased within the delivery agent. Advantageously , encap
lactosamine (GalNAc ), and antibody targeted approaches sulation may be determined by measuring the escape or the
(Kolhatkar et al., Curr Drug Discov Technol. 2011 8 : 197 activity of the pharmaceutical composition or compound of
206 ; Musacchio and Torchilin , Front Biosci. 2011 16 : 1388 the disclosure using fluorescence and/or electron micro
1412 ; Yu et al ., MolMembr Biol. 2010 27 :286-298 ; Patil et 10 graph . For example, at least 1, 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 ,
al., Crit Rev Ther Drug Carrier Syst. 2008 25 : 1-61; Benoit 80 , 85 , 90 , 95 , 96 , 97 , 98 , 99 , 99.9 , 99.99 or greater than
et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., 99.99 % of the pharmaceutical composition or compound of
Expert Opin Drug Deliv . 2008 5 :309-319 ; Akinc et al., Mol the disclosure are encapsulated in the delivery agent.
Ther. 2010 18 : 1357-1364; Srinivasan et al., Methods Mol
In some embodiments , the controlled release formulation
Biol. 2012 820 : 105-116 ; Ben - Arie et al.,MethodsMol Biol. 15 may include , but is not limited to , tri-block co -polymers . As
2012 757:497-507; Peer 2010 J Control Release . 20 :63-68 ; a non - limiting example , the formulation may include two
Peer et al., Proc Natl Acad Sci USA . 2007 104 : 4095-4100 ; different types of tri-block co -polymers (International Pub.
Kim et al., Methods Mol Biol. 2011 721: 339-353; Subra
No. WO2012131104 and WO2012131106 , the contents of
manya et al.,Mol Ther. 2010 18 :2028-2037 ; Song et al., Nat each of which are incorporated herein by reference in their
Biotechnol. 2005 23 :709-717 ; Peer et al., Science . 2008 20 entirety ).
319 :627-630 ; Peer and Lieberman , Gene Ther. 2011
In some embodiments, the RNA (e.g., mRNA ) vaccines
18 : 1127-1133 , the contents of each of which are incorpo may be encapsulated into a lipid nanoparticle or a rapidly
eliminated lipid nanoparticle and the lipid nanoparticles or a
rated herein by reference in their entirety ).
In some embodiments, the RNA ( e.g.,mRNA ) vaccine is rapidly eliminated lipid nanoparticle may then be encapsu
formulated as a solid lipid nanoparticle . A solid lipid nano- 25 lated into a polymer, hydrogel and /or surgical sealant
particle (SLN ) may be spherical with an average diameter described herein and/ or known in the art. As a non - limiting
between 10 to 1000 nm . SLN possess a solid lipid core example, the polymer, hydrogel or surgical sealant may be
matrix that can solubilize lipophilic molecules and may be
stabilized with surfactants and /or emulsifiers . In some
PLGA , ethylene vinyl acetate (EVAc), poloxamer,
GELSITE® (Nanotherapeutics , Inc. Alachua , Fla.), HYL
embodiments , the lipid nanoparticle may be a self-assembly 30 ENEX® ( Halozyme Therapeutics, San Diego Calif.), surgi
lipid -polymer nanoparticle (see Zhang et al ., ACS Nano , cal sealants such as fibrinogen polymers ( Ethicon Inc. Cor
2008 , 2 (8 ), pp 1696-1702 ; the contents of which are herein nelia , Ga.), TISSELL® (Baxter International, Inc Deerfield ,
incorporated by reference in their entirety ). As a non
I11.), PEG - based sealants , and COSEAL® Baxter Interna
limiting example , the SLN may be the SLN described in tional, Inc Deerfield , ill .).
International Patent Publication No. WO2013105101, the 35 In some embodiments , the lipid nanoparticle may be
contents of which are herein incorporated by reference in
their entirety . As another non - limiting example , the SLN
may be made by the methods or processes described in
International Patent Publication No. WO2013105101, the
contents of which are herein incorporated by reference in 40
their entirety .
Liposomes, lipoplexes, or lipid nanoparticles may be used
to improve the efficacy of polynucleotides directed protein
production as these formulationsmaybe able to increase cell
encapsulated into any polymer known in the art which may
form a gel when injected into a subject. As another non
limiting example , the lipid nanoparticle may be encapsu
lated into a polymer matrix which may be biodegradable.
In some embodiments , the RNA ( e.g., mRNA ) vaccine
formulation for controlled release and / or targeted delivery
may also include at least one controlled release coating .
Controlled release coatings include, but are not limited to ,
OPADRY® , polyvinylpyrrolidone /vinyl acetate copolymer,
transfection by the RNA ( e.g., mRNA ) vaccine; and/or 45 polyvinylpyrrolidone, hydroxypropyl methylcellulose,
increase the translation of encoded protein . One such hydroxypropyl cellulose , hydroxyethyl cellulose ,
example involves the use of lipid encapsulation to enable the EUDRAGIT RL® , EUDRAGIT RS® and cellulose deriva
effective systemic delivery of polyplex plasmid DNA tives such as ethylcellulose aqueous dispersions (AQUA
(Heyes et al., Mol Ther. 2007 15:713-720 ; the contents of COAT® and SURELEASE? ).
which are incorporated herein by reference in their entirety ). 50 In some embodiments , the RNA (e.g. , mRNA ) vaccine
The liposomes, lipoplexes, or lipid nanoparticles may also
be used to increase the stability of the polynucleotide.
In some embodiments, the RNA (e.g., mRNA ) vaccines of
the present disclosure can be formulated for controlled
release and /or targeted delivery . As used herein , “ controlled 55
release ” refers to a pharmaceutical composition or com
pound release profile that conforms to a particular pattern of
release to effect a therapeutic outcome. In some embodi
ments , the RNA (e.g. , mRNA ) vaccines may be encapsu
lated into a delivery agent described herein and /or known in 60
the art for controlled release and/or targeted delivery . As
used herein , the term “ encapsulate ” means to enclose, sur
round or encase . As it relates to the formulation of the
compounds of the disclosure , encapsulation may be substan
tial, complete or partial. The term “ substantially encapsu- 65
lated ” means that at least greater than 50 , 60 , 70, 80 , 85 , 90 ,
95 , 96 , 97, 98 , 99, 99.9, 99.9 or greater than 99.999 % of the
controlled release and /or targeted delivery formulation may
comprise at least one degradable polyester which may
contain polycationic side chains . Degradeable polyesters
include, but are not limited to , poly ( serine ester), poly (L
lactide -co -L -lysine ), poly (4 -hydroxy -L -proline ester), and
combinations thereof. In some embodiments, the degradable
polyesters may include a PEG conjugation to form a PEGy.
lated polymer.
In some embodiments , the RNA (e.g. , mRNA ) vaccine
controlled release and /or targeted delivery formulation com
prising at leastone polynucleotide may comprise at leastone
PEG and/or PEG related polymer derivatives as described in
U.S. Pat. No. 8,404,222 , the contents of which are incorpo
rated herein by reference in their entirety .
In some embodiments , the RNA (e.g., mRNA ) vaccine
controlled release delivery formulation comprising at least
one polynucleotide may be the controlled release polymer
US 10,702,600 B1
91
92
system described in US20130130348, the contents of which
In some embodiments , the therapeutic nanoparticle com
are incorporated herein by reference in their entirety .
prises a diblock copolymer. In some embodiments, the
In some embodiments, the RNA ( e.g. ,mRNA ) vaccines of diblock copolymer may include PEG in combination with a
the present disclosure may be encapsulated in a therapeutic polymer such as, but not limited to , polyethylenes, polycar
nanoparticle, referred to herein as “ therapeutic nanoparticle 5 bonates, polyanhydrides, polyhydroxyacids, polypropyl
fumerates, polycaprolactones, polyamides , polyacetals ,
RNA (e.g. , mRNA ) vaccines.” Therapeutic nanoparticles polyethers
, polyesters , poly ( orthoesters ), polycyanoacry
may be formulated by methods described herein and known lates, polyvinyl
, polyurethanes, polyphosphazenes ,
in the art such as , but not limited to , International Pub Nos . polyacrylates, alcohols
polymethacrylates
, polycyanoacrylates ,
WO2010005740 , WO2010030763, WO2010005721,
WO2010005723, WO2012054923, U.S. Publication Nos. 10 polyureas
, polystyrenes
, polyamines
, polylysine
imine
), poly (serine
ester ), poly
(L -lactide, poly
-co -L(eth
-ly
US20110262491, US20100104645 , US20100087337, ylene
US20100068285, US20110274759 , US20100068286 , sine), poly (4 -hydroxy-L - proline ester) or combinations
thereof. In yet another embodiment, the diblock copolymer
US20120288541, US20130123351 and US20130230567 may
be a high - X diblock copolymer such as those described
and U.S. Pat. Nos . 8,206,747, 8,293,276 , 8,318,208 and 15 in International Patent Publication No. WO2013120052, the
8,318,211; the contents of each of which are herein incor
porated by reference in their entirety . In some embodiments,
contents of which are incorporated herein by reference in
their entirety .
As a non - limiting example the therapeutic nanoparticle
therapeutic polymer nanoparticles may be identified by the
comprises a PLGA-PEG block copolymer (see U.S. Publi
contents of which are herein incorporated by reference in 20 cation No. US20120004293 and U.S. Pat . No. 8,236,330 ,
methods described in US Pub No. US20120140790 , the
their entirety .
In some embodiments , the therapeutic nanoparticle RNA
(e.g., mRNA ) vaccine may be formulated for sustained
each of which is herein incorporated by reference in their
entirety). In another non - limiting example , the therapeutic
nanoparticle is a stealth nanoparticle comprising a diblock
copolymer of PEG and PLA or PEG and PLGA (see U.S.
months and years. As a non - limiting example , the sustained
non - limiting example , the therapeutic nanoparticle is a
2010075072 and US Pub No. US20100216804 ,
US20110217377 and US201202 859, the contents of each
rated by reference in their entirety .
In some embo
its , the therapeutic nanoparticle may
release . As used herein , “ sustained release ” refers to a
pharmaceutical composition or compound that conforms to 25 Pat . No. 8,246,968 and International Publication No.
a release rate over a specific period of time. The period of WO2012166923, the contents of each of which are herein
time may include , but is not limited to , hours, days , weeks, incorporated by reference in their entirety ). In yet another
release nanoparticle may comprise a polymer and a thera
stealth nanoparticle or a target- specific stealth nanoparticle
peutic agent such as, but not limited to , the polynucleotides 30 as described in U.S. Patent Publication No.
of the present disclosure (see International Pub No. US20130172406 , the contents of which are herein incorpo
of which are incorporated herein by reference in their
comprise a multiblock copolymer (see e.g., U.S. Pat. Nos .
release formulation may comprise agents which permit
US20130195987 , the contents of each of which are herein
entirety). In another non - limiting example, the sustained 35 8,263,665 and 8,287,910 and U.S. Patent Pub . No.
persistentbioavailability such as , but not limited to , crystals,
macromolecular gels and/or particulate suspensions (see
U.S. Patent Publication No US20130150295 , the contents of
each of which are incorporated herein by reference in their 40
entirety ).
In some embodiments, the therapeutic nanoparticle RNA
(e.g., mRNA ) vaccines may be formulated to be target
specific . As a non -limiting example , the therapeutic nano
particles may include a corticosteroid (see International Pub . 45
No.WO2011084518 , the contents of which are incorporated
herein by reference in their entirety ). As a non -limiting
example , the therapeutic nanoparticlesmay be formulated in
nanoparticles described in International Pub No.
WO2008121949,
WO2010005726 ,
incorporated by reference in their entirety ).
In yet another non - limiting example , the lipid nanopar
ticle comprises the block copolymer PEG -PLGA -PEG (see
e.g., the thermosensitive hydrogel (PEG - PLGA-PEG ) was
used as a TGF-betal gene delivery vehicle in Lee et al.
Thermosensitive Hydrogel as a Tgf-b1 Gene Delivery
Vehicle Enhances Diabetic Wound Healing . Pharmaceutical
Research , 2003 20 ( 12 ): 1995-2000 ; as a controlled gene
delivery system in Li et al. Controlled Gene Delivery
System Based on Thermosensitive Biodegradable Hydrogel.
Pharmaceutical Research 2003 20 (6 ): 884-888 ; and Chang et
al., Non - ionic amphiphilic biodegradable PEG -PLGA - PEG
copolymer enhances gene delivery efficiency in rat skeletal
WO2010005725, 50 muscle . J Controlled Release . 2007 118 :245-253, the con
WO2011084521 and US Pub No. US20100069426 ,
US20120004293 and US20100104655 , the contents of each
of which are incorporated herein by reference in their
entirety .
tents of each ofwhich are herein incorporated by reference
in their entirety ) . The RNA ( e.g., mRNA ) vaccines of the
present disclosure may be formulated in lipid nanoparticles
comprising the PEG -PLGA -PEG block copolymer.
lactide-co -L -lysine), poly (4 -hydroxy- L - proline ester) or
comprise at least one acrylic polymer. Acrylic polymers
include but are not limited to , acrylic acid , methacrylic acid ,
In some embodiments , the nanoparticles of the present 55 In some embodiments , the therapeutic nanoparticle may
disclosure may comprise a polymeric matrix . As a non comprise a multiblock copolymer (see e.g., U.S. Pat. Nos .
limiting example, the nanoparticle may comprise two or 8,263,665 and 8,287,910 and U.S. Patent Pub. No.
more polymers such as, but not limited to , polyethylenes , US20130195987 , the contents of each of which are herein
polycarbonates, polyanhydrides , polyhydroxyacids, poly
incorporated by reference in their entirety ).
propylfumerates , polycaprolactones, polyamides, polyac- 60 In some embodiments, the block copolymers described
etals, polyethers, polyesters , poly ( orthoesters ), polycyano herein may be included in a polyion complex comprising a
acrylates, polyvinyl alcohols , polyurethanes, non -polymeric micelle and the block copolymer . ( see e.g.,
polyphosphazenes, polyacrylates, polymethacrylates, poly
U.S. Publication No. 20120076836 , the contents of which
cyanoacrylates, polyureas, polystyrenes, polyamines, are herein incorporated by reference in their entirety ).
polylysine , poly (ethylene imine), poly (serine ester ), poly (L- 65 In some embodiments, the therapeutic nanoparticle may
combinations thereof.
US 10,702,600 B1
93
acrylic acid and methacrylic acid copolymers, methylmeth
acrylate copolymers , ethoxyethyl methacrylates, cyanoethyl
methacrylate , amino alkyl methacrylate copolymer, poly
(acrylic acid ), poly (methacrylic acid ), polycyanoacrylates
and combinations thereof .
94
5
In some embodiments , the therapeutic nanoparticles may
comprise at least one poly ( vinyl ester ) polymer. The poly
(vinyl ester ) polymer may be a copolymer such as a random
copolymer. As a non - limiting example, the random copoly
mer
may Application
have a structure
such as those described
Inter- 10
national
No. WO2013032829
or U.S.in Patent
Publication No US20130121954, the contents of each of
US Pub Nos. US20110020388 and US20110027217 , each of
which is herein incorporated by reference in their entireties ).
In some embodiments, the synthetic nanocarriers may be
formulated for controlled and/ or sustained release of the
polynucleotides described herein . As a non -limiting
example , the synthetic nanocarriers for sustained release
may be formulated by methods known in the art, described
herein and / or as described in International Pub No.
WO2010138192 and US Pub No. 20100303850, each of
which is herein incorporated by reference in their entirety.
In some embodiments, the RNA (e.g., mRNA ) vaccine
may be formulated for controlled and/or sustained release
wherein the formulation comprises at least one polymer that
is a crystalline side chain (CYSC ) polymer. CYSC polymers
are described in U.S. Pat. No. 8,399,007 , herein incorpo
rated by reference in its entirety.
which are herein incorporated by reference in their entirety .
In some embodiments , the poly (vinyl ester ) polymers may
be conjugated to the polynucleotides described herein . 15
In some embodiments, the therapeutic nanoparticle may
comprise at least one diblock copolymer. The diblock copo
In some embodiments , the synthetic nanocarrier may be
lymer may be, but it not limited to , a poly (lactic) acid -poly
(ethylene ) glycol copolymer (see , e.g., International Patent formulated for use as a vaccine . In some embodiments, the
Publication No. WO2013044219, the contents of which are 20 synthetic nanocarrier may encapsulate at least one poly
nucleotide which encode at least one antigen . As a non
herein incorporated by reference in their entirety ).
As a non - limiting example , the therapeutic nanoparticle limiting example, the synthetic nanocarrier may include at
may be used to treat cancer (see International publication least one antigen and an excipient for a vaccine dosage form
No. WO2013044219, the contents of which are herein
( see International Publication No. WO2011150264 and U.S.
incorporated by reference in their entirety ).
25 Publication No. US20110293723 , the contents of each of
In some embodiments , the therapeutic nanoparticles may which are herein incorporated by reference in their entirety ).
comprise at least one cationic polymer described herein As another non - limiting example, a vaccine dosage form
and /or known in the art .
may include at least two synthetic nanocarriers with the
In some embodiments , the therapeutic nanoparticles may same or different antigens and an excipient ( see International
comprise at least one amine-containing polymer such as, but 30 Publication No. W02011150249 and U.S. Publication No.
not limited to polylysine, polyethylene imine , polyamido
US20110293701, the contents of each of which are herein
Pat. No. 8,287,849, the contents of which are herein incor
porated by reference in their entirety) and combinations
dosage form may be selected by methods described herein ,
known in the art and / or described in International Publica
amine ) dendrimers , poly (beta -amino esters ) (see , e.g., U.S.
thereof.
incorporated by reference in their entirety ). The vaccine
35 tion No. WO2011150258 and U.S. Publication No.
In some embodiments , the nanoparticles described herein
may comprise an amine cationic lipid such as those
described in International Patent Application No.
WO2013059496 , the contents of which are herein incorpo
US20120027806 , the contents of each of which are herein
incorporated by reference in their entirety ) .
In some embodiments, the synthetic nanocarrier may
comprise at least one polynucleotide which encodes at least
rated by reference in their entirety. In some embodiments, 40 one adjuvant. As non- limiting example, the adjuvant may
the cationic lipids may have an amino -amine or an amino comprise dimethyldioctadecylammonium -bromide, dimeth
yldioctadecylammonium - chloride, dimethyldioctadecylam
amide moiety .
In some embodiments , the therapeutic nanoparticles may monium -phosphate or dimethyldioctadecylammonium -ac
comprise at least one degradable polyester which may etate (DDA ) and an apolar fraction or part of said apolar
contain polycationic side chains. Degradeable polyesters 45 fraction of a total lipid extractofa mycobacterium (see , e.g.,
include, but are not limited to , poly ( serine ester), poly (L
U.S. Pat. No. 8,241,610 , the content of which is herein
lactide- co -L -lysine), poly (4 -hydroxy -L -proline ester), and incorporated by reference in its entirety ). In some embodi
combinations thereof. In someembodiments, the degradable ments, the synthetic nanocarrier may comprise at least one
polyesters may include a PEG conjugation to form a PEGY polynucleotide and an adjuvant. As a non- limiting example ,
50 the synthetic nanocarrier comprising and adjuvant may be
lated polymer.
In some embodiments , the synthetic nanocarriers may
contain an immunostimulatory agent to enhance the immune
response from delivery of the synthetic nanocarrier. As a
non- limiting example, the synthetic nanocarrier may com
prise a Thl immunostimulatory agent, which may enhance 55
a Th1-based response of the immune system (see Interna
tional Pub No. WO2010123569 and U.S. Publication No.
US20110223201, the contents of each of which are herein
incorporated by reference in their entirety ).
In some embodiments, the synthetic nanocarriers may be 60
formulated for targeted release. In some embodiments , the
synthetic nanocarrier is formulated to release the polynucle
otides at a specified pH and /or after a desired time interval.
As a non - limiting example, the synthetic nanoparticle may
be formulated to release the RNA (e.g., mRNA ) vaccines 65
after 24 hours and / or at a pH of 4.5 ( see International
Publication Nos. WO2010138193 and WO2010138194 and
formulated by the methods described in International Pub
lication No. WO2011150240 and U.S. Publication No.
US20110293700, the contents of each of which are herein
incorporated by reference in their entirety .
In some embodiments, the synthetic nanocarrier may
encapsulate at least one polynucleotide that encodes a pep
tide, fragment or region from a virus. As a non -limiting
example , the synthetic nanocarrier may include, but is not
limited to, any of the nanocarriers described in International
Publication No. WO2012024621 , WO201202629 ,
WO2012024632 and U.S. Publication No. US20120064110 ,
US20120058153 and US20120058154 , the contents of each
of which are herein incorporated by reference in their
entirety .
In some embodiments , the synthetic nanocarrier may be
coupled to a polynucleotide which may be able to trigger a
humoral and/or cytotoxic T lymphocyte (CTL ) response
US 10,702,600 B1
95
( see, e.g. , International Publication No. WO2013019669,
the contents of which are herein incorporated by reference in
their entirety).
In some embodiments, the RNA ( e.g. , mRNA ) vaccine
96
about 5 nm to about 20 nm , from about 5 nm to about 30 nm ,
from about 5 nm to about 40 nm , from about 5 nm to about
50 nm , from about 5 nm to about 60 nm , from about 5 nm
to about 70 nm , from about 5 nm to about 80 nm , from about
may be encapsulated in , linked to and /or associated with 5 5 nm to about 90 nm , about 10 to about 50 nm , from about
zwitterionic lipids. Non -limiting examples of zwitterionic 20 to about 50 nm , from about 30 to about 50 nm , from about
lipids and methods of using zwitterionic lipids are described 40 to about 50 nm , from about 20 to about 60 nm , from about
in U.S. Patent Publication No. US20130216607, the con
30 to about 60 nm , from about 40 to about 60 nm , from about
tents of which are herein incorporated by reference in their 20 to about 70 nm , from about 30 to about 70 nm , from about
entirety.
10 40 to about 70 nm , from about 50 to about 70 nm , from about
In some aspects, the zwitterionic lipidsmay be used in the 60 to about 70 nm , from about 20 to about 80 nm , from about
liposomes and lipid nanoparticles described herein .
30 to about 80 nm , from about 40 to about 80 nm , from about
In some embodiments , the RNA (e.g., mRNA ) vaccine 50 to about 80 nm , from about 60 to about 80 nm , from about
may be formulated in colloid nanocarriers as described in 20 to about 90 nm , from about 30 to about 90 nm , from about
U.S. Patent Publication No. US20130197100 , the contents 15 40 to about 90 nm , from about 50 to about 90 nm , from about
of which are herein incorporated by reference in their 60 to about 90 nm and / or from about 70 to about 90 nm .
entirety.
In some embodiments , such LNPs are synthesized using
In some embodiments, the nanoparticle may be optimized methods comprising microfluidic mixers . Examples of
for oral administration . The nanoparticle may comprise at microfluidic mixers may include, but are not limited to , a slit
least one cationic biopolymer such as, but not limited to , 20 interdigital micromixer including , but not limited to those
chitosan or a derivative thereof. As a non - limiting example , manufactured by Microinnova (Allerheiligen bei Wildon ,
the nanoparticle may be formulated by the methods Austria ) and /or a staggered herringbone micromixer (SHM )
described in U.S. Publication No. 20120282343 , the con
(Zhigaltsev, I. V. et al., Bottom -up design and synthesis of
tents of which are herein incorporated by reference in their limit size lipid nanoparticle systems with aqueous and
entirety .
25 triglyceride cores using millisecond microfluidic mixing
In some embodiments, LNPs comprise the lipid KL52 (an have been published (Langmuir. 2012. 28 : 3633-40 ; Bel
amino - lipid disclosed in U.S. Application Publication No. liveau, N. M. et al., Microfluidic synthesis of highly potent
2012/0295832 , the contents of which are herein incorpo
limit- size lipid nanoparticles for in vivo delivery of siRNA.
rated by reference in their entirety . Activity and/or safety (as Molecular Therapy-Nucleic Acids. 2012. 1:e37 ; Chen , D. et
measured by examining one or more of ALT /AST, white 30 al., Rapid discovery of potent siRNA -containing lipid nano
blood cell count and cytokine induction , for example ) of particles enabled by controlled microfluidic formulation . J
LNP administration may be improved by incorporation of Am Chem Soc . 2012. 134 ( 16 ):6948-51, the contents of each
such lipids. LNPs comprising KL52 may be administered of which are herein incorporated by reference in their
intravenously and /or in one ormore doses. In some embodi entirety ). In some embodiments,methods of LNP generation
ments, administration of LNPs comprising KL52 results in 35 comprising SHM , further comprise the mixing of at least
equal or improved mRNA and /or protein expression as two input streams wherein mixing occurs by microstructure
compared to LNPs comprising MC3 .
induced chaotic advection (MICA ). According to this
In some embodiments , RNA ( e.g.,mRNA ) vaccine may method , fluid streams flow through channels present in a
be delivered using smaller LNPs. Such particles may com
herringbone pattern causing rotational flow and folding the
prise a diameter from below 0.1 um up to 100 nm such as, 40 fluids around each other. This method may also comprise a
but not limited to , less than 0.1 um , less than 1.0 um , less surface for fluid mixing wherein the surface changes orien
than 5 um , less than 10 um , less than 15 um , less than 20 um , tations during fluid cycling . Methods of generating LNPs
less than 25 um , less than 30 um , less than 35 um , less than using SHM include those disclosed in U.S. Application
40 um , less than 50 um , less than 55 um , less than 60 um , Publication Nos . 2004/0262223 and 2012/0276209, the con
less than 65 um , less than 70 um , less than 75 um , less than 45 tents of each ofwhich are herein incorporated by reference
80 um , less than 85 um , less than 90 um , less than 95 um , in their entirety.
less than 100 um , less than 125 um , less than 150 um , less
In some embodiments, the RNA (e.g., mRNA ) vaccine of
than 175 um , less than 200 um , less than 225 um , less than the present disclosure may be formulated in lipid nanopar
250 um , less than 275 um , less than 300 um , less than 325 ticles created using a micromixer such as , butnot limited to ,
um , less than 350 um , less than 375 um , less than 400 um , 50 a Slit Interdigital Microstructured Mixer (SIMM -V2) or a
less than 425 um , less than 450 um , less than 475 um , less Standard Slit Interdigital Micro Mixer (SSIMM ) or Cater
than 500 um , less than 525 um , less than 550 um , less than pillar (CPMM ) or Impinging- jet (IJMM ) from the Institut
575 um , less than 600 um , less than 625 um , less than 650
fiir Mikrotechnik Mainz GmbH , Mainz Germany ).
um , less than 675 um , less than 700 um , less than 725 um ,
In some embodiments , the RNA ( e.g.,mRNA ) vaccines of
less than 750 um , less than 775 um , less than 800 um , less 55 the present disclosure may be formulated in lipid nanopar
than 825 um , less than 850 um , less than 875 um , less than ticles created using microfluidic technology ( see, e.g., Whi
900 um , less than 925 um , less than 950 um , less than 975 tesides, George M. The Origins and the Future of Micro
um , or less than 1000 um .
fluidics. Nature, 2006 442 : 368-373 ; and Abraham et al.
In some embodiments, RNA (e.g., mRNA ) vaccines may Chaotic Mixer forMicrochannels. Science , 2002 295 : 647
be delivered using smaller LNPs, which may comprise a 60 651; each of which is herein incorporated by reference in its
diameter from about 1 nm to about 100 nm , from about 1 nm
entirety ). As a non - limiting example, controlled microfluidic
to about 10 nm , about 1 nm to about 20 nm , from about 1 formulation includes a passive method for mixing streamsof
nm to about 30 nm , from about 1 nm to about 40 nm , from
steady pressure- driven flows in micro channels at a low
about 1 nm to about 50 nm , from about 1 nm to about60 nm , Reynolds number ( see , e.g., Abraham et al. Chaotic Mixer
from about 1 nm to about 70 nm , from about 1 nm to about 65 for Microchannels. Science , 2002 295: 647-651, the con
80 nm , from about 1 nm to about 90 nm , from about 5 nm
tents of which are herein incorporated by reference in their
to about from 100 nm , from about 5 nm to about 10 nm , entirety ).
US 10,702,600 B1
97
98
In some embodiments, the RNA (e.g., mRNA ) vaccines of
the present disclosure may be formulated in lipid nanopar
phosphatidylcholine (POPC ). In some embodiments , the
limit size lipid nanoparticle may comprise a polyethylene
glycol- lipid such as, but not limited to, DLPE - PEG , DMPE
PEG , DPPC -PEG and DSPE - PEG .
In some embodiments , the RNA ( e.g., mRNA ) vaccines
may be delivered , localized and /or concentrated in a specific
location using the delivery methods described in Interna
tional Patent Publication No. WO2013063530 , the contents
of which are herein incorporated by reference in their
entirety . As a non - limiting example , a subject may be
administered an empty polymeric particle prior to , simulta
neously with or after delivering the RNA (e.g., mRNA )
vaccines to the subject. The empty polymeric particle under
goes a change in volume once in contact with the subject and
becomes lodged , embedded , immobilized or entrapped at a
specific location in the subject.
In some embodiments , the RNA (e.g.,mRNA ) vaccines
may be formulated in an active substance release system
( see , e.g., U.S. Patent Publication No. US20130102545 , the
contents of which are herein incorporated by reference in
their entirety ). The active substance release system may
comprise 1 ) at least one nanoparticle bonded to an oligo
nucleotide inhibitor strand which is hybridized with a cata
lytically active nucleic acid and 2 ) a compound bonded to at
least one substrate molecule bonded to a therapeutically
ticles created using a micromixer chip such as, but not
limited to , those from Harvard Apparatus (Holliston , Mass .)
or Dolomite Microfluidics (Royston , UK ). A micromixer 5
chip can be used for rapid mixing of two or more fluid
streams with a split and recombine mechanism .
In some embodiments, the RNA (e.g.,mRNA ) vaccines of
the disclosure may be formulated for delivery using the drug
encapsulating microspheres described in International Pat- 10
ent Publication No. WO2013063468 or U.S. Pat. No. 8,440,
614 , the contents of each of which are herein incorporated
by reference in their entirety. The microspheres may com
prise a compound of the formula (I), ( II), (III), (IV ), ( V ) or
(VI) as described in International Patent Publication No. 15
WO2013063468, the contents of which are herein incorpo
rated by reference in their entirety . In some embodiments,
the amino acid , peptide , polypeptide, lipids (APPL ) are
useful in delivering the RNA ( e.g.,mRNA ) vaccines of the
disclosure to cells (see International Patent Publication No. 20
WO2013063468, the contents of which are herein incorpo
rated by reference in their entirety ).
In some embodiments, the RNA (e.g., mRNA ) vaccines of
the disclosure may be formulated in lipid nanoparticles
having a diameter from about 10 to about 100 nm such as , 25
but not limited to , about 10 to about 20 nm , about 10 to about active substance (e.g., polynucleotides described herein ),
30 nm , about 10 to about 40 nm , about 10 to about 50 nm , where the therapeutically active substance is released by the
about 10 to about 60 nm , about 10 to about 70 nm , about 10 cleavage of the substrate molecule by the catalytically active
to about 80 nm , about 10 to about 90 nm , about 20 to about nucleic acid .
30 nm , about 20 to about 40 nm , about 20 to about 50 nm , 30 In some embodiments , the RNA (e.g., mRNA ) vaccines
about 20 to about 60 nm , about 20 to about 70 nm , about 20 may be formulated in a nanoparticle comprising an inner
to about 80 nm , about 20 to about 90 nm , about 20 to about core comprising a non -cellular material and an outer surface
100 nm , about 30 to about 40 nm , about 30 to about 50 nm , comprising a cellular membrane. The cellular membrane
about 30 to about 60 nm , about 30 to about 70 nm , about 30 may be derived from a cell or a membrane derived from a
to about 80 nm , about 30 to about 90 nm , about 30 to about 35 virus . As a non- limiting example , the nanoparticle may be
100 nm , about 40 to about 50 nm , about 40 to about 60 nm ,
about 40 to about 70 nm , about 40 to about 80 nm , about 40
to about 90 nm , about 40 to about 100 nm , about 50 to about
made by the methods described in International Patent
Publication No. WO2013052167 , the contents of which are
herein incorporated by reference in their entirety. As another
60 nm , about 50 to about 70 nm about 50 to about 80 nm , non -limiting example, the nanoparticle described in Inter
about 50 to about 90 nm , about 50 to about 100 nm , about 40 national Patent Publication No. WO2013052167 , the con
60 to about 70 nm , about 60 to about 80 nm , about 60 to tents of which are herein incorporated by reference in their
about 90 nm , about 60 to about 100 nm , about 70 to about entirety , may be used to deliver the RNA (e.g. , mRNA )
80 nm , about 70 to about 90 nm , about 70 to about 100 nm ,
about 80 to about 90 nm , about 80 to about 100 nm and/ or
about 90 to about 100 nm .
In some embodiments , the lipid nanoparticles may have a
In some embodiments , the lipid nanoparticle may have a
diameter greater than 100 nm , greater than 150 nm , greater
than 200 nm , greater than 250 nm , greater than 300 nm ,
greater than 350 nm , greater than 400 nm , greater than 450
nm , greater than 500 nm , greater than 550 nm , greater than
600 nm , greater than 650 nm , greater than 700 nm , greater
diameter from about 10 to 500 nm .
than 750 nm , greater than 800 nm , greater than 850 nm ,
vaccines described herein .
In some embodiments , the RNA (e.g., mRNA ) vaccines
45 may be formulated in porous nanoparticle -supported lipid
bilayers (protocells ). Protocells are described in Interna
tional Patent Publication No. WO2013056132 , the contents
of which are herein incorporated by reference in their
entirety .
50 In some embodiments , the RNA (e.g., mRNA ) vaccines
described herein may be formulated in polymeric nanopar
ticles as described in or made by the methods described in
U.S. Pat. Nos . 8,420,123 and 8,518,963 and European Patent
No. EP2073848B1, the contents of each of which are herein
greater than 900 nm , greater than 950 nm or greater than 55 incorporated by reference in their entirety .As a non - limiting
example , the polymeric nanoparticle may have a high glass
1000 nm .
In some embodiments , the lipid nanoparticle may be a transition temperature such as the nanoparticles described in
limit size lipid nanoparticle described in International Patent or nanoparticles made by the methods described in U.S. Pat.
Publication No. WO2013059922 , the contents of which are No. 8,518,963, the contents of which are herein incorporated
herein incorporated by reference in their entirety. The limit 60 by reference in their entirety . As another non - limiting
size lipid nanoparticle may comprise a lipid bilayer sur
example, the polymer nanoparticle for oral and parenteral
rounding an aqueous core or a hydrophobic core ; where the formulations may be made by the methods described in
lipid bilayer may comprise a phospholipid such as, but not European Patent No. EP2073848B1, the contents of which
limited to , diacylphosphatidylcholine, a diacylphosphatidy
are herein incorporated by reference in their entirety .
lethanolamine , a ceramide , a sphingomyelin , a dihy- 65 In some embodiments , the RNA ( e.g., mRNA ) vaccines
drosphingomyelin , a cephalin , a cerebroside, a C8 - C20 fatty described herein may be formulated in nanoparticles used in
acid diacylphophatidylcholine , and l-palmitoyl- 2-oleoyl imaging . The nanoparticles may be liposome nanoparticles
US 10,702,600 B1
99
such as those described in U.S. Patent Publication No
100
In some embodiments the nanoparticles of the present
US20130129636 , herein incorporated by reference in its disclosure may be developed by the methods described in
entirety . As a non - limiting example, the liposome may U.S. Patent Publication No. US20130172406 , the contents
comprise gadolinium ( III)2- {4,7 -bis -carboxymethyl - 10 -[(N , of which are herein incorporated by reference in their
N -distearylamidomethyl-N '-amido -methyl)-1,4,7,10 - tetra 5 entirety .
azacyclododec- 1 -yl) -acetic acid and a neutral, fully satu
In some embodiments , the nanoparticles of the present
rated phospholipid component (see , e.g., U.S. Patent disclosure are stealth nanoparticles or target-specific stealth
Publication No US20130129636 , the contents of which are nanoparticles such as, but not limited to , those described in
U.S. Patent Publication No. US20130172406 , the contents
herein incorporated by reference in their entirety ).
which are herein incorporated by reference in their
In some embodiments , the nanoparticles which may be 10 of
entirety
nanoparticles of the present disclosure may be
used in the present disclosure are formed by the methods made by. The
the
methods
in U.S. Patent Publication
described in U.S. Patent Application No. US20130130348 , No. US20130172406 , described
the
contents
of which are herein
the contents ofwhich are herein incorporated by reference in incorporated by reference in their entirety
.
their entirety .
15
In
some
embodiments
,
the
stealth
or
target
-specific stealth
The nanoparticles of the present disclosure may further nanoparticles may comprise a polymeric matrix
. The poly
include nutrients such as, but not limited to , those which meric matrix may comprise two or more polymers such as,
deficiencies can lead to health hazards from anemia to neural but not limited to , polyethylenes, polycarbonates , polyan
tube defects (see, e.g., the nanoparticles described in Inter hydrides , polyhydroxyacids, polypropylfumerates, poly
national Patent Publication No WO2013072929 , the con- 20 caprolactones, polyamides, polyacetals, polyethers, polyes
tents of which are herein incorporated by reference in their
entirety ). As a non - limiting example , the nutrient may be
iron in the form of ferrous, ferric salts or elemental iron ,
ters , poly (orthoesters), polycyanoacrylates, polyvinyl
alcohols , polyurethanes, polyphosphazenes, polyacrylates,
polymethacrylates, polycyanoacrylates, polyureas, polysty
renes, polyamines, polyesters, polyanhydrides , polyethers,
iodine , folic acid , vitamins or micronutrients .
In some embodiments ,the RNA (e.g.,mRNA ) vaccines of 25 polyurethanes, polymethacrylates , polyacrylates, polycya
the present disclosure may be formulated in a swellable noacrylates or combinations thereof.
nanoparticle . The swellable nanoparticle may be, but is not
In some embodiments, the nanoparticle may be a nano
limited to , those described in U.S. Pat. No. 8,440,231 , the particle -nucleic acid hybrid structure having a high density
contents of which are herein incorporated by reference in nucleic acid layer. As a non - limiting example , the nanopar
their entirety . As a non - limiting embodiment, the swellable 30 ticle -nucleic acid hybrid structure may made by the methods
nanoparticle may be used for delivery of the RNA (e.g., described in U.S. Patent Publication No. US20130171646 ,
mRNA ) vaccines of the present disclosure to the pulmonary the contents ofwhich are herein incorporated by reference in
system (see , e.g., U.S. Pat . No. 8,440,231 , the cor nts of their entirety. The nanoparticle may comprise a nucleic acid
which are herein incorporated by reference in their entirety ). such as, but not limited to , polynucleotides described herein
The RNA (e.g.,mRNA ) vaccines of the present disclosure 35 and /or known in the art .
may be formulated in polyanhydride nanoparticles such as,
At least one of the nanoparticles of the present disclosure
but not limited to , those described in U.S. Pat. No. 8,449, may be embedded in in the core a nanostructure or coated
916 , the contents of which are herein incorporated by with a low density porous 3 - D structure or coating which is
capable of carrying or associating with at least one payload
reference in their entirety .
The nanoparticles and microparticles of the present dis- 40 within or on the surface of the nanostructure. Non - limiting
closure may be geometrically engineered to modulate mac examples of the nanostructures comprising at least one
rophage and /or the immune response. In some embodiments, nanoparticle are described in International Patent Publica
the geometrically engineered particles may have varied tion No. WO2013123523 , the contents of which are herein
shapes , sizes and /or surface charges in order to incorporated incorporated by reference in their entirety .
the polynucleotides of the present disclosure for targeted 45 In some embodiments the RNA ( e.g., mRNA) vaccine
delivery such as, butnot limited to , pulmonary delivery (see, may be associated with cationic or polycationic com
e.g., International Publication No WO2013082111 , the con
pounds, including protamine, nucleoline, spermine or sper
tents of which are herein incorporated by reference in their midine, or other cationic peptides or proteins, such as
entirety ) . Other physical features the geometrically engi poly -L -lysine (PLL ), polyarginine , basic polypeptides , cell
neering particles may have include, but are not limited to , 50 penetrating peptides (CPPS), including HIV -binding pep
fenestrations, angled arms, asymmetry and surface rough
tides, HIV -1 Tat (HIV ), Tat -derived peptides, Penetratin ,
ness, charge which can alter the interactions with cells and VP22 derived or analog peptides, Pestivirus Erns, HSV, VP22
tissues. As a non - limiting example, nanoparticles of the (Herpes simplex ), MAP, KALA or protein transduction
present disclosure may be made by the methods described in domains (PTDs), PpT620, prolin -rich peptides, arginine - rich
International Publication No WO2013082111, the contents 55 peptides, lysine-rich peptides, MPG -peptide(s), Pep -1,
of which are herein incorporated by reference in their L -oligomers, Calcitonin peptide(s ), Antennapedia -derived
entirety .
peptides (particularly from Drosophila antennapedia ),
In some embodiments, the nanoparticles of the present pAntp , pIsl, FGF, Lactoferrin , Transportan , Buforin -2 ,
disclosure may be water soluble nanoparticles such as, but Bac715-24 , SynB , SynB ( 1), pVEC , hCT-derived peptides,
not limited to , those described in International Publication 60 SAP, histones , cationic polysaccharides, for example chito
No. WO2013090601, the contents of which are herein san , polybrene, cationic polymers, e.g. polyethyleneimine
incorporated by reference in their entirety. The nanoparticles (PEI), cationic lipids, e.g. DOTMA: [ 1-(2,3 - sioleyloxy )
may be inorganic nanoparticles which have a compact and propyl)]- N ,N , N -trimethylammonium chloride, DMRIE ,
zwitterionic ligand in order to exhibit good water solubility. di-C14 -amidine, DOTIM , SAINT, DC -Chol, BGTC , CTAP,
The nanoparticlesmay also have small hydrodynamic diam- 65 DOPC , DODAP , DOPE : Dioleyl phosphatidylethanol
eters (HD ), stability with respect to time, pH , and salinity amine, DOSPA , DODAB , DOIC , DMEPC , DOGS : Diocta
decylamidoglicylspermin , DIMRI: Dimyristooxypropyl
and a low level of non -specific protein binding.
US 10,702,600 B1
101
102
dimethyl hydroxyethyl ammonium bromide, DOTAP : dio
leoyloxy - 3-(trimethylammonio )propane , DC -6-14 : 0,0
ditetradecanoyl-N -.alpha . -trimethylammonioacetyl )dietha
nolamine chloride, CLIP 1: rac - [(2,3 -dioctadecyloxypropyl)
( 2- hydroxyethyl) -dimethylammonium chloride, CLIP6: 5
rac-[2 ( 2,3 -dihexadecyloxypropyloxymethyloxy ) ethyl]
trimethylammonium , CLIP9 : rac-[2 ( 2,3 -dihexadecyloxy
propyloxysuccinyloxy )ethyl] -trimethylammonium , oligo
fectamine , or cationic or polycationic polymers , e.g. 10
each R is independently selected from the group consist
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
M and M ' are independently selected from C (O )O- ,
OC ( O ) - , -C (O )N (R ') — ,
-N (R ') C (O ) , C (O )
C ( S ) — , -C (S )S— , SC
(S ) - , -CH (OH ) — , -P (O )(OR ')O- , -S(O )2- , S
S an aryl group , and a heteroaryl group ;
R , is selected from the group consisting of C1-3 alkyl, C2-3
alkenyl, and H ; R , is selected from the group consisting of
and heterocycle ;
modified polyaminoacids, such as beta -aminoacid -polymers C3-6R ,carbocycle
is
selected
from the group consisting of H , CN , NO2,
or reversed polyamides , etc., modified polyethylenes, such
alkyl, OR , -S (O )2R , -S( O )2N (R ) 2, C2-6 alkenyl,
as PVP (poly (N - ethyl-4 -vinylpyridinium bromide )), etc., C1-6
and heterocycle ;
modified acrylates , such as PDMAEMA (poly (dimethylam C3-6eachcarbocycle
R
is
independently
selected from the group consist
inoethyl methylacrylate )), etc. , modified amidoamines such 15 ing of C1-3 alkyl, C2-3 alkenyl
, and H ;
as PAMAM (poly ( amidoamine )), etc., modified polybet
each R ' is independently selected from the group consist
aminoester (PBAE ), such as diamine end modified 1,4 ing of C -18 alkyl , C2-18 alkenyl, R * YR ", —YR ", and H ;
butanediol diacrylate -co -5 -amino -1 -pentanol polymers , etc.,
each R " is independently selected from the group con
9
dendrimers , such as polypropylamine dendrimers or
PAMAM based dendrimers, etc., polyimine(s ), such as PEI: 20
poly ( ethyleneimine ), poly (propyleneimine ), etc., polyally
lamine, sugar backbone based polymers , such as cyclodex
trin based polymers, dextran based polymers, chitosan , etc.,
silan backbone based polymers, such as PMOXA - PDMS
copolymers , etc., blockpolymers consisting of a combina- 25
tion of one or more cationic blocks ( e.g. selected from a
cationic polymer as mentioned above) and of one or more
hydrophilic or hydrophobic blocks (e.g. polyethylenegly
sisting of C3-14 alkyl and C3-14 alkenyl;
each R * is independently selected from the group con
sisting of C1-12 alkyl and C2-12 alkenyl ;
each Y is independently a C3-6 carbocycle ;
each X is independently selected from the group consist
ing of F , C1, Br, and I; and
m is selected from 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , and 13 .
In some embodiments, a subset of compounds of Formula
(I) includes those in which when R4 is – ( CH2) , (CH2)n
CHQR , CHQR , or CQ (R ) 2, then (i) Q is not —N (R ) 2
when n is 1, 2 , 3 , 4 or 5 , or ( ii ) Q is not 5 , 6 , or 7 -membered
cole ), etc.
In other embodiments the RNA (e.g., mRNA) vaccine is 30 heterocycloalkyl
when n is 1 or 2 .
In some embodiments , another subset of compounds of
not associated with a cationic or polycationic compounds. Formula
(I) includes those in which
In some embodiments , a nanoparticle comprises com
R
,
is
selected
from the group consisting of C5-30 alkyl,
pounds of Formula (I) :
C5-20 alkenyl, -R * YR " , —YR ", and - R "M'R ';
35
RA
Ri
Rs
R2
R7
R2 and R3 are independently selected from the group
consisting of H , C1-14 alkyl, C2-14 alkenyl, R * YR " ,
-YR " , and —R * OR " , or R2 and R3, togetherwith the atom
to which they are attached , form a heterocycle or carbocycle ;
R4 is selected from the group consisting of a C3-6 carbo
40 cycle , ( CH ), Q , ( CH ), CHQR ,
CHQR , CQ (R )2, and unsubstituted C1-6 alkyl, where Q
is selected from a C3-6 carbocycle, a 5- to 14 -membered
heteroarylhaving one or more heteroatoms selected from N ,
O , and S , OR ,
or a salt or isomer thereof, wherein :
R? is selected from the group consisting of C5-30 alkyl, 45 O (CH2) N (R )2, C (O )OR , OC (O ) R , CX3, CX2H ,
C5-20 alkenyl, R * YR " , —YR " , and --R" M'R ';
CXH , CN , C (O )N (R ) , -N (R ) C (O )R , N (R ) S
R2 and Rz are independently selected from the group (O ) , R , N (R )C ( O ) N (R )2, -N ( R ) C (S ) N ( R ) 2, CRN (R )2
consisting of H , C1-14 alkyl, C2-14 alkenyl, R * YR " , C (O )OR , —N (R )Rg, O (CH2),OR , — N (R CNR
)
,) N
-YR " , and —R *OR " , or R2 and Rz, together with the atom
(R ) 2, —N (R ) C ( = CHR , )N (R )2, OC (O ) N (R )2, -N (R ) C
to which they are attached , form a heterocycle or carbocycle; 50 (O )OR , -N (OR )C (O )R , N (OR )S (O ), R , N (OR )C (O )
M
Ró
R3,
17
R4 is selected from the group consisting of a C3-6 carbo OR , —N (OR ) C (O )N ( R )2, —N (OR )C (S )N (R )2, - N (OR )C
cycle , ( CH2), Q , (CH2) „ CHCR ,
ENR ,) N (R )2 , —NOR )CECHR , N (R ) 2, CNR , ) N
CHQR , CQ (R )2, and unsubstituted C1-6 alkyl, where (R ) 2, CENR ,)R , C (O )N (R )O R , and a 5- to
Qis selected from a carbocycle , heterocycle , OR , 14 -membered heterocycloalkylhaving one or more heteroa
O (CH2), N (R ) ,
C (O )OR ,
OCOR,
CX3, 55 toms selected from N , O , and S which is substituted with one
CXH , CN , —N (R ) , C (O ) N (R )2, -N (R ) or more substituents selected from oxo ( O ), OH , amino ,
CX , H , CXH2
C (O )R , -N (R )S (O ) R , N (R ) C (O )N (R )2, -N (R ) C ( S)N mono- or di -alkylamino , and C1-3 alkyl, and each n is
(R )2, -N (R )RE, O (CH2), OR , -N (R )CENR , N ( R ) , independently selected from 1, 2 , 3 , 4 , and 5 ;
-N (R )CECHR , N (R )2, OC (O )N (R )2, N (R )C (O )
each R? is independently selected from the group consist
OR , —N (OR )C (O ) R , —N (OR )S (O )2R , —N (OR )C (O )OR , 60 ing of C1-3 alkyl, C2-3 alkenyl, and H ;
N (OR )C (O )N ( R ) ,
N (OR )C ( S ) N ( R )
N (OR ) C
each R is independently selected from the group consist
(ANR ,)N (R )2, -N (OR ) C (= CHR ,)N (R )2, Ce= NR ,) N
ing ofC1-3 alkyl, C2-3 alkenyl, and H ;
(R ) , CONR )R , C (O ) N ( R )O R , and C (R ) N (R ) , C
M and M ' are independently selected from C ( O )O- ,
(O )OR , and each n is independently selected from 1, 2 , 3, 4 , 65 OC( O ) , C (O )N (R ') , -N (R ') C ( O ) , C ( O )
and 5 ;
C (S )
C (S )S
SC (S ) — , CH (OH ) — , -P( O )
each R , is independently selected from the group consist
(OR )O- , -S(O )2- , S — 5— , an aryl group, and a
9
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
heteroaryl group;
US 10,702,600 B1
103
104
R , is selected from the group consisting of C1-3 alkyl, C2-3
Rg is selected from the group consisting of C3-6 carbo
cycle and heterocycle;
R , is selected from the group consisting of H , CN , NO2,
C1-6 alkyl, OR , -S(O ) R , -S (O ), N (R )2, C2-6 alkenyl,
C3-6 carbocycle and heterocycle ;
each R is independently selected from the group consist
each R ' is independently selected from the group consist
ing of C1-18 alkyl, C2-18 alkenyl , -R * YR " , —YR ", and H ;
each R " is independently selected from the group con
sisting of C3-14 alkyl and C3-14 alkenyl;
each R * is independently selected from the group con
sisting of C -12 alkyl and C2-12 alkenyl;
each Y is independently a C3.6 carbocycle ;
each X is independently selected from the group consist
ing of F , C1, Br, and I; and
alkenyl, and H ;
each R is independently selected from the group consist
ing of C1-3 alkyl , C2-3 alkenyl, and H ;
each R ' is independently selected from the group consist
ing ofC1-18 alkyl, C2-18 alkenyl, -R * YR " , YR " , and H ;
each R " is independently selected from the group con
sisting of C3-14 alkyl and C3-14 alkenyl;
each R * is independently selected from the group con
sisting of C1-12 alkyl and C2-12 alkenyl;
each Y is independently a C3-6 carbocycle ;
each X is independently selected from the group consist
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
5
10
m is selected from 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , and 13 ,
or salts or isomers thereof.
15
In some embodiments, another subset of compounds of
Formula (I) includes those in which
R , is selected from the group consisting of C5-30 alkyl,
ing of F , C1, Br, and I; and
R2 and Rz are independently selected from the group
m is selected from 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , and 13 ,
20 consisting of H , C1-14 alkyl, C2-14 alkenyl, -R * YR " ,
or salts or isomers thereof.
-YR " , and - R * OR ", or R2 and Rz, together with the atom
In some embodiments , another subset of compounds of to which they are attached , form a heterocycle or carbocycle;
R4 is selected from the group consisting of a C3-6 carbo
Formula (I) includes those in which
R , is selected from the group consisting of C5-30 alkyl, cycle , (CH2),, ( CH2), CHQR ,
C5-20 alkenyl, R * YR " , —YR " , and —R "M'R ';
CHQR , CQ (R )2, and unsubstituted C1-6 alkyl, where Q
is selected from a C3-6 carbocycle , a 5- to 14 -membered
consisting of H , C1-14 alkyl, C2-14 alkenyl, R * YR" , heteroaryl having one or more heteroatoms selected from N ,
YR " , and -R * OR ", or R2 and Rz, together with the atom
O , and S , OR ,
to which they are attached , form a heterocycle or carbocycle ;
O (CH2), N (R )2
C ( O )OR , OC (O ) R , CX3,
R4 is selected from the group consisting of a C3-6 carbo- 30 CX H , CXH2, CN , C (O ) N (R ) 2,,, -N (R ) C ( O )R ,
cycle, (CH ), Q , (CH ), CHOR ,
N (R )S (O )2R , N (R ) C (O ) N (R )2, -N (R ) C (S )N (R )2,
CHQR , CQ (R )2, and unsubstituted C1-6 alkyl ,where Q
CRN ( R )2C (O )OR , N (R )R3, O ( CH2)NOR , —N (R )C
is selected from a C3-6 carbocycle , a 5- to 14 -membered ENR ,) N (R )2, -NN (R ) C ( = CHR , )N (R ) 2, OC (O ) N ( R )2,
heterocycle having one or more heteroatoms selected from
-N (R ) C (O )OR ,
-N (OR ) C (O )R ,
N (OR ) S (O ), R ,
N , O , and S , OR ,
35 - N (OR )C (O )OR , -N (OR )C ( O ) N (R )2, -N (OR ) C ( S ) N
O ( CH2), N (R )2,
C (O )OR ,
OC (O )R , CX3, (R )2, —N (ORCNR
)
,)N (R )2, —N (OR )C ( = CHR , )N (R )
CX , H , CXH , CN , -C ( O )N (R )2, —N ( R )C (O )R , 2 , CONR ) R , -C(O )N (R )OR , and CONR , )N (R ) 2,
N (R )S (O )2R , N (R ) C (O )N ( R )2, N (R ) C (S ) N (R )2, and each n is independently selected from 1 , 2 , 3 , 4 , and 5 ;
each R , is independently selected from the group consist
CRN (R ), C ( O )OR , N (R ) R ,
O (CH ), OR ,
N (R CNR
)
,) N ( R ) ,
-N (R )C 40 ing of C1-3 alkyl, C2-3 alkenyl, and H ;
N ( R ) C (O )OR ,
each R is independently selected from the group consist
CHR, )N (R ) ,
OC (O )N (R ) .
-N (OR )C (O )R , -N (OR ) S ( O ) , R ,
N (OR ) C (O )OR , ing of C1-3 alkyl, C2-3 alkenyl, and H ;
-N (OR ) C ( O )N ( R )2, N (OR )C (S )N (R ) ,
N (OR )C
M and M ' are independently selected from C (O )O- ,
ENR , N (R )2 , -N (OR )CECHR , N (R ) 2, CONR , R ,
OC (O ) - , -CC (O )N ( R ') — , -N (R ') C (O ) - , -C (O )
C (S ) , -C(S )S— , -SC ( S ) , CH (OH ) — , -P (O )
C (O )N (R )OR , and C = NR ,)N (R )2, and each n is 45C
independently selected from 1, 2, 3, 4 , and 5 ; and when Q (OR ')O- , -S( O )2- , SS an aryl group, and a
is a 5- to 14 -membered heterocycle and (i) R4 is – ( CH2) Q heteroaryl group;
in which n is 1 or 2 , or ( ii ) R4 is (CH2), CHQR in which
R , is selected from the group consisting of C1-3 alkyl, C2-3
n is 1, or ( iii ) R4 is - CHQR , and CQ (R )2, then Q is either alkenyl, and H ;
a 5- to 14 -membered heteroaryl or 8- to 14 -membered 50 Rg is selected from the group consisting of C3-6 carbo
heterocycloalkyl;
cycle and heterocycle ;
each R? is independently selected from the group consist
R , is selected from the group consisting of H , CN , NO2,
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
C1-6 alkyl, OR , -S (O )2R , -S (O ) 2N (R )2, C2-6 alkenyl,
each Ro is independently selected from the group consist
C3-6 carbocycle and heterocycle ;
55
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
each R is independently selected from the group consist
M and M ' are independently selected from - C (O )O- , ing of C1-3 alkyl, C2-3 alkenyl, and H ;
OC (O )
C (O ) N ( R ') , N (R ') C (O ) , C (O ) ,
each R ' is independently selected from the group consist
C (S ) , C (S ) S , SC ( S )
CH (OH ) , —P(O ) ing of C1-18 alkyl, C2-18 alkenyl, R * YR ", —YR ", and H ;
(OR ')O- , -S (O )2
S - S— , an aryl group , and a
each R " is independently selected from the group con
heteroaryl group ;
60 sisting of C3-14 alkyl and C3-14 alkenyl;
R , is selected from the group consisting of C - 3 alkyl, C2.3
each R * is independently selected from the group con
alkenyl, and H ;
sisting of C1-12 alkyl and C2-12 alkenyl;
R , is selected from the group consisting of C3-6 carbo
each Y is independently a C2-6 carbocycle ;
cycle and heterocycle ;
each X is independently selected from the group consist
R , is selected from the group consisting of H , CN , NO2, 65 ing of F , C1, Br, and I; and
C1-6 alkyl, OR , -S (O ) R , -S(O ) 2N (R )2, C2-6 alkenyl,
m is selected from 5 , 6 , 7 , 8 , 9, 10 , 11 , 12 , and 13 ,
or salts or isomers thereof.
C3-6 carbocycle and heterocycle ;
C5-20 alkenyl, R * YR " , YR " , and —R" M'R ';
R2 and Rz are independently selected from the group
2
9
25
US 10,702,600 B1
106
105
In some embodiments, another subset of compounds of
Formula (I) includes those in which
R , is selected from the group consisting of C5-30 alkyl,
C5-20 alkenyl, R * YR " , -YR " , and —R" M'R ';
m is selected from 5 , 6 , 7, 8 , 9, 10 , 11, 12 , and 13 ,
or salts or isomers thereof.
In some embodiments, a subset of compounds of Formula
R2 and Rz are independently selected from the group 5 (1) includes those of Formula (IA ):
consisting of H , C2-14 alkyl, C2-14 alkenyl, = R * YR " ,
-YR " , and -R * OR " , or R , and Rz, together with the atom
to which they are attached , form a heterocycle or carbocycle ;
R4 is (CH ) , or (CH ), CHOR , where Q is -N ( R )
and n is selected from 3 , 4 , and
each R , is independently selected from the group consist
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
2,
H
10
(IA )
M1 -R
R2
R4
177
M
each Ro is independently selected from the group consist
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
R3
M and M ' are independently selected from C (O )O- , 15
OC (O ) - , C ( O )N (R ') — , -N (R ')C (O ) - , -C(O ) - ,
or a salt or isomer thereof, wherein 1 is selected from 1,
C (S )
C (S ) S
SC (S ) — CH (OH ) — , -P(O ) 2, 3 , 4 , and 5 ; m is selected from 5 , 6 , 7 , 8, and 9 ; M , is a
S — 5— , an aryl group , and a bond or M '; R4 is unsubstituted C1-3 alkyl, or (CH ) , in
(OR )O- , -S (O )2
heteroaryl group;
which Q is OH , - NHC (S ) N (R )2, - NHC ( O ) N (R )2 , - N (R )
R , is selected from the group consisting ofC1-3 alkyl,C2-3 20 C (O ) R , -N (R )S (O )2R , -N (R )Rg, -NHC (= NR , )N (R )2,
NHC ( = CHR , )N (R )2, OC (O ) N (R )2, -N (R ) C (O )OR ,
each R is independently selected from the group consist
heteroaryl or heterocycloalkyl; M and M ' are independently
ing of C1-3 alkyl , C2-3 alkenyl, and H ;
selected
each R ' is independently selected from the group consist from C (O )O-, -OC (O ) , C (O )N (R ') — , P (O )
ing of C1-18 alkyl, C2-18 alkenyl, R * YR " , —YR " , and H ; 25 (OR )O- , -S - S—
, an aryl group , and a heteroaryl group ;
each R " is independently selected from the group con and R2 and R3 areS independently
selected from the group
sisting of C3-14 alkyl and C3-14 alkenyl;
consisting
of
H
,
C1-14
alkyl
,
and
C2-14
alkenyl.
each R * is independently selected from the group con
In
some
embodiments
,
a
subset
of
compounds
of Formula
sisting of C1-12 alkyl and C1-12 alkenyl;
30 (1) includes those of Formula (II):
each Y is independently a C3-6 carbocycle ;
each X is independently selected from the group consist
alkenyl, and H ;
ing of F , C1, Br, and I; and
m is selected from 6 , 7, 8 , 9 , 10 , 11 , 12 , and
M1
or salts or isomers thereof.
In some embodiments , another subset of compounds of 35
Formula (I) includes those in which
R? is selected from the group consisting of C5-30 alkyl,
C5-20 alkenyl, -R * YR " , -YR " , and —R " M'R ';
( II)
R
R2
M
R2 and Rz are independently selected from the group
R3
and - R *OR " , or R2 and Rz, together with the atom to which
they are attached , form a heterocycle or carbocycle ;
or isomer thereof, wherein 1 is selected from 1 ,
R4 is selected from the group consisting of (CH2) Q , 2 , or3 , a4,salt
and
; M is a bond or M '; R4 is unsubstituted C1-3
(CH2), CHQR , CHQR , and CQ (R )2, where is alkyl, or (5CH2
) Q , in which n is 2 , 3 , or 4 , and Q is OH ,
45
-N (R )2, and n is selected from 1, 2 , 3 , 4, and 5 ;
(S )N (R )2, - NHC (O )N (R )2, - N (R )C (O )R , -N (R )
each R , is independently selected from the group consist S (NHC
O )2R , -N ( R )RE, -NHCNR ,) N ( R )2, NHC
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
ECHR
(R )2, OC (O ) N ( R )2, -N (R )C (O )OR , het
each Ro is independently selected from the group consist eroaryl ,or) Nheterocycloalkyl
; M and M ' are independently
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
selected
M and M ' are independently selected from C (0 ) 0 , 50
OC (O ) - , -CC ( O )N ( R ') , -N (R ') C (O ) - , -C(O ) , from -C (O )O- , OC (0 ) , C (O )N (R ') — , —P (O )
P ( O ) (OR ')O- , -SS- , an aryl group , and a heteroaryl group ;
C (S ) , C ( S )S— , SC ( S ) , _CH (OH -)
S — 5— , an aryl group , and a and R , and Rz are independently selected from the group
(OR ') 0 % , -S(O )2
consisting of H , C1-14 alkyl, and C2-14 alkenyl.
heteroaryl group:
R , is selected from the group consisting ofC1-3 alkyl, C2-3 55 In some embodiments, a subset of compounds of Formula
alkenyl, and H ;
(I) includes those of Formula (IIa ), (IIb ), (Ilc ), or ( Ile ):
each R is independently selected from the group consist
ing of C1-3 alkyl, C2-3 alkenyl, and H ;
(IIa )
each R ' is independently selected from the group consist
consisting of C - 14 alkyl, C2.14 alkenyl, R * YR" , -YR ", 40
9
ing of C1-18 alkyl, C2-18 alkenyl, -R * YR", —YR", and H ; 60
each R " is independently selected from the group con
sisting of C3-14 alkyl and C3-14 alkenyl;
each R * is independently selected from the group con
sisting of C1-12 alkyl and C1-12 alkenyl;
each Y is independently a C3-6 carbocycle ;
each X is independently selected from the group consist
ing of F , C1, Br, and I; and
65
R4
US 10,702,600 B1
107
108
-continued
-continued
(IIb )
(IIb )
5
R4
R4
10
( IIC )
15
(IIC )
or
R4
20
or
R4
(IId )
25
R4
30
(IId )
or a salt or isomer thereof, wherein R4 is as described
herein .
35
In some embodiments , a subsetof compounds of Formula
(1) includes those of Formula ( IId ):
R4
(IId ) 40
R
R"
or a salt or isomer thereof, wherein R4 is as described
HO
45
R5
herein .
R3,
RO
In some embodiments , a subset of compounds of Formula
R2
(I) includes those of Formula (IId ):
50
or a salt or isomer thereof, wherein n is 2 , 3 , or 4 ; and m ,
R ', R " , and R , through Ro are as described herein . For
example, each of R , and Rz may be independently selected
from the group consisting of C5-14 alkyl and C5-14 alkenyl.
In some embodiments , a subset of compounds ofFormula 55
(I) includes those of Formula (Ila ), (IIb ), (IIC ), or (Ile ):
(IId )
R
„R"
HO
R5
(IIa )
60
R3,
R6
R2
R4
or a salt or isomer thereof, wherein n is 2 , 3 , or 4 ; and m ,
65 R ', R " , and R , through Ro are as described herein . For
example, each of R2 and R , may be independently selected
from the group consisting of C5-14 alkyl and C5-14 alkenyl.
US 10,702,600 B1
110
109
In some embodiments , the compound of Formula (I) is
selected from the group consisting of:
( Compound 1)
HO
( Compound 2 )
HO
( Compound 3)
HO
( Compound 4 )
HO
( Compound 5)
HO
HO
w
( Compound 6 )
(Compound 7)
HO .
(Compound 8 )
US 10,702,600 B1
112
111
-continued
( Compound 9)
en
( Compound 10 )
HO
(Compound 11 )
HO
( Compound 12 )
Holland
( Compound 13 )
HO
( Compound 14 )
( Compound 15)
US 10,702,600 B1
113
114
-continued
( Compound 16 )
(Compound 17 )
an
(Compound 18 )
HO
( Compound 19 )
??.
( Compound 20 )
HO
(Compound 21)
HO
(Compound 22 )
OH
w
US 10,702,600 B1
116
115
-continued
(Compound 23)
HO
(Compound 24 )
HO
(Compound 25 )
HO
( Compound 26 )
HO
(Compound 27)
HO
(Compound 28 )
HO
( Compound 29 )
HO
US 10,702,600 B1
118
117
-continued
( Compound 30 )
HO
( Compound 31 )
HO
(Compound 32)
HO
(Compound 33)
HO
Compound 34 )
HO
( Compound 35 )
HO
(Compound 36 )
HO
US 10,702,600 B1
119
120
-continued
(Compound 37 )
( Compound 38 )
???
tua
yua
( Compound 39 )
( Compound 40 )
( Compound 41 )
????
quo
( Compound 42 )
H
S
( Compound 43)
HN .
US 10,702,600 B1
122
121
quo
H?N .
-continued
( Compound 44 )
( Compound 45 )
H2N
B
( Compound 46 )
NH2
N
( Compound 47)
HO
(Compound 48 )
HO
(Compound 49)
HO
( Compound 50 )
HO
van
20
US 10,702,600 B1
124
123
-continued
(Compound 51)
HO
( Compound 52 )
HO
(Compound 53 )
HO
(Compound 54 )
HO
( Compound 55 )
HO
(Compound 56 )
HO
( Compound 57 )
HO
US 10,702,600 B1
125
126
-continued
( Compound 58 )
HO
(Compound 59 )
HO
(Compound 60 )
HO
( Compound 61)
HO
In further embodiments , the compound of Formula (I) is 40
selected from the group consisting of:
(Compound 62 )
45
HO
( Compound 63)
50
55
HO
( Compound 64 )
60
HO
65
US 10,702,600 B1
127
128
In some embodiments , the compound of Formula (I) is
selected from the group consisting of:
( Compound 65)
?? .
(Compound 66 )
?? .
N
(Compound 67)
??.
(Compound 68 )
HO .
( Compound 69 )
?? ,
N
HO
(Compound 70 )
HO .
N
US 10,702,600 B1
130
129
-continued
(Compound 71)
HO .
( Compound 72 )
HO .
(Compound 73 )
HO .
( Compound 74 )
?? .
( Compound 75 )
HO .
( Compound 76 )
?? .
N
US 10,702,600 B1
132
131
-continued
( Compound 77 )
HO .
N
(Compound 78)
?? ,
N
( Compound 79 )
HO .
N
( Compound 80 )
HO .
N
(Compound 81)
?? .
N
HO .
( Compound 82 )
US 10,702,600 B1
134
133
-continued
(Compound 83 )
HO .
N
(Compound 84 )
HO .
N
(Compound 85 )
?? .
N
( Compound 86 )
HO .
N
(Compound 87)
HO
N
( Compound 88 )
HO .
N
US 10,702,600 B1
135
136
-continued
( Compound 89 )
HO .
( Compound 90 )
HO .
(Compound 91)
HO .
(Compound 92)
HO .
(Compound 93 )
HO .
N
(Compound 94 )
US 10,702,600 B1
137
138
-continued
( Compound 95)
Meo
(Compound 96 )
HO .
(Compound 97 )
?? .
(Compound 98 )
Me0
N
( Compound 99 )
HO .
N
US 10,702,600 B1
140
139
-continued
(Compound 100 )
HO .
N
oly wyni
( Compound 101 )
( Compound 102 )
( Compound 103)
Compound 104)
?? .
N
US 10,702,600 B1
142
141
-continued
( Compound 105 )
?? .
NH2
(Compound 106 )
OH
( Compound 107)
F
F
( Compound 108 )
( Compound 109 )
(Compound 110 )
N
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