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 1

US010898574B2

( 12 ) deUnited
States Patent
Fougerolles et al .

( 10 ) Patent No.: US 10,898,574 B2
(45 ) Date of Patent :
* Jan . 26 , 2021

( 54 ) DELIVERY AND FORMULATION OF

( 58 ) Field of Classification Search
None
See application file for complete search history .

ENGINEERED NUCLEIC ACIDS

( 71 ) Applicant: Moderna TX , Inc. , Cambridge, MA

References Cited
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( 56)

(US )

(72) Inventors : Antonin de Fougerolles , Waterloo

( BE ) ; Sayda M. Elbashir , Cambridge,
MA ( US )

5,034,506 A
5,426,180 A

5,489,677
5,512,439
5,591,722
5,637,459
5,639,873
5,641,400
5,789,578
5,808,039
5,989,911
6,022,715
6,022,737
6,248,268
6,303,378
6,423,492

( 73 ) Assignee : Moderna TX , Inc. , Cambridge , MA
( US )

( * ) Notice:

Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154 (b ) by 0 days.
This patent is subject to a terminal dis
claimer .

(21 ) Appl. No .: 15 /927,730
Mar. 21 , 2018
(22) Filed :
Prior Publication Data
( 65 )
US 2019/0060458 A1

6,511,832 B1
6,521,411 B2

7,691,569 B2
8,075,780 B2

8,093,367
8,664,194
8,680,069
8,691,750
8,710,200
8,716,465
8,802,438

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( 60 ) Continuation of application No. 15 / 379,284 , filed on

May 18 , 2013 , now abandoned, which is
continuation of application No. 13 /437,034 , filed on
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A61K 31/7088

( 2017.01)

( 2006.01 )

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A61K 47/22 ( 2013.01 ) ; A61K 47/28 ( 2013.01 ) ;
CO7K 14/535 (2013.01 ) ; C12N 9/644
(2013.01 ) ; C12N 15867 ( 2013.01 ) ; C12N
15/87 (2013.01 ) ; A61K 48/00 ( 2013.01 ) ; C12N
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Primary Examiner Robert M Kelly
( 74 ) Attorney, Agent, or Firm — Clark & Elbing LLP
( 57 )

ABSTRACT

Provided are formulations, compositions and methods for
delivering biological moieties such as modified nucleic acids
into cells to modulate protein expression. Such compositions
and methods include the delivery of biological moieties, and
are useful for production of proteins.
10 Claims , 20 Drawing Sheets
Specification includes a Sequence Listing .

US 10,898,574 B2
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* cited by examiner

U.S. Patent

Jan. 26 , 2021

Sheet 1 of 20

US 10,898,574 B2

98N12-5 ( TETA5 - LAP )
HO

OH

H

HON

OH

??

DLin - K- DMA ( 2,2-Dilinoleyl -4 - dimethylaminomethyl- ( 1,3 ) -dioxolane)

ge
DLin - KC2 - DMA

88
DLin - MC3.DMA
ocus

you
GoH21

OH

min

Cotar

???21

a

OHC10H21

OH

JOH

C10H21
PRIOR ART

U.S. Patent

Sheet 2 of 20

Jan. 26 , 2021

US 10,898,574 B2

FIG . 2

Only single cutters are shown in the map
BUSHI (3006

)

Asel ( 182 )

ASISI (385 )
*****

PN01:64818

haMSH optHs2

Apal (2072 )

txn terminator

Rpn txi terminator

Smal ( 1153 )

Hindill ( 1187)

ECORV (1728)
HincII ( 1710 ) Sphl ( 1614 ).
Noti ( 1599 )

Sac? ( 1370)

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 3 of 20

HEK293, 24- well , 250 ng
Modified RNA/ well

TH

untreaNPAted -001-1NPA-001-1aPNPA-002-1 NPA-003-1 NPA-005-1NPA-005-1aPNPA-006-1
FIG . 3B

HepG2 , 24 - well , 250 ng
Modified RNA /well
25,00

untreatNPAed -001-1NPA-001-1aPNPA-002-1NPA-002-1ap VIN-1-600deNPA-005.1NPA-005-1aPNPA-006-1NPA-006-12P

U.S. Patent

Jan. 26 , 2021

Sheet 4 of 20

US 10,898,574 B2

HEK293 , 24 - well , 250 ng
Modified RNA /well

untreated

WPA-002-1

NPA-003-1ap
NP4003-1

HepG2 , 24 - well , 250 ng
Modified RNA/ well

untreatNPAed -001-1NPA-001-1ap NPA-002-1apNPA-003-1

NPA-OOG-Iap
NPA-005-1
NPA-005-1aNPA-006-1

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 5 of 20

FIG . SA

HEK293, NPA-005, 24-well , n =4
2

1

1.
HEK293 , NPA - 003, 24-well , n =4

3

*****

2

1.5

1
0

control 250ng

U.S. Patent

Jan. 26 , 2021

Sheet 6 of 20

US 10,898,574 B2

HEK293, NPA -003, 24 -well, n = 4
8

6

0

NPA -003-250ng NPA -003-100ng NPA - 003-30ng

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 7 of 20

HEK293 , 96 - well , 60 ng Modified RNA/ well

untreated

NBA-003

?NPA01315
NPA-015

HEK293 , 62.5 ng/well
25000

MmChFerIy

intreated

NPA-015
NPA-006
NPA-013
NPA-008
NPA-003
NPA-007
NPA-010
NPA-012
NPA-009
NPA-005
NPA-001
NPA-014
NPA-002

U.S. Patent

Jan. 26 , 2021

Sheet 8 of 20

US 10,898,574 B2

HEK293,62.5ng / well

untreated

NPA-007NPA-012

NPA-003-2NPA-018NPA-010NPA-015NPA-019

HepG2 , 62.5 ng / well

TTTT

untreated

NPA-009
NPANPA
-002-002-2
NPA-013
NPA-014
NPA-001
NPA-008
NPA-012
NPA-005
NPANPA
-003-003-2
NPA-007
NPA-006
NPA-010
NPA-015

U.S. Patent

Jan. 26 , 2021

Sheet 9 of 20

US 10,898,574 B2

HepG2 , 62.5ng / well
35000

untreatecdontrol

ill

NPA-007NPA-012NPA-017NPA-003-2NPA-018NPA-010NPA-015NPA-019

U.S. Patent

Jan. 26 , 2021

Sheet 10 of 20

US 10,898,574 B2

Human EPO Protein in Mouse Serum

( I.M. Injection Route)

10 0

Pgimi

Epo -Lipoplex
Luc-Lipoplex

Epo - Saline

Epo-LipoplexLuc-LipoplexEpo-SalineLucSaline F.Buf er
Treatment Groups

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 11 of 20

FIG . 7B

Human EPO Protein in Mouse Serum

( S.C. Injection Route )
Luc -Lipoplex

Epo - Saline

ml
/
Pg

Epo-LipoplexLuc-LipoplexEpo-SalineLuc-Saline FBuffer
Treatment Groups

U.S. Patent

Jan. 26 , 2021

Sheet 12 of 20

US 10,898,574 B2

In vivo Biophotoic Imaging
( I.M. Injection- Left)
5ug

Biolumnesc

Leit, 2 hrs
Left 8 hrs

Left 24 hrs

Photon
(
sec
/
)
5.0x105

Aqa

luc
Naked-LUCLipoplex
Lipoplex-Cytokine

FormulationBuffer

Treatment

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 13 of 20

FIG . 8B

In vivo Biophotoic Imaging
( I.M. Injection - Right)
50ug

Biolumnesc

101

1x1006

Photon
(
sec
/
)

luc
Naked-LUCLipoplex
Lipoplex-Cytokine

Right 2 hrs

Right, 8 hrs

Right, 24 hrs

FormulationBuffer

Treatment

U.S. Patent

Jan. 26 , 2021

Sheet 14 of 20

US 10,898,574 B2

In vivo Biophotoic Imaging
( S.C.Injection Route)
50ug

Biolumnesc

001

b)(esjuoj ud

dhe
Naked-LucLipoplexLipoplex
-luc -G-CSF

FormulationBuffer

Treatment

U.S. Patent

Jan. 26 , 2021

Sheet 15 of 20

US 10,898,574 B2

In vivo Biophotoic Imaging
(1.V. Injection Route)
50ug
Spleen

Blouminesc

T
)
sec
/
Photon
(

-Naked-LucLipoplex-lucLipoplex-G-CSF

FormulatioBnuf er

Treatment

U.S. Patent

Jan. 26 , 2021

Sheet 16 of 20

Human EPO Protein

( IM Injection Route)

US 10,898,574 B2

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 17 of 20

Avers

LUCBuf er

unsp

. Averg
N1
CSF
G

.' .

Avers
CSF
G

TGreaotmupns

Avers

G-CSFinSHeurmuamn (1.M.,I.V.S.C.IRoute)njection

5
20
4
3
2
1

S.C.
Avers
N1
CSF
G

Averg

120.0 10 .0 80.0 7W/8d40.0 20.0

U.S. Patent

Jan. 26 , 2021

Sheet 18 of 20

US 10,898,574 B2

Human G - CSF Protein in Mouse Serum

( 1.M. Injection Route)

FBuffer
Luc
Saline
Luc
Lipoplex
G2
-CSF
-Gen2-Saline
G--Lipoplex
CSFG
-Gent
Saline
-CSF--Gen
-Lipoplex
G-CSF-Gent
Treatment Groups

U.S. Patent

Jan. 26 , 2021

Sheet 19 of 20

US 10,898,574 B2

FIG . 11B

Human G - CSF Protein in Mouse Serum
( S.C. Injection Route )

mL
/
pg

Anagrams

FBuffer
Luc
saline
Luc
lipoplex
-Gen2-saline
GCSF-saline
GenGCSF
2-lipoplex
-GentGCSF-Gen1-GCSFlipoplex

U.S. Patent

Jan. 26 , 2021

US 10,898,574 B2

Sheet 20 of 20

Human EPO Protein in Mouse Serum
( IM Injection Route )

FBuffer
+30Epo
%.RNAIMAX
+10%Epo
RNAIMAX
+50%RNAIMAX
Epo#10%LucRNAIMAX
+30%Luc
RNA?MAX
+50%LucRNAIMAX
Treatment Groups

US 10,898,574 B2
1

2

DELIVERY AND FORMULATION OF
ENGINEERED NUCLEIC ACIDS

modulation of intracellular translation and processing of
nucleic acids encoding polypeptides and therefore optimiz
ing protein expression from the delivered modalities .
The present invention addresses this need by delivering

This application is a continuation of U.S. patent application Ser. No. 15 /379,284 , filed Dec. 14 , 2016 , entitled 5 pharmaceutical compositions which can contain modified

Delivery and Formulation of Engineered Nucleic Acids , nucleic acids such as modified mRNA (mmRNA ) and may
which is a continuation of U.S. patent application Ser. No. further include formulations to avoid the problems in the art.
14 /337,513 , filed Jul . 22 , 2014 , entitled Delivery and For
SUMMARY OF THE INVENTION
mulation of Engineered Nucleic Acids , which is a continu
ation of U.S. patent application Ser. No. 13 / 897,362 , filed 10 Described herein are compositions and methods for deliv
May 18 , 2013 , entitled Modified Polynucleotides for the ery of biological moieties , such as modified nucleic acids ,
Production of Factor IX , which is a continuation of U.S.
engineered messenger RNA and isolated polynucleotides
patent application Ser. No. 13 /437,034 , filed Apr. 2 , 2012 , into
in order to modulate protein expression .
now issued U.S. Pat. No. 8,710,200 , entitled Delivery and 15 Ancells
isolated polynucleotide may comprise a sequence such
Formulation of Engineered Nucleic Acids which claims
as , but not limited to , SEQ ID NO : 4 , 7 , 8 and 12. The
priority to U.S. Provisional Patent Application No. 61/470 , polynucleotide
further comprise a 5'Cap1 structure and
451 , filed Mar. 31 , 2011 , entitled Delivery and Formulation a polyA tail ofmay
approximately 160 nucleotides in length .
of Engineered Nucleic Acids the contents, the contents of Further, the isolated
polynucleotide may be formulated in a
each is incorporated by reference in its entirety .
20 pharmaceutical composition. A polypeptide of interest may
be produced in a cell , tissue or bodily fluid in a subject in
REFERENCE TO SEQUENCE LISTING
need thereof by administering to the subject a pharmaceu
The present application is being filed along with a tical composition comprising a polynucleotide. The poly
Sequence Listing in electronic format. The Sequence Listing nucleotide may comprise a sequence selected from the group
is provided as a file entitled MO03USSQLST.txt created on 25 consisting of SEQ ID NO : 4 , 7 , 8 and 12. The polynucleotide
May 17 , 2013 which is 17,058 bytes in size . The information may further comprise a 5'Cap1 structure and a poly -A tail of
in electronic format of the sequence listing is incorporated approximately 160 nucleotides in length .
The pharmaceutical composition may be formulated
herein by reference in its entirety .
where the formulation may be selected from , but is not
30 limited to , saline or a lipid formulation . The pharmaceutical
FIELD OF THE INVENTION
composition may be administered by any route of adminis
The invention relates to delivery methods. These methods tration such as , but not limited to , intravenous, intramuscu
are specifically useful in therapeutic delivery of modified lar, subcutaneous, and local administration. The lipid for
mulation may be selected from , but is not limited to , such as ,
nucleic acids such as modified mRNA (mmRNA ).
35 but not limited to , liposomes , lipoplexes , copolymers such
BACKGROUND OF THE INVENTION
as PLGA and lipid nanoparticles
The pharmaceutical composition may be administered at
There are multiple problems with prior methodologies of a total dose of about 0.1 mg /kg to about 40 mg /kg. The total
delivering pharmaceutical compositions in order to achieve dose may be administered by multiple administrations. The
effective protein expression both for therapeutics and bio- 40 administration and / or the multiple administration may occur
processing applications. For example, introduced DNA can on a schedule such as , but not limited to , three time a day,
integrate into host cell genomic DNA at some frequency, twice a day, once a day, every other day, every third day,
resulting in alterations and /or damage to the host cell weekly, biweekly, every three weeks, every four weekly , and
genomic DNA . Alternatively, the heterologous deoxyribo- monthly
nucleic acid (DNA ) introduced into a cell can be inherited by 45 The modified polypeptide may include a polynucleotide
daughter cells (whether or not the heterologous DNA has modification such as , but not limited to , a nucleoside modi
integrated into the chromosome) or by offspring.
fication . The nucleoside modification may include , but is not
In addition, there are multiple steps which must occur limited to , pyridin - 4 -one ribonucleoside, 5 - aza -uridine ,
after delivery but before the encoded protein is made which 2 - thio - 5 -aza -uridine, 2 - thiouridine, 4 - thio -pseudouridine,
can effect protein expression. Once inside the cell, DNA 50 2 -thio -pseudouridine, 5 -hydroxyuridine, 3 -methyluridine ,
must be transported into the nucleus where it is transcribed 5 - carboxymethyl -uridine, 1 -carboxymethyl-pseudouridine,
into RNA . The RNA transcribed from DNA must then enter 5 -propynyl -uridine, 1 -propynyl -pseudouridine, 5-taurinom
the cytoplasm where it is translated into protein . Not only do ethyluridine, 1 - taurinomethyl -pseudouridine, 5- taurinom
the multiple processing steps from administered DNA to ethyl-2 -thio - uridine,
1 - taurinomethyl - 4 - thio - uridine,
protein create lag times before the generation of the func- 55 5 -methyl-uridine, 1 -methyl-pseudouridine, 4 - thio -1 -methyl
tional protein , each step represents an opportunity for error pseudouridine, 2 -thio -1 -methyl-pseudouridine, 1 -methyl- 1
and damage to the cell . Further, it is known to be difficult to deaza -pseudouridine, 2 -thio - 1-methyl- 1-deaza-pseudouri
obtain DNA expression in cells as frequently DNA enters a dine, dihydrouridine, dihydropseudouridine, 2 -thio
2 - thio -dihydropseudouridine,
cell but is not expressed or not expressed at reasonable rates dihydrouridine,
or concentrations. This can be a particular problem when 60 2 -methoxyuridine, 2 -methoxy - 4 -thio -uridine, 4 -methoxy
DNA is introduced into primary cells or modified cell lines . pseudouridine, 4 -methoxy - 2 -thio -pseudouridine, 5 -aza -cyti
Assuming the proper management of the foregoing, effec- dine , pseudoisocytidine, 3 -methyl -cytidine, N4 -acetylcyti
tive delivery and achievement of therapeutically relevant dine ,
5 - formylcytidine,
N4 -methylcytidine,
levels of proteins for a time sufficient to product clinical 5 -hydroxymethylcytidine, 1 -methyl -pseudoisocytidine, pyr
65 rolo - cytidine, pyrrolo -pseudoisocytidine, 2 -thio -cytidine,
outcomes remains a significant hurdle .
Consequently, there is a need in the art for the delivery of 2 -thio -5 -methyl-cytidine, 4 -thio -pseudoisocytidine, 4 -thio
biological modalities to address pitfalls surrounding the 1 -methyl -pseudoisocytidine, 4 -thio - 1 -methyl-1 -deaza -pseu

US 10,898,574 B2
3

4

doisocytidine, 1 -methyl- 1 -deaza -pseudoisocytidine, zebularine , 5 - aza -zebularine, 5 -methyl-zebularine, 5 -aza- 2-thio-

after purification ) that contain mCherry mmRNA . FIG . 4A
shows the screening results in HEK293 cells and FIG . 4B
zebularine, 2 - thio -zebularine, 2 -methoxy -cytidine, shows the screening results in HepG2 cells .
FIGS . 5A , 5B , and 5C are histograms showing in vitro
2 -methoxy - 5 -methyl -cytidine, 4 -methoxy -pseudoisocyti
dine, 4 -methoxy -1-methyl-pseudoisocytidine, 2-aminopu- 5 screening results for nanoparticle formulations of DLin
KC2 -DMA and 98N12-15 before and after purification. FIG .
rine , 2 , 6 -diaminopurine, 7 -deaza -adenine, 7 -deaza -8 -aza 5A
shows the screening results of 98N15-2 in HEK293 cells ,
adenine,
7 -deaza - 2 -aminopurine,
7 -deaza - 8 - aza - 2
FIGS . 5B and 5C shows the screening results of
aminopurine, 7 -deaza - 2,6 -diaminopurine, 7 - deaza - 8 - aza - 2 , and
DLin
-KC2 -DMA in HEK293 cells .
6 - diaminopurine, 1 -methyladenosine, N6 -methyladenosine,
FIGS
. 6A , 6B , 6C , and 6D are histograms showing in
N6 - isopentenyladenosine, N6-(cis - hydroxyisopentenyl )ad- 10 vitro screening
for nanoparticle formulations of
enosine , 2 -methylthio -N6- (cis -hydroxyisopentenyl) adenos DLin - DMA , DLinresults
K
DMA
, DLin -KC2 -DMA, 98N12-5 ,
ine , N6 - glycinylcarbamoyladenosine, N6 - threonylcarbam C12-200 and DLin -MC3 - DMA
that contain mCherry
oyladenosine, 2 -methylthio -N6 -threonylcarbamoylade mmRNA . FIG . 6A shows the mean fluorescence
intensity of
nosine , N6 , N6 -dimethyladenosine, 7 -methyladenine, mCherry in HEK293 cells containing 60 ng of modified
2 -methylthio - adenine, and 2 -methoxy -adenine, inosine, 15 mCherry mRNA per well . FIGS . 6B and 6C show the mean
1 -methyl- inosine, wyosine, wybutosine, 7 -deaza - guanosine, fluorescence intensity of mCherry in HEK293 cells which
7 -deaza - 8 -aza - guanosine, 6 - thio - guanosine, 6 - thio - 7 -deaza- contained nanoparticles formulations having a concentration
guanosine, 6 - thio -7 -deaza - 8 -aza - guanosine, 7 -methyl- of 62.5 ng /well of modified mCherry mRNA . FIGS . 6D and
guanosine, 6 -thio -7 -methyl -guanosine, 7 -methylinosine, 6E show the mean fluorescence intensity of mCherry in
6 -methoxy -guanosine, 1 -methylguanosine, N2 -methyl- 20 HepG2 cells which contained nanoparticle formulations
guanosine, N2 ,N2 -dimethylguanosine, 8 -oxo - guanosine,
7 -methyl - 8 -oxo - guanosine, 1 -methyl-6 - thio - guanosine,

having a concentration of 62.5 ng /well of modified mCherry
mRNA .
N2 -methyl - 6 -thio - guanosine, and N2,N2-dimethyl-6 -thioFIGS . 7A and 7B are histograms showing in vivo screen
guanosine, and combinations thereof.
ing results of human erythropoietin in serum after the
An increase in the level of a polypeptide of interest can be 25 administration of modified human erythropoietin mmRNA
observed in tissue such as , but not limited to , the liver, or luciferase mmRNA in mice . FIG . 7A shows the concen
spleen , kidney, lung , heart, peri - renal adipose tissue , thymus tration in pg/ml of human erythropoietin after intramuscular
and muscle and / or in a bodily fluid such as , but not limited administration . FIG . 7B shows the concentration in pg/ml of
to , peripheral blood, serum , plasma , ascites, urine, cerebro- human erythropoietin after subcutaneous administration .
spinal fluid (CSF ) , sputum , saliva , bone marrow , synovial 30 FIGS . 8A , 8B , 8C , and 8D are histograms of in vivo

fluid , aqueous humor, amniotic fluid , cerumen , breast milk ,
broncheoalveolar lavage fluid , semen, prostatic fluid, cowper's fluid or pre - ejaculatory fluid , sweat , fecal matter, hair,
tears , cyst fluid, pleural and peritoneal fluid , pericardial

screening results from biophotoic imaging. FIG . 8A is a
histogram of bioluminescence (photon / sec) from the intra
muscular injection of 5 ug in the left hind leg . FIG . 8B is a
histogram of bioluminescence from the intramuscular injec

fluid , lymph, chyme, chyle , bile , interstitial fluid , menses , 35 tion of 50 ug in the right hind leg . FIG . 8C is a histogram
pus , sebum , vomit , vaginal secretions, mucosal secretion, showing in vivo screening results from biophotoic imaging
stool water, pancreatic juice , lavage fluids from sinus cavi- after a subcutaneous injection of 50 ug . FIG . 8D is a

ties , bronchopulmonary aspirates , blastocyl cavity fluid, and

histogram showing in vivo screening results from biopho

umbilical cord blood . The increased level can be observed in

toic imaging after a administration of 50 ug intravenously.

the present invention . Shown are the structures for 98N12-5
( TETA5 -LAP ), DLin - DMA, DLin- K - DMA ( 2,2 -Dilinoleyl4 -dimethylaminomethyl- [ 1,3 ) -dioxolane ),
DLin -KC2

human G - CSF in serum after the administration of modified
G -CSF subcutaneously.

the tissue and /or bodily fluid of the subject within 2 , 8 and / or 40 FIG . 9 is a histogram showing in vivo screening results
24 hours after administration . Further, the increased level for modified human G - CSF mmRNA administered intra
can be determined from the level of a modified polypeptide muscularly, subcutaneously or intravenously in mice .
in an exosome .
FIG . 10 is a histogram showing in vivo screening results
The details of various embodiments of the invention are for modified G - CSF administered intramuscularly, subcuta
set forth in the description below. Other features, objects , 45 neously or intravenously.
and advantages of the invention will be apparent from the
FIGS . 11A and 11B are histograms showing in vivo
description and the drawings, and from the claims .
screening results of modified human G - CSF mmRNA
administered intramuscularly or subcutaneously in mice .
BRIEF DESCRIPTION OF THE FIGURES
FIG . 11A shows the concentration in pg /ml of human G - CSF
50 in serum after the administration of modified G - CSF intra
FIG . 1 illustrates lipid structures in the prior art useful in muscularly. FIG . 11B shows the concentration in pg /ml of
DMA , DLin -MC3 - DMA and C12-200 .

FIG . 12 is a histogram showing in vivo screening results
55 of human erythropoietin in serum after the administration of
modified human erythropoietin mmRNA or luciferase
mmRNA administered intramuscularly in mice .
Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as those com
60 monly understood to one of ordinary skill in the art to which
this invention pertains.

FIG . 2 is a representative plasmid useful in the IVT
reactions taught herein . The plasmid contains Insert 64818 ,
designed by the instant inventors.
FIGS . 3A and 3B are histograms showing in vitro screening results for nanoparticle formulations of DLin-KC2DMA and 98N12-15 (before and after purification ) that
contain mCherry mmRNA . FIG . 3A shows the screening
DETAILED DESCRIPTION
results in HEK293 cells and FIG . 3B shows the screening
results in HepG2 cells .
FIGS . 4A and 4B are histograms showing in vitro screen- 65 Described herein are compositions and methods for the
ing results for mean fluorescence intensity for nanoparticle delivery of modified mRNA molecules in order to modulate
formulations of DLin -KC2 -DMA and 98N12-15 ( before and protein expression .

US 10,898,574 B2
5

6

As described herein and as in copending, co - owned
applications International Application PCT /US2011 / 046861
filed Aug. 5 , 2011 and PCT /US2011 / 054636 filed Oct. 3 ,
2011 , the contents of which are incorporated by reference

500 , 600 , 700 , 800 , 900 , 1,000 , 1,100 , 1,200 , 1,300 , 1,400 ,
1,500 , 1,600 , 1,700 , 1,800 , 1,900 , 2,000 , 2,500 , and 3,000 ,
4,000 , 5,000 , 6,000 , 7,000 , 8,000 , 9,000 , 10,000 , 20,000 ,
30,000 , 40,000 , 50,000 , 60,000 , 70,000 , 80,000 , 90,000 or
herein in their entirety, these modified nucleic acid mol- 5 up to and including 100,000 nucleotides ).
ecules are capable of reducing the innate immune activity of
In some embodiments , the modified mRNA of the present
a population of cells into which they are introduced, thus invention includes from about 30 to about 100,000 nucleo
increasing the efficiency of protein production in that cell tides (e.g. , from 30 to 50 , from 30 to 100 , from 30 to 250 ,
population.
from 30 to 500 , from 30 to 1,000 , from 30 to 1,500 , from 30
10 to 3,000 , from 30 to 5,000 , from 30 to 7,000 , from 30 to
Modified mRNAs (mmRNAs)
This invention provides nucleic acids , including RNAs, 10,000 , from 30 to 25,000 , from 30 to 50,000 , from 30 to
specifically mRNAs, that encode at least one polypeptide 70,000 , from 100 to 250 , from 100 to 500 , from 100 to
and contain one or more modified nucleosides ( termed 1,000 , from 100 to 1,500 , from 100 to 3,000 , from 100 to
“ modified nucleic acids” or “ modified nucleic acid mol- 5,000 , from 100 to 7,000 , from 100 to 10,000 , from 100 to
ecules " or " engineered nucleic acids” ), which have useful 15 25,000 , from 100 to 50,000 , from 100 to 70,000 , from 100

properties including the lack of a substantial induction of the to 100,000 , from 500 to 1,000 , from 500 to 1,500 , from 500
innate immune response of a cell into which the mRNA is to 2,000 , from 500 to 3,000 , from 500 to 5,000 , from 500 to
introduced . Because these mmRNAs enhance the efficiency 7,000 , from 500 to 10,000 , from 500 to 25,000 , from 500 to
of protein production, intracellular retention of nucleic 50,000 , from 500 to 70,000 , from 500 to 100,000 , from
acids , and viability of contacted cells , as well as possess 20 1,000 to 1,500 , from 1,000 to 2,000 , from 1,000 to 3,000 ,
reduced immunogenicity, these nucleic acids having these from 1,000 to 5,000 , from 1,000 to 7,000 , from 1,000 to
properties are termed " enhanced ” nucleic acids or modified 10,000 , from 1,000 to 25,000 , from 1,000 to 50,000 , from
RNAs herein .
1,000 to 70,000 , from 1,000 to 100,000 , from 1,500 to 3,000 ,
The term “ nucleic acid ,” in its broadest sense , includes from 1,500 to 5,000 , from 1,500 to 7,000 , from 1,500 to
any compound and / or substance that comprise a polymer of 25 10,000 , from 1,500 to 25,000 , from 1,500 to 50,000 , from
nucleotides linked via a phosphodiester bond . These poly- 1,500 to 70,000 , from 1,500 to 100,000 , from 2,000 to 3,000 ,
mers are often referred to as oligonucleotides.
Exemplary nucleic acids include ribonucleic acids

from 2,000 to 5,000 , from 2,000 to 7,000 , from 2,000 to
10,000 , from 2,000 to 25,000 , from 2,000 to 50,000 , from

(RNAs), deoxyribonucleic acids ( DNAs ), threose nucleic
acids ( TNAs), glycol nucleic acids (GNAs ), peptide nucleic 30
acids ( PNAs), locked nucleic acids ( LNAs) or hybrids
thereof. They may also include RNAi - inducing agents ,
RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs,
ribozymes , catalytic DNA , RNA, RNAs that induce triple
helix formation , aptamers, vectors , etc.
35
In preferred embodiments, the nucleic acid is one or more
modified messenger RNAs (mmRNAs ). As described
herein , in some embodiments the mmRNAs of the invention
do not substantially induce an innate immune response of a
cell into which the mRNA is introduced .
40
The mmRNA of the present invention may encode one or
more polypeptides . Generally the polypeptides of interest
are those which are naturally occurring in the mammalian
genome.
According to the present invention, the shortest length of 45
a modified mRNA , herein “ mmRNA , ” of the present disclosure can be the length of an mRNA sequence that may be
sufficient to encode for a dipeptide. In another embodiment,
the length of the mRNA sequence may be sufficient to
encode for a tripeptide. In another embodiment, the length of 50
an mRNA sequence may be sufficient to encode for a
tetrapeptide. In another embodiment, the length of an
mRNA sequence may be sufficient to encode for a pentapeptide. In another embodiment, the length of an mRNA
sequence may be sufficient to encode for a hexapeptide. In 55
another embodiment, the length of an mRNA sequence may
be sufficient to encode for a heptapeptide. In another
embodiment, the length of an mRNA sequence may be
sufficient to encode for an octapeptide. In another embodiment, the length of an mRNA sequence may be sufficient to 60
encode for a nonapeptide. In another embodiment, the length

2,000 to 70,000 , and from 2,000 to 100,000) .
Polypeptide Variants
The mmRNA of the present invention may encode variant
polypeptides, which have a certain identity with a reference
polypeptide sequence, for example a wild type mRNA . The
term “ identity ” as known in the art, refers to a relationship
between the sequences of two or more peptides , as deter
mined by comparing the sequences . In the art, “ identity ”
also means the degree of sequence relatedness between
peptides, as determined by the number of matches between
strings of two or more amino 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 com
puter program ( i.e. , “ algorithms” ). Identity of related pep
tides can be readily calculated by known methods. Such
methods include , but are not limited to , those described in
Computational Molecular Biology , Lesk , A. M. , ed . , Oxford
University Press, New York , 1988 ; Biocomputing: Informat
ics and Genome Projects, Smith, D. W., ed . , Academic Press ,
New York , 1993 ; Computer Analysis of Sequence Data , Part
1 , Griffin , A. M. , and Griffin , H. G. , eds . , Humana Press,
New Jersey, 1994 ; Sequence Analysis in Molecular Biology,
von Heinje , G. , Academic Press , 1987 ; Sequence Analysis
Primer, Gribskov , M. and Devereux, J. , eds . , M. Stockton
Press, New York , 1991 ; and Carillo et al . , SIAM J. Applied
Math . 48 , 1073 ( 1988 ) .
In some embodiments, the polypeptide variant has the
same or a similar activity as the reference polypeptide.
Alternatively, the variant has an altered activity ( e.g. ,
increased or decreased ) relative to a reference polypeptide.
Generally, variants of a particular polynucleotide or poly
peptide of the invention will have at least about 40 % , 45 % ,

decapeptide.

92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 99 % or more

of an mRNA sequence may be sufficient to encode for a

Generally, the length of a modified mRNA of the present
at least or greater than about 35 , 40 , 45 , 50 , 55 , 60 , 70 , 80 ,

50% , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90% , 91 % ,

sequence identity to that particular reference polynucleotide
grams and parameters described herein and known to those

invention is greater than about 30 nucleotides in length ( e.g. , 65 or polypeptide as determined by sequence alignment pro
90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 ,

skilled in the art.

US 10,898,574 B2
7

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 this invention .

8

approach, Oxford [ Oxfordshire ), Washington , D.C .: IRL

Press, 1984 ; and Herdewijn , P. ( ed .) Oligonucleotide syn
thesis : methods and applications, Methods in Molecular

For example, provided herein is any protein fragment of a Biology, v. 288 ( Clifton , N.J. ) Totowa, N.J .: Humana Press ,
reference protein (meaning a polypeptide sequence at least 5 2005 ; both of which are incorporated herein by reference ).
one amino acid residue shorter than a reference polypeptide
sequence but otherwise identical) 10 , 20 , 30 , 40 , 50 , 60 , 70 ,
80 , 90 , 100 or greater than 100 amino acids in length In

The modified nucleosides and nucleotides used in the
synthesis of modified RNAs disclosed herein can be pre
pared from readily available starting materials using the

which are about 40 % , about 50 % , about 60 % , about 70 % ,
about 80 % , about 90 % , about 95 % , or about 100 % identical

reaction temperatures, times , mole ratios of reactants , sol
vents , pressures , etc.) are given ; other process conditions can
also be used unless otherwise stated . Optimum reaction
conditions may vary with the particular reactants or solvent

The modified nucleic acids and mmRNA may encode a

et al . Nucleic Acids Research 22 ( 1 ) : 72-78 , 1994 ; Fukuhara

way to permit the expression of the complex when introduced into the cell . As used herein , “ charged protein ” refers
to a protein that carries a positive , negative or overall neutral

the entire length of the molecule . Different nucleotide modi
fications and / or backbone structures may exist at various
positions in the nucleic acid . One of ordinary skill in the art

another example, any protein that includes a stretch of about following general methods and procedures . It is understood
20 , about 30 , about 40 , about 50 , or about 100 amino acids 10 that where typical or preferred process conditions (i.e. ,

to any of the sequences described herein can be utilized in
accordance with the invention . In certain embodiments, a
protein sequence to be utilized in accordance with the 15 used, but such conditions can be determined by one skilled
invention includes 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or more mutations in the art by routine optimization procedures.
as shown in any of the sequences provided or referenced
The manufacturing process herein can be monitored
herein .
according to any suitable method known in the art. For
Targeting Moieties
example , product formation can be monitored by spectro
In
embodiments
of
the
invention
,
mmRNAs
are
provided
20
scopic means, such as nuclear magnetic resonance spectros
to express a protein -binding partner or a receptor on the copy (e.g. , ' H or 13C ) infrared spectroscopy, spectropho
surface of the cell , which functions to target the cell to a tometry (e.g. , UV - visible ), or mass spectrometry , or by
specific tissue space or to interact with a specific moiety, chromatography such as high performance liquid chroma
either in vivo or in vitro . Suitable protein -binding partners tography ( HPLC ) or thin layer chromatography.
include antibodies and functional fragments thereof, scaffold 25 Modification of mRNAs
proteins, or peptides .
Provided are mmRNAs containing a translatable region
Cell Penetrating Peptides
and one , two , or more than two different modifications.
The mmRNAs disclosed herein may encode a cell -penIn some embodiments, the chemical modifications can be
etrating polypeptide. As used herein , “ cell- penetrating poly- located on the nucleobase of the nucleotide .
peptide ” refers to a polypeptide which may facilitate the 30 In some embodiments, the chemical modifications can be
cellular uptake of molecules . It is known in the art that located on the sugar moiety of the nucleotide .
“ CPP ” refers to cell-penetration polypeptides and cell-penIn some embodiments, the chemical modifications can be
etrating peptides . When used herein , it will be clarified as to located on the phosphate backbone of the nucleotide .
which of either cell-penetrating polypeptides or cell -penPreparation of modified nucleosides and nucleotides used
etrating peptides the abbreviation CPP refers to .
35 in the manufacture or synthesis of modified RNAs of the
A cell-penetrating polypeptide of the present invention present invention can involve the protection and deprotec
may contain one or more detectable labels . The polypeptides tion of various chemical groups. The need for protection and
may be partially labeled or completely labeled throughout. deprotection , and the selection of appropriate protecting
The mmRNA may encode the detectable label completely, groups can be readily determined by one skilled in the art.
partially or not at all . The cell-penetrating peptide may also 40 The chemistry of protecting groups can be found, for
include a signal sequence . As used herein , a “ signal example, in Greene, et al . , Protective Groups in Organic
sequence” refers to a sequence of amino acid residues bound Synthesis, 2d . Ed . , Wiley & Sons , 1991 , which is incorpo
at the amino terminus of a nascent protein during protein rated herein by reference in its entirety.
translation . The signal sequence may be used to signal the
Modified nucleosides and nucleotides can be prepared
secretion of the cell -penetrating polypeptide.
45 according to the synthetic methods described in Ogata et al .
Fusion Proteins
Journal of Organic Chemistry 74 : 2585-2588 , 2009 ; Purmal

fusion protein . The fusion protein may be created by oper- et al . Biochemistry 1 ( 4 ) : 563-568 , 1962 ; and Xu et al .
ably linking a charged protein to a therapeutic protein . As Tetrahedron 48 ( 9 ) : 1729-1740 , 1992 , each of which are
used herein , “ operably linked ” refers to the therapeutic 50 incorporated by reference in their entirety .
protein and the charged protein being connected in such a
Modified mRNAs need not be uniformly modified along

electrical charge. Preferably , the therapeutic protein may be 55 will appreciate that the nucleotide analogs or other modifi
covalently linked to the charged protein in the formation of cation (s) may be located at any position( s ) of a nucleic acid
the fusion protein . The ratio of surface charge to total or such that the function of the nucleic acid is not substantially
surface amino acids may be approximately 0.1 , 0.2 , 0.3 , 0.4 ,

decreased . A modification may also be a 5 ' or 3 ' terminal
modification . The nucleic acids may contain at a minimum
Synthesis of Modified mRNAs
60 one and at maximum 100 % modified nucleotides , or any
Nucleic acids for use in accordance with the invention intervening percentage , such as at least 50 % modified
may be prepared according to any available technique nucleotides , at least 80 % modified nucleotides, or at least
including , but not limited to chemical synthesis, enzymatic 90% modified nucleotides .
synthesis, which is generally termed in vitro transcription ,
For example, the mmRNAs may contain a modified
enzymatic or chemical cleavage of a longer precursor, etc. 65 pyrimidine such as uracil or cytosine . In some embodiments,
Methods of synthesizing RNAs are known in the art ( see , at least 5 % , at least 10 % , at least 25 % , at least 50% , at least
e.g. , Gait , M. J. (ed . ) Oligonucleotide synthesis: a practical 80% , at least 90% or 100 % of the uracil in the nucleic acid
0.5 , 0.6 , 0.7 , 0.8 or 0.9 .

US 10,898,574 B2
10

9
may be replaced with a modified uracil. The modified uracil
can be replaced by a compound having a single unique
structure , or can be replaced by a plurality of compounds

tidine , 5 '- 0-( 1 - Thiophosphate ) -Guanosine, 5-0-( 1 - Thio
phosphate ) -Uridine or 5 '-0-( 1 - Thiophosphate )-Pseudo
uridine .
Further examples of modified nucleotides and modified

having different structures ( e.g. , 2 , 3 , 4 or more unique
structures ). In some embodiments, at least 5 % , at least 10 % , 5
at least 25 % , at least 50 % , at least 80 % , at least 90 % or nucleotide combinations are provided below in Table 1 .
100% of the cytosine in the nucleic acid may be replaced
TABLE 1
with a modified cytosine . The modified cytosine can be Modified Nucleotides
Modified Nucleotide Combinations
replaced by a compound having a single unique structure , or 10
can be replaced by a plurality of compounds having different 6 -aza -cytidine
a -thio -cytidine/ 5 -iodo -uridine
2 - thio -cytidine
a - thio -cytidine /N1-methyl -pseudo -uridine
structures (e.g. , 2 , 3 , 4 or more unique structures ).
a - thio -cytidine
a - thio -cytidine / a -thio - uridine
In some embodiments, modified nucleosides include pyri Pseudo
- iso - cytidine
a - thio -cytidine/ 5 -methyl -uridine
din - 4 - one ribonucleoside, 5 -aza -uridine, 2 -thio - 5 - aza-uri 5 - aminoallyl
-uridine
a -thio -cytidine/pseudo -uridine
Pseudo -iso -cytidine / 5 -iodo - uridine
dine , 2 - thiouridine, 4 - thio -pseudouridine, 2- thio -pseudouri- 15 5 - iodo - uridine
dine, 5 -hydroxyuridine , 3 -methyluridine, 5 -carboxymethyl-

N1 -methyl-pseudouridine Pseudo - iso -cytidine /N1-methyl-pseudo

uridine,

5,6 -dihydrouridine

1 -carboxymethyl-pseudouridine,

5 -propynyl

uridine, 1 -propynyl -pseudouridine, 5 -taurinomethyluridine,
1 -taurinomethyl -pseudouridine, 5 - taurinomethyl- 2- thio -uri-

a -thio -uridine
4 -thio -uridine

dine , 1 - taurinomethyl - 4 - thio -uridine, 5 -methyl-uridine, 20

5 - hydroxy -uridine
Deoxy
-thymidine

1 -methyl-pseudouridine,

4 - thio - 1 -methyl -pseudouridine,

6 -aza -uridine

2 - thio - 1 -methyl-pseudouridine, 1 -methyl- 1-deaza -pseudou- Pseudo -uridine
ridine, 2 - thio - 1 -methyl - 1 -deaza - pseudouridine, dihydrouri Inosine
thio - guanosine
dine, dihydropseudouridine, 2 -thio -dihydrouridine, 2 -thio a8 --oxo
- guanosine
dihydropseudouridine, 2 -methoxyuridine, 2 -methoxy -4- 25 06 -methyl-guanosine
7 -deaza - guanosine
thio - uridine, 4 -methoxy -pseudouridine, and 4 -methoxy -2 No
modification
thio -pseudouridine. In some embodiments, modified N1 -methyl
- adenosine
nucleosides include 5 - aza -cytidine, pseudoisocytidine,

3 -methyl-cytidine, N4 - acetylcytidine, 5 - formylcytidine,
N4 -methylcytidine, 5 -hydroxymethylcytidine, 1 -methyl 30
pseudoisocytidine, pyrrolo -cytidine, pyrrolo -pseudoisocyti
dine, 2 - thio - cytidine , 2 - thio - 5 -methyl -cytidine, 4 -thio -pseu
doisocytidine, 4 - thio - 1 -methyl -pseudoisocytidine, 4 - thio - 1
methyl - 1 - deaza -pseudoisocytidine,
1 -methyl- 1 -deaza 35
pseudoisocytidine, zebularine , 5 - aza - zebularine , 5 -methyl
zebularine,

5 - aza - 2 - thio - zebularine ,

2 - amino - 6 - Chloro -purine

uridine

Pseudo - iso -cytidine / a -thio -uridine
Pseudo - iso -cytidine / 5 -methyl -uridine
Pseudo - iso -cytidine /Pseudo -uridine
Pyrrolo - cytidine
Pyrrolo - cytidine /5 - iodo -uridine
Pyrrolo -cytidine/N1-methyl-pseudo -uridine
Pyrrolo -cytidine / a - thio - uridine
Pyrrolo -cytidine / 5 -methyl -uridine
Pyrrolo -cytidine /Pseudo -uridine
5 -methyl- cytidine / 5 - iodo -uridine

5 -methyl-cytidine /N1-methyl-pseudo - uridine

5 -methyl-cytidine / a - thio -uridine
5 -methyl -cytidine / 5 -methyl -uridine
5 -methyl -cytidine /Pseudo -uridine

5 -methyl-cytidine

N6 -methyl- 2 -amino -purine 25 % Pseudo - iso -cytidine
6 - Chloro -purine
25 % N1-methyl-pseudo-uridine
N6 -methyl -adenosine
25 % N1-Methyl-pseudo -uridine /75 %
pseudo -uridine
5 -methyl-uridine
a -thio - adenosine
8 - azido - adenosine

7 - deaza- adenosine

5 - iodo- cytidine

2 - thio -zebularine,

In some embodiments, at least 25 % of the cytosines are

yisopentenyl ) adenosine, 2-methylthio -N6-(cis-hydroxyisoN6 - threonylcarbamoyladenosine, 2 -methylthio -N6 -threonyl
carbamoyladenosine, N6 , N - dimethyladenosine, 7 -methyl-

45 % , at least about 50 % , at least about 55 % , at least about
60% , at least about 65 % , at least about 70 % , at least about
75 % , at least about 80 % , at least about 85 % , at least about
50
90% , at least about 95 % , or about 100 % ) .
In some embodiments , at least 25 % of the cytosines and
25 % of the uracils are replaced by a compound of Formula
I - a ( e.g. , at least about 30 % , at least about 35 % , at least
55 about 40 % , at least about 45 % , at least about 50 % , at least
about 55 % , at least about 60% , at least about 65 % , at least
about 70 % , at least about 75 % , at least about 80 % , at least
about 85 % , at least about 90 % , at least about 95 % , or about
100 % ).
60 Other components of nucleic acid are optional , and are

2 -methoxy -cytidine,
2 -methoxy - 5 -methyl -cytidine, replaced by a compound of Formula I - a ( e.g. , at least about
4 -methoxy -pseudoisocytidine, and 4 -methoxy - 1-methyl- 30% , at least about 35 % , at least about 40 % , at least about
pseudoisocytidine.
40 45 % , at least about 50 % , at least about 55 % , at least about
In other embodiments, modified nucleosides include 60% , at least about 65 % , at least about 70 % , at least about
2 -aminopurine , 2 , 6 -diaminopurine, 7 -deaza - adenine , 75 % , at least about 80 % , at least about 85 % , at least about
7 -deaza - 8 -aza -adenine, 7 -deaza - 2 -aminopurine, 7 - deaza -8- 90% , at least about 95 % , or about 100 % ) .
aza - 2 - aminopurine, 7 -deaza -2,6 -diaminopurine, 7 -deaza - 8In some embodiments, at least 25 % of the uracils are
aza - 2,6 -diaminopurine, l -methyladenosine, N6 -methylad- 45 replaced by a compound of Formula 1 - a (e.g. , at least about
enosine, N6 - isopentenyladenosine, N6- (cis-hydrox 30% , at least about 35 % , at least about 40 % , at least about
pentenyl) adenosine, N6 - glycinylcarbamoyladenosine,

adenine , 2 -methylthio -adenine, and 2 -methoxy -adenine.
In other embodiments, modified nucleosides include inos
ine , 1 -methyl - inosine, wyosine , wybutosine, 7-deaza
guanosine, 7 - deaza - 8 - aza -guanosine, 6 - thio -guanosine,

6 - thio - 7 - deaza -guanosine, 6 - thio - 7 -deaza - 8 -aza - guanosine ,
7 -methyl- guanosine, 6 -thio - 7 -methyl -guanosine, 7 -methylinosine , 6 -methoxy - guanosine, 1 -methylguanosine,
N2 -methylguanosine, N2 ,N2 - dimethylguanosine, 8 -oxo-

guanosine, 7 -methyl - 8 -oxo - guanosine, 1 -methyl- 6 -thioethyl -6 - thio - guanosine.

guanosine, N2-methyl -6 - thio -guanosine, and N2 N2-dim

beneficial in some embodiments. For example, a 5 ' untrans
lated region (UTR ) and /or a 3'UTR are provided, wherein
In some embodiments, the nucleotide can be modified on either or both may independently contain one or more
the major groove face and can include replacing hydrogen different nucleoside modifications . In such embodiments ,
on C - 5 of uracil with a methyl group or a halo group .
65 nucleoside modifications may also be present in the trans
In specific embodiments , a modified nucleoside is 5'- 0- latable region. Also provided are nucleic acids containing a
( 1 - Thiophosphate)-Adenosine, 5'-0-( 1- Thiophosphate )-Cy Kozak sequence .

US 10,898,574 B2
11

12

Linkers and Payloads
The nucleobase of the nucleotide, which may be incorporated into a mmRNA , can be covalently linked at any
chemically appropriate position to a payload, e.g. detectable
agent or therapeutic agent. For example , the nucleobase can
be deaza - adenosine or deaza -guanosine and the linker can be
attached at the C - 7 or C - 8 positions of the deaza - adenosine
or deaza -guanosine. In other embodiments , the nucleobase
can be cytosine or uracil and the linker can be attached to the

Labels , other than those described herein , are contem
plated by the present disclosure, including , but not limited
to , other optically -detectable labels. Labels can be attached
to the modified nucleotide of the present disclosure at any
position using standard chemistries such that the label can be
removed from the incorporated base upon cleavage of the

5

cleavable linker.

Terminal Architecture Modifications: 5 ' - Capping

10
N - 3 or C - 5 positions of cytosine or uracil.
The term “ linker” as used herein refers to a group of
atoms , e.g. , 10-1,000 atoms , and can be comprised of the
atoms or groups such as , but not limited to , carbon , amino ,
alkylamino , oxygen , sulfur, sulfoxide, sulfonyl, carbonyl, 15

Linker

and imine . The linker can be attached to a modified nucleo

side or nucleotide on the nucleobase or sugar moiety at a first
end , and to a payload , e.g. , detectable or therapeutic agent,
at a second end . The linker may be of sufficient length as to
not interfere with incorporation into a nucleic acid sequence . 20
Examples of chemical groups that can be incorporated
into the linker include, but are not limited to , an alkyl, an
alkene , an alkyne, an amido , an ether, a thioether or an ester
group . The linker chain can also comprise part of a saturated ,
unsaturated or aromatic ring, including polycyclic and het- 25
eroaromatic rings wherein the heteroaromatic ring may be
an aryl group containing one to four heteroatoms, N , O or S.
Specific examples of linkers include , but are not limited to ,
unsaturated alkanes, polyethylene glycols , and dextran polymers .
30
For example , the linker can include, but is not limited to ,

ethylene or propylene glycol monomeric units, e.g. , diethylene glycol , dipropylene glycol, triethylene glycol , tripro-

pylene glycol , tetraethylene glycol , or tetraethylene glycol .

In some embodiments, the linker can include , but is not 35

limited to , a divalent alkyl, alkenyl, and / or alkynyl moiety .
The linker can include an ester, amide , or ether moiety.
Other examples include , but are not limited to , cleavable
moieties within the linker, such as , for example, a disulfide
bond ( S S ) or an azo bond ( N = N— ), which can be
cleaved using a reducing agent or photolysis . When a
cleavable bond which has been incorporated into the linker
and attached to a modified nucleotide, is cleaved , a short
“ scar ” or chemical modification on the nucleotide may
result . For example, after cleaving , the resulting scar on a
nucleotide base , which formed part of the modified nucleotide , and is incorporated into a polynucleotide strand , is
unreactive and does not need to be chemically neutralized .
This increases the ease with which a subsequent nucleotide
can be incorporated during sequencing of a nucleic acid
polymer template. For example , conditions include the use
of tris ( 2 -carboxyethyl)phosphine ( TCEP ), dithiothreitol
( DTT ) and /or other reducing agents for cleavage of a disulfide bond . A selectively severable bond that includes an
amido bond can be cleaved for example by the use of TCEP
or other reducing agents, and / or photolysis. A selectively
severable bond that includes an ester bond can be cleaved for
example by acidic or basic hydrolysis.
Detectable Agents
The mmRNAs of the present invention may also be linked
or conjugated to one or more detectable agents. Examples of
detectable substances include , but are not limited to , various
organic small molecules , inorganic compounds, nanoparticles , enzymes or enzyme substrates, fluorescent materials,
luminescent materials, bioluminescent materials, chemiluminescent materials, radioactive materials , and contrast
agents.

40

45

50

55
60
65

Endogenous eukaryotic cellular messenger RNA (mRNA )
molecules contain a 5 ' - cap structure on the 5 ' - end of a
mature mRNA molecule . The 5 ' - cap contains a 5'- 5'- triphos
phate linkage between the 5 ' -most nucleotide and guanine
nucleotide . The conjugated guanine nucleotide is methylated
at the N7 position . Additional modifications include meth
ylation of the ultimate and penultimate most 5 '-nucleotides
on the 2 '-hydroxyl group. The 5 ' - cap structure is responsible
for binding the mRNA Cap Binding Protein ( CBP ) , which is
responsibility for mRNA stability in the cell and translation
competency .
Multiple distinct 5 '-cap structures can be used to generate
the 5 ' - cap of a synthetic mRNA molecule . Many chemical
cap analogs are used to co - transcriptionally cap a synthetic
mRNA molecule . For example, the Anti-Reverse Cap Ana
log (ARCA) cap contains a 5 '- 5 ' -triphosphate guanine- gua
nine linkage where one guanine contains an N7 methyl
group as well as a 3 ' - O -methyl group . While chemical cap
analogs allow for the concomitant capping of an RNA
molecule , up 20 % of transcripts remain uncapped and the
synthetic cap analog is not identical to an endogenous 5 ' - cap
structure of an authentic cellular mRNA . This may lead to
reduced
translationally -competency and reduced cellular
stability .
Synthetic mRNA molecules may also be capped post
transcriptionally using enzymes responsible for generating a
more authentic 5 '-cap structure . As used herein the phrase
“ more authentic” refers to a feature that closely mirrors or
mimics , either structurally or functionally an endogenous or
wild type feature . More authentic 5 ' - cap structures of the
present invention are those which , among other things , have
enhanced binding of cap binding proteins , increased half
life , reduced susceptibility to 5 ' endonucleases and / or
reduced 5'decapping. For example, recombinant Vaccinia
Virus Capping Enzyme and recombinant 2 - O -methyltrans
ferase enzyme can create a canonical 5 '- 5 '-triphosphate
linkage between the 5 ' -most nucleotide of an mRNA and a
guanine nucleotide where the guanine contains an N7 meth
ylation and the ultimate 5 ' - nucleotide contains a 2 '- O -methyl
generating the Capl structure . This results in a cap with
higher translational - competency and cellular stability and
reduced activation of cellular pro - inflammatory cytokines .
Because the synthetic mRNA is capped post-transcription
ally, nearly 100 % of the mRNA molecules are capped in
contrast to ~ 80% of synthetic mRNAs containing a chemical
cap analog .
Terminal Architecture Modifications: Poly -A Tails
During RNA processing , a long chain of adenine nucleo
tides (poly - A tail ) is normally added to a messenger RNA
(mRNA ) molecules to increase the stability of the molecule .
Immediately after transcription, the 3 ' end of the transcript
is cleaved to free a 3 ' hydroxyl. Then poly -A polymerase
adds a chain of adenine nucleotides to the RNA . The
process, called polyadenylation, adds a poly - A tail that is
between 100 and 250 residues long .
It has been discovered that unique poly - A tail lengths
provide certain advantages to the modified RNAs of the
present invention .

US 10,898,574 B2
13
Generally , the length of a poly -A tail of the present

invention is greater than 30 nucleotides in length . In another

embodiment, the poly - A tail is greater than 35 nucleotides in

14

such that the mmRNA is localized into one or more cells of

the cell population and the recombinant polypeptide is
translated in the cell from the nucleic acid .

length . In another embodiment, the length is at least 40
An effective amount of the composition is provided based ,
nucleotides . In another embodiment, the length is at least 45 5 at least in part, on the target tissue , target cell type , means
nucleotides . In another embodiment, the length is at least 55 of administration , physical characteristics of the nucleic acid
nucleotides . In another embodiment, the length is at least 60 (e.g. , size , and extent of modified nucleosides ), and other
nucleotides . In another embodiment, the length is at least 60 determinants. In general, an effective amount of the com
nucleotides. In another embodiment, the length is at least 80 position provides efficient protein production in the cell ,
nucleotides. In another embodiment, the length is at least 90 10 preferably more efficient than a composition containing a
nucleotides . In another embodiment, the length is at least corresponding unmodified nucleic acid . Increased efficiency
100 nucleotides . In another embodiment, the length is at may be demonstrated by increased cell transfection (i.e. , the

least 120 nucleotides. In another embodiment, the length is
at least 140 nucleotides. In another embodiment, the length
is at least 160 nucleotides . In another embodiment, the 15
length is at least 180 nucleotides . In another embodiment,
the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides . In another
embodiment, the length is at least 300 nucleotides . In
another embodiment, the length is at least 350 nucleotides. 20
In another embodiment, the length is at least 400 nucleotides . In another embodiment, the length is at least 450
nucleotides . In another embodiment, the length is at least
500 nucleotides. In another embodiment, the length is at
least 600 nucleotides. In another embodiment, the length is 25
at least 700 nucleotides . In another embodiment, the length
is at least 800 nucleotides. In another embodiment, the
length is at least 900 nucleotides. In another embodiment,
the length is at least 1000 nucleotides.
In one embodiment, the poly -A tail is designed relative to 30
the length of the overall modified RNA molecule . This
design may be based on the length of the coding region of
the modified RNA , the length of a particular feature or
region of the modified RNA ( such as the mRNA ), or based
on the length of the ultimate product expressed from the 35
modified RNA . In this context the poly -A tail may be 10 , 20 ,
30 , 40 , 50 , 60 , 70 , 80 , 90 or 100% greater in length than the
modified RNA or feature thereof. The poly -A tail may also
be designed as a fraction of the modified RNA to which it
belongs. In this context, the poly -A tail may be 10 , 20 , 30 , 40
40 , 50 , 60 , 70 , 80 , or 90 % or more of the total length of the
construct or the total length of the construct minus the

percentage of cells transfected with the nucleic acid ),
increased protein translation from the nucleic acid ,
decreased nucleic acid degradation (as demonstrated , e.g. ,
by increased duration of protein translation from a
mmRNA ), or reduced innate immune response of the host
cell .
Aspects of the invention are directed to methods of
inducing in vivo translation of a recombinant polypeptide in
a mammalian subject in need thereof. Therein , an effective
amount of a composition containing a mmRNA that has at
least one nucleoside modification and a translatable region
encoding the recombinant polypeptide is administered to the
subject using the delivery methods and split dosing regimens
described herein . The mmRNA is provided in an amount and
under other conditions such that the nucleic acid is localized
into a cell of the subject and the recombinant polypeptide is

and in kits and devices.

production of one or more recombinant polypeptides that

translated in the cell from the mmRNA . The cell in which

the mmRNA is localized , or the tissue in which the cell is

present, may be targeted with one or more than one rounds
of mmRNA administration .
The subject to whom the therapeutic agent is administered
suffers from or is at risk of developing a disease , disorder, or

deleterious condition . Provided are methods of identifying,
diagnosing, and classifying subjects on these bases , which
may include clinical diagnosis , biomarker levels , genome

wide association studies (GWAS ), and other methods known
in the art.
In certain embodiments , the administered mmRNA

directs production of one or more recombinant polypeptides
that provide a functional activity which is substantially
poly -A tail .
absent in the cell in which the recombinant polypeptide is
Use of Modified mRNAS
translated . For example, the missing functional activity may
The mmRNAs of the present invention may find uses in 45 be enzymatic, structural, or gene regulatory in nature. In
many areas of research , discovery, therapeutics, diagnostics related embodiments , the administered mmRNA directs
Therapeutics

increases ( e.g. , synergistically ) a functional activity which is
The mmRNAs (modified RNAs) and the proteins trans- present but substantially deficient in the cell in which the
lated from the mmRNAs described herein can be used as 50 recombinant polypeptide is translated .
therapeutic agents. For example, an mmRNA described
In other embodiments, the administered mmRNA directs
herein can be administered to a subject, wherein the production of one or more recombinant polypeptides that
mmRNA is translated in vivo to produce a therapeutic replace a polypeptide (or multiple polypeptides ) that is
polypeptide in the subject. Provided are compositions, meth- substantially absent in the cell in which the recombinant

ods , kits, and reagents for treatment or prevention of disease 55 polypeptide is translated. Such absence may be due to
or conditions in humans and other mammals . The active

therapeutic agents of the invention include mmRNAs, cells
containing mmRNAs or polypeptides translated from the

genetic mutation of the encoding gene or regulatory pathway

thereof. In some embodiments, the recombinant polypeptide
increases the level of an endogenous protein in the cell to a

desirable level ; such an increase may bring the level of the
mmRNAs, polypeptides translated from mmRNAs.
Provided herein are methods of inducing translation of a 60 endogenous protein from a subnormal level to a normal level
recombinant polypeptide in a cell population using the or from a normal level to a super -normal level.
mmRNAs described herein . Such translation can be in vivo ,
Alternatively, the recombinant polypeptide functions to
ex vivo , in culture , or in vitro . The cell population is antagonize the activity of an endogenous protein present in ,
contacted with an effective amount of a composition con- on the surface of, or secreted from the cell . Usually, the
taining a mmRNA that has at least one nucleoside modifi- 65 activity of the endogenous protein is deleterious to the
cation , and a translatable region encoding the recombinant subject; for example, do to mutation of the endogenous
polypeptide . The population is contacted under conditions protein resulting in altered activity or localization . Addition

US 10,898,574 B2
15

16

ally, the recombinant polypeptide antagonizes, directly or
indirectly, the activity of a biological moiety present in , on
the surface of, or secreted from the cell . Examples of
antagonized biological moieties include lipids ( e.g. , choles-

(e.g. , TLR7 and TLR8 ) . Reduction of innate immune
response can also be measured by decreased cell death
following one or more administrations of modified RNAs to
a cell population ; e.g. , cell death is 10% , 25 % , 50% , 75 % ,

terol), a lipoprotein (e.g. , low density lipoprotein ), a nucleic 5 85 % , 90 % , 95 % , or over 95 % less than the cell death
acid, a carbohydrate, a protein toxin such as shiga and frequency observed with a corresponding unmodified

tetanus toxins , or a small molecule toxin such as botulinum , nucleic acid . Moreover, cell death may affect fewer than
cholera, and diphtheria toxins. Additionally , the antagonized 50% , 40% , 30 % , 20 % , 10 % , 5 % , 1 % , 0.1 % , 0.01 % or fewer
biological molecule may be an endogenous protein that than 0.01 % of cells contacted with the mmRNAs.
exhibits an undesirable activity, such as a cytotoxic or 10 The invention provides therapeutic methods for the
cytostatic activity.
repeated introduction ( e.g. , transfection ) of mmRNAs into a
The polypeptides encoded by the mmRNA described target cell population , e.g. , in vitro, ex vivo , or in vivo . The
herein are engineered for localization within the cell , poten- step of contacting the cell population may be repeated one
tially within a specific compartment such as the nucleus, or or more times ( such as two, three, four, five or more than five
are engineered for secretion from the cell or translocation to 15 times ) . In some embodiments, the step of contacting the cell
the plasma membrane of the cell .
population with the mmRNAs is repeated a number of times
In one embodiment of the invention are bifunctional sufficient such that a predetermined efficiency of protein
mmRNA . As the name implies, bifunctional mmRNA are translation in the cell population is achieved . Given the
reduced cytotoxicity of the target cell population provided
those having or capable of at least two functions.
The multiple functionalities of bifunctional mmRNAs 20 by the nucleic acid modifications, such repeated transfec
may be encoded by the mRNA ( the function may not tions are achievable in a diverse array of cell types.
manifest until the encoded product is translated ) or may be Protein Production
a property of the RNA itself. It may be structural or
The methods provided herein are useful for enhancing
chemical. Bifunctional modified RNAs may comprise a protein product yield in a cell culture process. In a cell

function that is covalently associated with the RNA or 25 culture containing a plurality of host cells , introduction of

electrostatically associated.
the modified mRNAs described herein results in increased
In some embodiments , modified mRNAs and their protein production efficiency relative to a corresponding
encoded polypeptides in accordance with the present inven- unmodified nucleic acid . Such increased protein production
tion may be used for treatment of any of a variety of efficiency can be demonstrated , e.g. , by showing increased
diseases , disorders, and / or conditions , including but not 30 cell transfection , increased protein translation from the
limited to one or more of the following: autoimmune dis- nucleic acid , decreased nucleic acid degradation, and / or
orders ( e.g. diabetes, lupus, multiple sclerosis, psoriasis, reduced innate immune response of the host cell . Protein

rheumatoid arthritis ); inflammatory disorders (e.g. arthritis,
pelvic inflammatory disease) ; infectious diseases (e.g. viral
infections (e.g. , HIV , HCV, RSV) , bacterial infections, fungal infections, sepsis ) ; neurological disorders (e.g. Alzheimer's disease , Huntington's disease ; autism ; Duchenne muscular dystrophy ); cardiovascular disorders ( e.g.
atherosclerosis, hypercholesterolemia , thrombosis, clotting
disorders, angiogenic disorders such as macular degeneration ); proliferative disorders (e.g. cancer, benign neoplasms ) ; respiratory disorders (e.g. chronic obstructive pulmonary disease ) ; digestive disorders (e.g. inflammatory
bowel disease, ulcers ) ; musculoskeletal disorders (e.g. fibromyalgia , arthritis ); endocrine , metabolic , and nutritional
disorders (e.g. diabetes , osteoporosis ) ; urological disorders
( e.g. renal disease) ; psychological disorders (e.g. depression ,
schizophrenia ); skin disorders ( e.g. wounds, eczema ); blood
and lymphatic disorders ( e.g. anemia , hemophilia ); etc.
Avoidance of the Innate Immune Response
The term “ innate immune response” includes a cellular
response to exogenous single stranded nucleic acids , generally of viral or bacterial origin , which involves the induction of cytokine expression and release , particularly the
interferons, and cell death . Protein synthesis is also reduced
during the innate cellular immune response . While it is
advantageous to eliminate the innate immune response in a
cell , the invention provides modified mRNAs that substantially reduce the immune response , including interferon

production can be measured by EI A , and protein activity
can be measured by various functional assays known in the
35 art. The protein production may be generated in a continuous
or a fed -batch mammalian process .
Additionally, it is useful to optimize the expression of a
specific polypeptide in a cell line or collection of cell lines
of potential interest, particularly an engineered protein such
40 as a protein variant of a reference protein having a known

activity. In one embodiment, provided is a method of opti
mizing expression of an engineered protein in a target cell ,
by providing a plurality of target cell types , and indepen
dently contacting with each of the plurality of target cell
45 types a modified mRNA encoding an engineered polypep

tide. Additionally, culture conditions may be altered to
increase protein production efficiency. Subsequently, the
presence and / or level of the engineered polypeptide in the
plurality of target cell types is detected and / or quantitated ,
50 allowing for the optimization of an engineered polypeptide's
expression by selection of an efficient target cell and cell
culture conditions relating thereto . Such methods are par
ticularly useful when the engineered polypeptide contains
one or more post - translational modifications or has substan
55 tial tertiary structure , situations which often complicate
efficient protein production .
Gene Silencing
The modified mRNAs described herein are useful to
silence (i.e. , prevent or substantially reduce ) expression of

signaling, without entirely eliminating such a response . In 60 one or more target genes in a cell population. A modified
some embodiments, the immune response is reduced by mRNA encoding a polypeptide capable of directing

10 % , 20 % , 30 % , 40 % , 50 % , 50 % , 70% , 80% , 90% , 95 % , sequence - specific histone H3 methylation is introduced into
99 % , 99.9 % , or greater than 99.9 % as compared to the the cells in the population under conditions such that the
immune response induced by a corresponding unmodified polypeptide is translated and reduces gene transcription of a
nucleic acid . Such a reduction can be measured by expres- 65 target gene via histone H3 methylation and subsequent
sion or activity level of Type 1 interferons or the expression heterochromatin formation . In some embodiments, the
of interferon - regulated genes such as the toll - like receptors silencing mechanism is performed on a cell population

US 10,898,574 B2
17

18

present in a mammalian subject. By way of non- limiting ways that modulate cell fate determination . Such agoniza
example , a useful target gene is a mutated Janus Kinase - 2 tion is reversible or, alternatively, irreversible .
family member, wherein the mammalian subject expresses Cellular Nucleic Acid Delivery
the mutant target gene suffers from a myeloproliferative
Methods of the present invention enhance nucleic acid
5 delivery into a cell population, in vivo , ex vivo , or in culture .
disease resulting from aberrant kinase activity.
Co - administration of modified mRNAs and siRNAs are For example, a cell culture containing a plurality of host
( e.g. , eukaryotic cells such as yeast or mammalian
also provided herein . As demonstrated in yeast, sequence cells
specific trans silencing is an effective mechanism for alter cells ) is contacted with a composition that contains an
ing cell function . Fission yeast require two RNAi complexes enhanced nucleic acid having at least one nucleoside modi
for
siRNA -mediated heterochromatin assembly: the RNA- 10 fication
, optionally
, a translatable
regionreagent
. The orcompo
sition alsoandgenerally
contains
a transfection
other
induced transcriptional silencing (RITS ) complex and the compound
increases the efficiency of enhanced nucleic
RNA -directed RNA polymerase complex ( RDRC ) (Mot acid uptakethat
into the host cells . The enhanced nucleic acid
amedi et al . Cell 2004 , 119 , 789-802 ) . In fission yeast , the exhibits enhanced
retention in the cell population , relative to
RITS
complex
contains
the
siRNA
binding
Argonaute
fam15
a
corresponding
unmodified
nucleicacid. The retention of
ily protein Agol, a chromodomain protein Chpl, and Tas3 . the enhanced nucleic acid is greater
than the retention of the
The fission yeast RDRC complex is composed of an RNA unmodified nucleic acid . In some embodiments
least
dependent RNA Polymerase Rdp1, a putative RNA helicase about 50% , 75 % , 90 % , 95 % , 100 % , 150 % , 200, it%isoratmore
Hrr1, and a polyA polymerase family protein Cid12 . These than 200 % greater than the retention of the unmodified
two complexes require the Dicer ribonuclease and Clr4 20 nucleic acid. Such retention advantage may be achieved by
histone H3 methyltransferase for activity . Together, Ago 1 one round of transfection with the enhanced nucleic acid, or
binds siRNA molecules generated through Dicer-mediated may be obtained following repeated rounds of transfection .
cleavage of Rdpl co - transcriptionally generated dsRNA
In some embodiments , the enhanced nucleic acid is deliv
transcripts and allows for the sequence - specific direct asso- ered to a target cell population with one or more additional
ciation of Chpl, Tas3 , Hrrl, and Clr4 to regions of DNA 25 nucleic acids . Such delivery may be at the same time , or the
destined for methylation and histone modification and sub- enhanced nucleic acid is delivered prior to delivery of the
sequent compaction into transcriptionally silenced heterochromatin . While this mechanism functions in cis- with
centromeric regions of DNA , sequence -specific trans silenc-

one or more additional nucleic acids . The additional one or
more nucleic acids may be modified nucleic acids or
unmodified nucleic acids . It is understood that the initial

ing is possible through co - transfection with double - stranded 30 presence of the enhanced nucleic acids does not substan
siRNAs for specific regions of DNA and concomitant RNAi- tially induce an innate immune response of the cell popu
directed silencing of the siRNA ribonuclease Eril (Buhler et
al . Cell 2006 , 125 , 873-886 ) .
Modulation of Biological Pathways

lation and, moreover, that the innate immune response will
not be activated by the later presence of the unmodified
nucleic acids . In this regard , the enhanced nucleic acid may
The rapid translation of modified mRNAs introduced into 35 not itself contain a translatable region , if the protein desired
cells provides a desirable mechanism of modulating target to be present in the target cell population is translated from
biological pathways. Such modulation includes antagonism the unmodified nucleic acids .
or agonism of a given pathway. In one embodiment, a Expression of Ligand or Receptor on Cell Surface
method is provided for antagonizing a biological pathway in
In some aspects and embodiments of the aspects

a cell by contacting the cell with an effective amount of a 40 described herein , the modified RNAs can be used to express
composition comprising a modified nucleic acid encoding a a ligand or ligand receptor on the surface of a cell (e.g. , a
recombinant polypeptide , under conditions such that the homing moiety ). A ligand or ligand receptor moiety attached
nucleic acid is localized into the cell and the recombinant to a cell surface can permit the cell to have a desired
polypeptide is capable of being translated in the cell from the biological interaction with a tissue or an agent in vivo . A

nucleic acid, wherein the recombinant polypeptide inhibits 45 ligand can be an antibody, an antibody fragment, an aptamer,
the activity of a polypeptide functional in the biological

a peptide , a vitamin , a carbohydrate, a protein or polypep

pathway. Exemplary biological pathways are those defective tide, a receptor, e.g. , cell- surface receptor, an adhesion
in an autoimmune or inflammatory disorder such as multiple molecule, a glycoprotein , a sugar residue, a therapeutic
sclerosis, rheumatoid arthritis, psoriasis , lupus erythemato- agent, a drug, a glycosaminoglycan, or any combination
sus , ankylosing spondylitis colitis , or Crohn's disease; in 50 thereof. For example , a ligand can be an antibody that
particular, antagonism of the IL - 12 and IL - 23 signaling recognizes a cancer- cell specific antigen, rendering the cell
pathways are of particular utility . ( See Kikly K , Liu L , Na capable of preferentially interacting with tumor cells to
S , Sedgwick J D (2006 ) Curr. Opin . Immunol. 18 ( 6 ) : permit tumor -specific localization of a modified cell . A
670-5) .
ligand can confer the ability of a cell composition to
Further, provided are modified nucleic acids encoding an 55 accumulate in a tissue to be treated , since a preferred ligand
antagonist for chemokine receptors ; chemokine receptors may be capable of interacting with a target molecule on the
CXCR - 4 and CCR- 5 are required for, e.g. , HIV entry into external face of a tissue to be treated . Ligands having limited
cross -reactivity to other tissues are generally preferred .
host cells (et al , ( 1996 ) October 3 ; 383 ( 6599 ) : 400 ) .
Alternatively, provided are methods of agonizing a bioIn some cases , a ligand can act as a homing moiety which
logical pathway in a cell by contacting the cell with an 60 permits the cell to target to a specific tissue or interact with
effective amount of a modified nucleic acid encoding a a specific ligand. Such homing moieties can include , but are
recombinant polypeptide under conditions such that the not limited to , any member of a specific binding pair,
nucleic acid is localized into the cell and the recombinant antibodies , monoclonal antibodies, or derivatives or analogs
polypeptide is capable of being translated in the cell from the thereof, including without limitation : Fv fragments, single

nucleic acid, and the recombinant polypeptide induces the 65 chain Fv ( scFv) fragments, Fab ' fragments , F ( ab ' )2 frag

activity of a polypeptide functional in the biological pathway. Exemplary agonized biological pathways include path-

ments , single domain antibodies, camelized antibodies and
antibody fragments, humanized antibodies and antibody

US 10,898,574 B2
19

20

fragments, and multivalent versions of the foregoing; mul- totic when formed into trimers or higher order of structures.
tivalent binding reagents including without limitation : As monomers , they may serve as antiapoptotic agents by
monospecific or bispecific antibodies, such as disulfide competing with the trimers for binding to the death recep
stabilized Fv fragments, scFv tandems ( ( SCFV) 2 frag- tors .
ments ), diabodies , tribodies or tetrabodies, which typically 5 In one embodiment, the modified nucleic acid molecule
are covalently linked or otherwise stabilized ( i.e. , leucine composition encodes for a death receptor (e.g. , Fas , TRAIL ,
zipper or helix stabilized ) scFv fragments; and other homing TRAMO , TNFR , TLR etc ). Cells made to express a death
moieties include for example, aptamers, receptors, and receptor by transfection of modified RNA become suscep
fusion proteins.
tible to death induced by the ligand that activates that
In some embodiments, the homing moiety may be a 10 receptor. Similarly, cells made to express a death ligand, e.g. ,
surface - bound antibody, which can permit tuning of cell on their surface, will induce death of cells with the receptor
targeting specificity. This is especially useful since highly when the transfected cell contacts the target cell . In another
specific antibodies can be raised against an epitope of embodiment, the modified RNA composition encodes for a
interest for the desired targeting site . In one embodiment, death receptor ligand (e.g. , FasL , TNF, etc ) . In another
multiple antibodies are expressed on the surface of a cell , 15 embodiment, the modified RNA composition encodes a
and each antibody can have a different specificity for a caspase ( e.g. , caspase 3 , caspase 8 , caspase 9 etc ). Where
desired target. Such approaches can increase the avidity and cancer cells often exhibit a failure to properly differentiate to
specificity of homing interactions .
a non - proliferative or controlled proliferative form , in
A skilled artisan can select any homing moiety based on another embodiment, the synthetic, modified RNA compo
the desired localization or function of the cell , for example 20 sition encodes for both a death receptor and its appropriate
an estrogen receptor ligand , such as tamoxifen , can target activating ligand. In another embodiment, the synthetic ,
cells to estrogen -dependent breast cancer cells that have an modified RNA composition encodes for a differentiation
increased number of estrogen receptors on the cell surface . factor that when expressed in the cancer cell , such as a
Other non - limiting examples of ligand/ receptor interactions cancer stem cell , will induce the cell to differentiate to a

include CCRI (e.g. , for treatment of inflamed joint tissues or 25 non - pathogenic or nonself- renewing phenotype ( e.g. ,

brain in rheumatoid arthritis , and /or multiple sclerosis ),
CCR7 , CCR8 ( e.g. , targeting to lymph node tissue ) , CCR6 ,
CCR9 , CCR10 (e.g. , to target to intestinal tissue ) , CCR4 ,

reduced cell growth rate , reduced cell division etc ) or to
induce the cell to enter a dormant cell phase (e.g. , G. resting
phase ).

CCR10 ( e.g. , for targeting to skin) , CXCR4 (e.g. , for general
One of skill in the art will appreciate that the use of
enhanced transmigration ), HCELL ( e.g. , for treatment of 30 apoptosis - inducing techniques may require that the modified
inflammation and inflammatory disorders, bone marrow ), nucleic acid molecules are appropriately targeted to e.g. ,
Alpha4beta 7 (e.g. , for intestinal mucosa targeting ), VLA - 4 / tumor cells to prevent unwanted wide - spread cell death .

VCAM - 1 ( e.g. , targeting to endothelium ). In general, any
hus, one can use a delivery mechanism ( e.g. , attached
receptor involved in targeting ( e.g. , cancer metastasis ) can ligand or antibody, targeted liposome etc) that recognizes a
be harnessed for use in the methods and compositions 35 cancer antigen such that the modified nucleic acid molecules
described herein .
are expressed only in cancer cells .
Mediators of Cell Death
Formulations of Modified mRNAS
In one embodiment, a modified nucleic acid molecule
Provided herein are formulations containing an effective
composition can be used to induce apoptosis in a cell ( e.g. , amount of an mmRNA .
a cancer cell ) by increasing the expression of a death 40 In certain embodiments, the formulations include one or
receptor, a death receptor ligand or a combination thereof. more cell penetration agents, e.g. , transfection agents . In one
This method can be used to induce cell death in any desired specific embodiment, an mmRNA is mixed or admixed with
cell and has particular usefulness in the treatment of cancer a transfection agent ( or mixture thereof) and the resulting
where cells escape natural apoptotic signals .
mixture is employed to transfect cells . Preferred transfection
Apoptosis can be induced by multiple independent sig- 45 agents are cationic lipid compositions, particularly monova
naling pathways that converge upon a final effector mecha- lent and polyvalent cationic lipid compositions, more par
nism consisting of multiple interactions between several ticularly LIPOFECTIN® , LIPOFECTACE® , LIPO
“ death receptors ” and their ligands, which belong to the FECTAMINETM , CELLFECTIN® , DMRIE - C , DMRIE ,
tumor necrosis factor ( TNF) receptor /ligand superfamily. DOTAP, DOSPA , and DOSPER , and dendrimer composi
The best - characterized death receptors are CD95 (“ Fas ” ), 50 tions , particularly G5 - G10 dendrimers, including dense star
TNFRI ( p55 ), death receptor 3 (DR3 or Apo3 / TRAMO ), dendrimers, PAMAM dendrimers , grafted dendrimers, and
DR4 and DR5 (apo 2 -TRAIL -R2). The final effector mecha- dendrimers known as dendrigrafts and SUPERFECT® .
nism of apoptosis may be the activation of a series of
In a second specific transfection method, a ribonucleic

proteinases designated as caspases . The activation of these acid is conjugated to a nucleic acid -binding group , for
caspases results in the cleavage of a series of vital cellular 55 example a polyamine and more particularly a spermine,
proteins and cell death . The molecular mechanism of death which is then introduced into the cell or admixed with a
receptors / ligands -induced apoptosis is well known in the art. transfection agent ( or mixture thereof) and the resulting
For example, Fas /FasL - mediated apoptosis is induced by mixture is employed to transfect cells . In a third specific
binding of three Fasl molecules which induces trimerization

embodiment, a mixture of one or more transfection - enhanc

of Fas receptor via C - terminus death domains ( DDs ) , which 60 ing peptides, proteins , or protein fragments, including fus

in turn recruits an adapter protein FADD (Fas - associated agenic peptides or proteins, transport or trafficking peptides
protein with death domain) and Caspase - 8 . The oligomer- or proteins, receptor- ligand peptides or proteins , or nuclear
ization of this trimolecular complex , Fas /FAIDD /caspase - 8, localization peptides or proteins and / or their modified ana
results in proteolytic cleavage of proenzyme caspase - 8 into logs (e.g. , spermine modified peptides or proteins) or com
active caspase - 8 that, in turn , initiates the apoptosis process 65 binations thereof are mixed with and complexed with a
by activating other downstream caspases through proteoly- ribonucleic acid to be introduced into a cell , optionally being
sis , including caspase - 3 . Death ligands in general are apop- admixed with transfection agent and the resulting mixture is

US 10,898,574 B2
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22

employed to transfect cells . Further, a component of a

According to the present invention , complexes , micelles,

In other embodiments, provided are sustained -release
delivery depots , such as for administration of a mmRNA to

is not limited to , DLin - DMA , DLin - K - DMA, DLin -KC2
DMA , 12-5 , C12-200 ( including variants and deriva

transfection agent (e.g. , lipids , cationic lipids or dendrimers ) liposomes or particles can be prepared containing these
is covalently conjugated to selected peptides , proteins , or lipidoids and therefore, result in an effective delivery of
protein fragments directly or via a linking or spacer group . mmRNA, as judged by the production of an encoded pro
Of particular interest in this embodiment are peptides or 5 tein , following the injection of an mmRNA - formulated
proteins that are fusagenic, membrane -permeabilizing, lipidoids via localized and systemic routes of administration .
transport or trafficking, or which function for cell- targeting. Modified mRNA - lipidoid complexes can be administered by
The peptide- or protein -transfection agent complex is com- various means disclosed herein .
bined with a ribonucleic acid and employed for transfection .
The characteristics of optimized lipidoid formulations for
In certain embodiments, the formulations include a phar- 10 intramuscular or subcutaneous routes may vary significantly
maceutically acceptable carrier that causes the effective depending on the target cell type and the ability of formu
amount of mmRNA to be substantially retained in a target lations to diffuse through the extracellular matrix into the
tissue containing the cell .
blood stream . While a particle size of less than 150 nm may
In certain embodiments, the formulation may include at be desired for effective hepatocyte delivery due to the size
least an mmRNA and a delivery agent. In some embodi- 15 of the endothelial fenestrae ( see , Akinc et al . , Mol Ther.
ments, the delivery agent may comprise lipidoid -based 2009 17 : 872-879 herein incorporated by reference ), use of
formulations allowed for localized and systemic delivery of lipidoid oligonucleotides to deliver the formulation to other
mmRNA .
cells types including , but not limited to , endothelial cells ,
Also provided are compositions for generation of an in myeloid cells , and muscle cells may not be similarly size
vivo depot containing an engineered ribonucleotide . For 20 limited .
example , the composition contains a bioerodible , biocomIn one aspect , effective delivery to myeloid cells , such as
patible polymer, a solvent present in an amount effective to monocytes, lipidoid formulations may have a similar com
plasticize the polymer and form a gel therewith , and an ponent molar ratio . Different ratios of lipidoids and other
engineered ribonucleic acid . In certain embodiments the components including , but not limited to , disteroylphospha
composition also includes a cell penetration agent as 25 tidyl choline , cholesterol and PEG - DMG , may be used to
described herein . In other embodiments, the composition optimize the formulation of the mmRNA molecule for
also contains a thixotropic amount of a thixotropic agent delivery to different cell types including , but not limited to ,
mixable with the polymer so as to be effective to form a hepatocytes, myeloid cells , muscle cells , etc. For example,
thixotropic composition. Further compositions include a the component molar ratio may include, but is not limited to ,
stabilizing agent, a bulking agent, a chelating agent, or a 30 50% lipid, 10% disteroylphosphatidyl choline, 38.5 % cho
buffering agent.
lesterol, and % 1.5 PEG . The lipid may be selected from , but
an environment (meaning an organ or tissue site) in a patient.
Such depots generally contain a mmRNA and a flexible 35
chain polymer where both the mmRNA and the flexible
chain polymer are entrapped within a porous matrix of a
crosslinked matrix protein. Usually, the pore size is less than
1 mm , such as 900 nm , 800 nm , 700 nm , 600 nm , 500 nm ,
400 nm , 300 nm , 200 nm , 100 nm , or less than 100 nm . 40
Usually the flexible chain polymer is hydrophilic. Usually
the flexible chain polymer has a molecular weight of at least
50 kDa , such as 75 kDa , 100 kDa , 150 kDa , 200 kDa, 250
kDa , 300 kDa , 400 kDa, 500 kDa, or greater than 500 kDa .
Usually the flexible chain polymer has a persistence length 45
of less than 10 % , such as 9 , 8 , 7 , 6 , 5 , 4 , 3 , 2 , 1 or less than
1 % of the persistence length of the matrix protein . Usually
the flexible chain polymer has a charge similar to that of the
matrix protein . In some embodiments, the flexible chain
polymer alters the effective pore size of a matrix of cross- 50
linked matrix protein to a size capable of sustaining the
diffusion of the mmRNA from the matrix into a surrounding
tissue comprising a cell into which the mmRNA is capable
of entering
Formulation Using Lipidoids
55
The pharmaceutical compositions described herein
include lipidoid -based formulations allowing for localized
and systemic delivery of mmRNA . The synthesis of lipidoids has been extensively described and formulations con-

tives ) , DLin -MC3 - DMA and analogs thereof. The use of
lipidoid formulations for the localized delivery of nucleic
acids to cells ( such as , but not limited to , adipose cells and
muscle cells ) via either subcutaneous or intramuscular deliv
ery , may also not require all of the formulation components
which may be required for systemic delivery, and as such
may comprise the lipidoid and the mmRNA .
In a further embodiment, combinations of different lipi
doids may be used to improve the efficacy of mmRNA
directed protein.
According to the present invention , modified mRNA may
be formulated by mixing the mmRNA with the lipidoid at a
set ratio prior to addition to cells . In vivo formulations may
require the addition of extra ingredients to facilitate circu
lation throughout the body. To test the ability of these
lipidoids to form particles suitable for in vivo work , a
standard formulation process used for siRNA - lipidoid for
mulations may be used as a starting point. Initial mmRNA
lipidoid formulations consist of particles composed of 42 %
lipidoid, 48 % cholesterol and 10 % PEG , with further opti
mization of ratios possible . After formation of the particle ,
In vivo delivery of nucleic acids may be affected by many
parameters, including, but not limited to , the formulation

21 : 1448-1454 ; Schroeder et al . , J Intern Med . 2010 267: 921 ; Akinc et al . , Nat Biotechnol. 2008 26 : 561-569 ; Love et

eters such as particle size (Akinc et al . , Mol Ther. 2009
17 : 872-879 ; herein incorporated by reference in its entirety ).

mmRNA is added and allowed to integrate with the com
plex . The encapsulation efficiency is determined using a
standard dye exclusion assays .

taining these compounds are particularly suited for delivery 60 composition, nature of particle PEGylation, degree of load
of polynucleotides ( see Mahon et al . , Bioconjug Chem . 2010 ing , oligonucleotide to lipid ratio , and biophysical param
al . , Proc Natl Acad Sci USA . 2010 107 : 1864-1869 ; Siegwart

As an example, small changes in the anchor chain length of

which are incorporated herein by reference in their entire-

effects on in vivo efficacy. Formulations with the different
lipidoids , including , but not limited to penta [ 3-( 1 -laurylami

et al . , Proc Natl Acad Sci USA . 2011 108 :12996-3001 ; all of 65 poly ( ethylene glycol) (PEG ) lipids may result in significant

ties) .

US 10,898,574 B2
23

24

nopropionyl) ] - triethylenetetramine hydrochloride ( TETA5LAP ; aka 98N12-5 , see Murugaiah et al . , Analytical Biochemistry, 401 : 61 ( 2010 ) ) , C12-200 ( including derivatives
and variants ), MD1 , DLin - DMA , DLin - K - DMA, DLin-

depending on the body weight of the animal . At various
points in time points following the administration of the
mmRNA - lipidoid , serum , tissues , and tissue lysates can be
obtained and the level of the mmRNA -encoded product
KC2 - DMA and DLin -MC3 - DMA ( see FIG . 1 ) , can be tested 5 determined . The ability of lipidoid -formulated mmRNA to
for in vivo activity.
express the desired protein product can be confirmed by
The lipidoid referred to herein as “ 98N12-5 ” is disclosed luminescence for luciferase expression , flow cytometry, and

by Akinc et al . , Mol Ther. 2009 17 : 872-879 and is incor-

by ELISA .

or more components in addition to polynucleotide, primary
construct, or mmRNA. As an example, formulations with

then determining whether expression level is affected by
factors such as immunogenicity ).

porated by reference in its entirety. ( See FIG . 1 )
Additional studies for a multi -dose regimen can also be
The lipidoid referred to herein as “ C12-200 ” is disclosed 10 performed to determine the maximal expression using
by Love et al . , Proc Natl Acad Sci USA . 2010 107 : 1864- mmRNA , to evaluate the saturability of the mmRNA - driven
1869 ( see FIG . 1 ) and Liu and Huang, Molecular Therapy. expression (achieved by giving a control and active
2010 669-670 ( see FIG . 1 ) ; both of which are herein mmRNA formulation in parallel or in sequence ), and to
incorporated by reference in their entirety. The lipidoid determine the feasibility of repeat drug administration (by
formulations can include particles comprising either 3 or 4 15 giving mmRNA in doses separated by weeks or months and
certain lipidoids, include, but are not limited to , 98N12-5
and may contain 42 % lipidoid , 48 % cholesterol and 10 %

Administration
The present invention provides methods comprising

lations with certain lipidoids , include , but are not limited to ,
C12-200 and may contain 50% lipidoid , 10 % disteroylphosphatidyl choline , 38.5 % cholesterol, and 1.5 % PEG - DMG .
The ratio of mmRNA to lipidoid used to test for in vitro
transfection is tested empirically at different lipidoid : 25
mmRNA ratios. Previous work using siRNA and lipidoids
have utilized 2.5 : 1 , 5 : 1 , 10 : 1 , and 15 : 1 lipidoid :siRNA wt:wt
ratios . Given the longer length of mmRNA relative to
siRNA , a lower wt : wt ratio of lipidoid to mmRNA is likely
to be effective. In addition, for comparison mmRNA are also 30
formulated using RNAiMax ( Invitrogen, Carlsbad, Calif.) or
TRANSIT -mRNA (Mirus Bio , Madison Wis .) cationic lipid
delivery vehicles.
The ability of lipidoid - formulated mmRNA to express the
desired protein product can be confirmed by luminescence 35
for luciferase expression, flow cytometry for expression, and
by ELISA for secretion .
The expression of mmRNA - encoded proteins can be
assessed both within the muscle or subcutaneous tissue and
systemically in blood and other organs and fluids such as the 40
liver and spleen, urine, saliva, etc.
For example, single dose studies allow an assessment of
the magnitude, dose responsiveness, and longevity of
expression of the desired product. After formulation of
mmRNA with the lipidoid formulations, as described pre- 45
viously, animals are divided into groups receiving either a
saline formulation, or a lipidoid - formulation containing one
of several different mmRNA . Prior to injection , mmRNAcontaining lipidoid formulations are diluted in PBS and

dance with the invention to a subject in need thereof.
mmRNA or complexes, or pharmaceutical, imaging , diag
nostic , or prophylactic compositions thereof, may be admin
istered to a subject using any amount and any route of
administration which may be effective for preventing, treat
ing , diagnosing, or imaging a disease , disorder, and / or
condition (e.g. , a disease , disorder, and / or condition relating

PEG (C14 alkyl chain length ). As another example, formu- 20 administering modified mRNAs and or complexes in accor

to working memory deficits ). The exact amount required
will vary from subject to subject, depending on factors such
as , but not limited to , the species , age , and general condition
of the subject, the severity of the disease , the particular

composition, its mode of administration , its mode of activity,
and the like .
mmRNA to be delivered and / or pharmaceutical, prophy
lactic , diagnostic, or imaging compositions thereof may be
administered to animals, such as mammals (e.g. , humans,
domesticated animals, cats , dogs , mice , rats , etc. ) . In some
embodiments , pharmaceutical, prophylactic , diagnostic , or
imaging compositions thereof are administered to humans .
mmRNA may be administered by any route . In some
embodiments, mmRNA are administered by one or more of
a variety of routes , including , but not limited to , local , oral ,
intravenous, intramuscular, intra - arterial, intramedullary,
intrathecal, subcutaneous, intraventricular, transdermal,
interdermal, rectal, intravaginal, intraperitoneal, topical (e.g.
by powders, ointments, creams, gels , lotions , and / or drops ),
mucosal, nasal , buccal , enteral, vitreal, intratumoral, sublin
gual ; by intratracheal instillation , bronchial instillation , and /
or inhalation ; as an oral spray, nasal spray , and / or aerosol,
animals administered a single intramuscular dose of formu- 50 and /or through a portal vein catheter.
lated mmRNA ranging from 50 mg/kg to doses as low as 1
In some embodiments , mmRNA are administered by
ng /kg with a preferred range to be 10 mg/kg to 100 ng /kg. systemic intravenous injection . In specific embodiments,
If the animal tested is a mouse the maximum dose can be mmRNA may be administered intravenously and / or orally.
roughly 1 mg mmRNA or as low as 0.02 ng mmRNA if In specific embodiments, mmRNA may be administered in
administered once into the hind limb. Likewise for subcu- 55 a way which allows the mmRNA to cross the blood -brain
taneous administration , mmRNA - containing lipidoid formu- barrier, vascular barrier, or other epithelial barrier.
lations are diluted in PBS before the animals are adminisInjectable preparations, for example, sterile injectable
tered a single subcutaneous dose of formulated mmRNA aqueous or oleaginous suspensions may be formulated
ranging from 400 mg/kg- to doses as low as 1 ng /kg. A according to the known art using suitable dispersing agents,
preferred dosage range comprises 80 mg /kg to 100 ng /kg. If 60 wetting agents, and / or suspending agents. Sterile injectable
the animal tested is a mouse , the maximum dose adminis- preparations may be sterile injectable solutions , suspen
tered can be roughly 8 mg mmRNA or as low as 0.02 ng sions , and / or emulsions in nontoxic parenterally acceptable
mmRNA if the dose is administered once subcutaneously.

diluents and / or solvents, for example, as a solution in

ous injection is maximally 0.2 ml for a 20 gram mouse . The

and isotonic sodium chloride solution . Sterile , fixed oils are

It is preferred that the volume of a single intramuscular 1,3 - butanediol. Among the acceptable vehicles and solvents
injection is maximally 0.025 ml and of a single subcutane- 65 that may be employed are water, Ringer's solution , U.S.P.,
dose of the mmRNA administered to the animal is calculated

conventionally employed as a solvent or suspending

US 10,898,574 B2
25

26
embodiments, the desired dosage may be delivered using

medium . For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. Fatty
acids
such. as oleic acid can be used in the preparation of
injectables

multiple administrations ( e.g. , two , three, four, five , six ,
seven , eight, nine , ten, eleven, twelve , thirteen , fourteen , or

filtration through a bacterial - retaining filter, and / or by incor
porating sterilizing agents in the form of sterile solid com
positions which can be dissolved or dispersed in sterile
water or other sterile injectable medium prior to use .

herein may be used .
According to the present invention , it has been discovered
that administration of mmRNA in split -dose regimens pro

more administrations ). When multiple administration is
Injectable formulations can be sterilized, for example, by 5 employed , split dosing regimens such as those described

duce higher levels of proteins in mammalian subjects . As
or total daily dose into two or more doses . As used herein ,
a “ single unit dose ” is a dose of any therapeutic administered

Dosageofforms
for local, topical
and/or transdermal
istration
a composition
may include
ointments, adminpastes , 10 used herein, a “ split dose” is the division of single unit dose

creams, lotions , gels , powders , solutions , sprays, inhalants

and / or patches. Additionally, the present invention contem
plates the use of transdermal patches, which often have the

in one dose /at one time/ single route / single point of contact,

added advantage of providing controlled delivery of a com- 15 i.e. , single administration event. As used herein, a “total
pound to the body. Such dosage forms may be prepared, for daily dose” is an amount given or prescribed in 24 hr period .

example, by dissolving and /or dispensing the compound in
the proper medium . Alternatively or additionally , rate may
be controlled by either providing a rate controlling membrane and / or by dispersing the compound in a polymer 20
matrix and /or gel .
Formulations suitable for topical administration include,

It may be administered as a single unit dose . In one
embodiment, the mmRNA of the present invention are
administered to a subject in split doses . The mmRNA may
be formulated in buffer only or in a formulation described
herein .
Modified nucleic acid molecules or complexes may be

mulations may, for example, comprise from about 1 % to
about 10 % ( w / w ) active ingredient, although the concentra
tion of active ingredient may be as high as the solubility
limit of the active ingredient in the solvent. Formulations for 30
topical administration may further comprise one or more of
the additional ingredients described herein .
A pharmaceutical composition may be prepared , pack
aged , and / or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in 35
the form of eye drops including, for example, a 0.1 / 1.0 %
( w / w ) solution and / or suspension of the active ingredient in
an aqueous or oily liquid excipient. Such drops may further

mulated for delivery together, although these methods of
delivery are within the scope of the present disclosure .
Compositions can be administered concurrently with , prior
to , or subsequent to , one or more other desired therapeutics
or medical procedures. In general, each agent will be admin

but are not limited to , liquid and / or semi liquid preparations used or administered in combination with one or more other
such as liniments, lotions , oil in water and /or water in oil therapeutic , prophylactic, diagnostic, or imaging agents. By
emulsions such as creams, ointments and / or pastes , and / or 25 “ in combination with ,” it is not intended to imply that the
solutions and / or suspensions. Topically -administrable for- agents must be administered at the same time and / or for

comprise buffering agents, salts , and / or one or more other of
any additional ingredients described herein . Other opthal- 40
mically -administrable formulations which are useful include
those which comprise the active ingredient in microcrystalline form and / or in a liposomal preparation. Ear drops and / or
eye drops are contemplated as being within the scope of this
invention .
45
In general the most appropriate route of administration
will depend upon a variety of factors including the nature of

the mmRNA to be delivered (e.g. , its stability in the environment of the gastrointestinal tract, bloodstream , etc. ), the
condition of the patient ( e.g. , whether the patient is able to 50
tolerate particular routes of administration ), etc. The invention encompasses the delivery of the mmRNA by any
appropriate route taking into consideration likely advances
in the sciences of drug delivery.
In certain embodiments, compositions in accordance with 55
the present invention may be administered at dosage levels
sufficient to deliver from about 0.0001 mg /kg to about 100
mg/kg, from about 0.01 mg/kg to about 50 mg /kg, from
about 0.1 mg/kg to about 40 mg /kg, from about 0.5 mg /kg
to about 30 mg /kg, from about 0.01 mg /kg to about 10 60
mg/kg, from about 0.1 mg /kg to about 10 mg /kg, or from
about 1 mg /kg to about 25 mg/kg, of subject body weight per
day, one or more times a day, to obtain the desired thera-

istered at a dose and /or on a time schedule determined for

that agent. In some embodiments, the present disclosure

encompasses
deliverycompositions
of pharmaceutical
, prophylactic
diagnostic, ortheimaging
in combination
with,

agents that may improve their bioavailability, reduce and /or
modify their metabolism , inhibit their excretion, and / or
modify their distribution within the body.
It will further be appreciated that therapeutically, prophy
lactically, diagnostically, or imaging active agents utilized in
combination may be administered together in a single com
position or administered separately in different composi
tions . In general, it is expected that agents utilized in
combination with be utilized at levels that do not exceed the
levels at which they are utilized individually. In some
embodiments, the levels utilized in combination will be
lower than those utilized individually. In one embodiment,
the combinations, each or together may be administered
according to the split dosing regimens described herein .
The particular combination of therapies ( therapeutics or
procedures) to employ in a combination regimen will take
into account compatibility of the desired therapeutics and /or
procedures and the desired therapeutic effect to be achieved .
It will also be appreciated that the therapies employed may
achieve a desired effect for the same disorder ( for example ,
a composition useful for treating cancer in accordance with
the invention may be administered concurrently with a
chemotherapeutic agent ), or they may achieve different
effects (e.g. , control of any adverse effects ).
Compositions containing mmRNAs are formulated for
administration intramuscularly, transarterially , intraocularly,
vaginally , rectally, intraperitoneally, intravenously, intrana

peutic , diagnostic or prophylactic effect. The desired dosage sally, subcutaneously, endoscopically, transdermally, intra
may be delivered three times a day, two times a day, once a 65 muscularly, intraventricularly, intradermally, intrathecally,
day, every other day, every third day, every week, every two topically ( e.g. by powders, ointments , creams, gels , lotions ,
weeks, every three weeks , or every four weeks . In certain and / or drops), mucosally, nasal , enterally, intratumorally , by

US 10,898,574 B2
27

28

intratracheal instillation , bronchial instillation , and / or inha- of interest in a substantial percentage of cells contained
lation ; nasal spray and / or aerosol, and /or through a portal within a predetermined volume of the target tissue . In some
vein catheter.
embodiments, the composition includes a plurality of dif
The compositions may also be formulated for direct ferent ribonucleic acids , where one or more than one of the
delivery to an organ or tissue in any of several ways in the 5 ribonucleic acids is engineered to avoid an innate immune
art including, but not limited to , direct soaking or bathing, response of a cell into which the ribonucleic acid enters , and
via a catheter, by gels , powder, ointments , creams , gels , where one or more than one of the ribonucleic acids encodes
lotions , and / or drops, by using substrates such as fabric or a polypeptide of interest. Optionally, the composition also
biodegradable materials coated or impregnated with the contains a cell penetration agent to assist in the intracellular
compositions , and the like . In some embodiments , the com- 10 delivery of the ribonucleic acid . A determination is made of
position is formulated for extended release . In specific the dose of the composition required to produce the poly
embodiments, mmRNA molecules or complexes, and / or peptide of interest in a substantial percentage of cells
pharmaceutical, prophylactic , diagnostic , or imaging com- contained within the predetermined volume of the target
positions thereof, may be administered in a way which tissue ( generally, without inducing significant production of
allows the mmRNA molecules or complex to cross the 15 the polypeptide of interest in tissue adjacent to the prede
blood- brain barrier, vascular barrier, or other epithelial bar- termined volume, or distally to the target tissue ) . Subsequent
rier.
to this determination , the determined dose is introduced
In some aspects of the invention, the nucleic acids (par- directly into the tissue of the mammalian subject.
ticularly ribonucleic acids encoding polypeptides) are spaFormulations which may be administered intramuscularly
tially retained within or proximal to a target tissue . Provided 20 and / or subcutaneously may include , but are not limited to ,
are method of providing a composition to a target tissue of polymers, copolymers, and gels. The polymers, copolymers
a mammalian subject by contacting the target tissue (which and / or gels may further be adjusted to modify release
contains one or more target cells ) with the composition kinetics by adjusting factors such as , but not limited to ,
under conditions such that the composition, in particular the molecular weight, particle size , payload and / or ratio of the
nucleic acid component ( s) of the composition, is substan- 25 monomers . As a non -limiting example, formulations admin
tially retained in the target tissue , meaning that at least 10 , istered intramuscularly and / or subcutaneously may include a
20 , 30 , 40 , 50 , 60 , 70 , 80 , 85 , 90 , 95 , 96 , 97 , 98 , 99 , 99.9 , copolymer such as poly (lactic - co - glycolic acid) .
99.99 or greater than 99.99 % of the composition is retained
Localized delivery of the compositions described herein
in the target tissue . Advantageously, retention is determined may be administered by methods such as , but not limited to ,
by measuring the amount of the nucleic acid present in the 30 topical delivery, ocular delivery, transdermal delivery, and
composition that enters one or more target cells . For the like . The composition may also be administered locally
example , at least 1 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 70 , 80 , 85 , 90 , to a part of the body not normally available for localized
95 , 96 , 97 , 98 , 99 , 99.9 , 99.99 or greater than 99.99 % of the delivery such as , but limited to , when a subject's body
nucleic acids administered to the subject are present intra- is open to the environment during treatment. The composi
cellularly at a period of time following administration . For 35 tion may further be delivered by bathing, soaking and / or
example , intramuscular injection to a mammalian subject is surrounding the body part with the composition.
performed using an aqueous composition containing a riboHowever, the present disclosure encompasses the delivery
nucleic acid and a transfection reagent, and retention of the of mmRNA molecules or complexes, and / or pharmaceutical,
composition is determined by measuring the amount of the prophylactic , diagnostic , or imaging compositions thereof,
40 by any appropriate route taking into consideration likely
ribonucleic acid present in the muscle cells .
Aspects of the invention are directed to methods of advances in the sciences of drug delivery .
providing a composition to a target tissue of a mammalian
The level or concentration of a mmRNA may be charac

subject, by contacting the target tissue (containing one or terized using exosomes . A level or concentration of the
more target cells ) with the composition under conditions mmRNA in exosomes can represent an expression level ,
such that the composition is substantially retained in the 45 presence , absence , truncation or alteration of the mmRNA .

target tissue . The composition contains an effective amount The level or concentration may be determined by a method
of a ribonucleic acid engineered to avoid an innate immune such as , but not limited to , an assay using construct specific
response of a cell into which the ribonucleic acid enters, probes , cytometry , qRT- PCR , realtime PCR , PCR , flow
where the ribonucleic acid contains a nucleotide sequence cytometry, electrophoresis, mass spectrometry, or combina
encoding a polypeptide of interest, under conditions such 50 tions thereof. Further, the level or concentration may be
that the polypeptide of interest is produced in at least one associated with a clinical phenotype. For analysis , the exo
target cell . The compositions generally contain a cell pen- some may be isolated by a method such as , but not limited
etration agent, although “naked ” nucleic acid ( such as to , immunohistochemcial methods such as enzyme linked
nucleic acids without a cell penetration agent or other agent) immunosorbant assay ( ELISA) methods, size exclusion
is also contemplated , and a pharmaceutically acceptable 55 chromatography, density gradient centrifugation, differential
carrier.
centrifugation, nanomembrane ultrafiltration, immunoabsor
In some circumstances, the amount of a protein produced bent capture, affinity purification, microfluidic separation, or
by cells in a tissue is desirably increased . Preferably , this combinations thereof.
increase in protein production is spatially restricted to cells Pharmaceutical Compositions
within the target tissue . Thus, provided are methods of 60 When administered to a subject the pharmaceutical com
increasing production of a protein of interest in a tissue of a positions described herein may provide proteins which have

mammalian subject. A composition is provided that contains been generated from modified mRNAs. Pharmaceutical
a ribonucleic acid that is engineered to avoid an innate compositions may optionally comprise one or more addi
immune response of a cell into which the ribonucleic acid tional therapeutically active substances. In accordance with
enters and encodes the polypeptide of interest and the 65 some embodiments, a method of administering pharmaceu
composition is characterized in that a unit quantity of tical compositions comprising one or more proteins to be
composition has been determined to produce the polypeptide delivered to a subject in need thereof is provided. In some

US 10,898,574 B2
29

30

embodiments , compositions are administered to human sub-

such as by producing any undesirable biological effect or

jects . In a further embodiment, the compositions are administered to a subject who is a patient.
Pharmaceutical compositions may optionally comprise

one or more additional therapeutically active substances .
In some embodiments, compositions are administered to
humans. For the purposes of the present disclosure, the
phrase “ active ingredient generally refers to a mmRNA to

otherwise interacting in a deleterious manner with any other
component ( s) of the pharmaceutical composition, its use is
contemplated to be within the scope of this invention .
5

In some embodiments , a pharmaceutically acceptable
excipient is at least 95 % , at least 96 % , at least 97 % , at least
98 % , at least 99 % , or 100 % pure . In some embodiments, an

excipient is approved for use in humans and for veterinary

be delivered as described herein .

use .

Although the descriptions of pharmaceutical composi- 10
tions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to
humans, it will be understood by the skilled artisan that such
compositions are generally suitable for administration to
animals of all sorts. Modification of pharmaceutical com- 15
positions suitable for administration to humans in order to
render the compositions suitable for administration to various animals is well understood , and the ordinarily skilled
veterinary pharmacologist can design and / or perform such
modification with merely ordinary, if any, experimentation. 20
Subjects to which administration of the pharmaceutical
compositions is contemplated include, but are not limited to ,
humans and / or other primates ; mammals, including commercially relevant mammals such as cattle, pigs , horses ,
sheep, cats, dogs , mice , and / or rats ; and / or birds , including 25
commercially relevant birds such as chickens, ducks, geese ,
and / or turkeys.
Formulations of the pharmaceutical compositions
described herein may be prepared by any method known or
hereafter developed in the art of pharmacology. In general, 30
such preparatory methods include the step of bringing the
active ingredient into association with an excipient and /or
if nec
one or more other accessory ingredients , and
essary and / or desirable , shaping and / or packaging the prod35
uct into a desired single- or multi - dose unit.
A pharmaceutical composition in accordance with the
invention may be prepared, packaged , and / or sold in bulk , as
a single unit dose , and / or as a plurality of single unit doses .
As used herein, a " unit dose” is discrete amount of the
pharmaceutical composition comprising a predetermined 40
amount of the active ingredient. The amount of the active
ingredient is generally equal to the dosage of the active
ingredient which would be administered to a subject and / or
a convenient fraction of such a dosage such as , for example,
one -half or one - third of such a dosage .
45
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and /or any additional ingredients in a pharmaceutical composition in accordance with
the invention will vary, depending upon the identity, size ,
and / or condition of the subject treated and further depending 50
upon the route by which the composition is to be administered. By way of example, the composition may comprise
between 0.1 % and 100% ( w / w ) active ingredient.
Pharmaceutical formulations may additionally comprise a
pharmaceutically acceptable excipient, which, as used 55
herein , includes any and all solvents, dispersion media ,
diluents, or other liquid vehicles, dispersion or suspension
aids , surface active agents, isotonic agents, thickening or

In some embodiments , an excipient is approved by United
States Food and Drug Administration. In some embodi
ments , an excipient is pharmaceutical grade. In some
embodiments, an excipient meets the standards of the United
States Pharmacopoeia ( USP ) , the European Pharmacopoeia
(EP ) , the British Pharmacopoeia, and / or the International
Pharmacopoeia .
Pharmaceutically acceptable excipients used in the manu
facture of pharmaceutical compositions include , but are not
limited to , inert diluents, dispersing and /or granulating
agents, surface active agents and /or emulsifiers, disintegrat
ing agents , binding agents, preservatives, buffering agents ,
lubricating agents, and / or oils . Such excipients may option
ally be included in pharmaceutical formulations. Excipients
such as cocoa butter and suppository waxes , coloring agents ,
coating agents, sweetening, flavoring, and / or perfuming
agents can be present in the composition, according to the
judgment of the formulator.
Exemplary diluents include, but are not limited to , cal
cium carbonate, sodium carbonate, calcium phosphate ,
dicalcium phosphate , calcium sulfate, calcium hydrogen
phosphate, sodium phosphate lactose , sucrose , cellulose ,
microcrystalline cellulose , kaolin, mannitol, sorbitol, inosi
tol , sodium chloride, dry starch , cornstarch , powdered sugar,
etc. , and / or combinations thereof.
Exemplary granulating and / or dispersing agents include ,
but are not limited to , potato starch , corn starch , tapioca
starch, sodium starch glycolate , clays , alginic acid , guar
gum , citrus pulp , agar, bentonite, cellulose and wood prod
ucts , natural sponge , cation -exchange resins , calcium car
bonate, silicates, sodium carbonate, cross - linked poly ( vinyl
pyrrolidone ) ( crospovidone), sodium carboxymethyl starch
( sodium starch glycolate ) , carboxymethyl cellulose , cross
linked sodium carboxymethyl cellulose ( croscarmellose ),

methylcellulose, pregelatinized starch ( starch 1500 ), micro

crystalline starch , water insoluble starch , calcium car
boxymethyl cellulose , magnesium aluminum silicate
( Veegum ), sodium lauryl sulfate , quaternary ammonium

compounds, etc. , and / or combinations thereof.
Exemplary surface active agents and / or emulsifiers

include, but are not limited to , natural emulsifiers (e.g.
acacia , agar, alginic acid, sodium alginate, tragacanth , chon
drux , cholesterol , xanthan , pectin, gelatin, egg yolk, casein ,
wool fat, cholesterol, wax , and lecithin ), colloidal clays (e.g.
bentonite [ aluminum silicate ) and Veegum [magnesium
aluminum silicate ] ) , long chain amino acid derivatives, high
molecular weight alcohols ( e.g. stearyl alcohol , cetyl alco
hol , oleyl alcohol , triacetin monostearate, ethylene glycol
distearate, glyceryl monostearate , and propylene glycol

emulsifying agents, preservatives, solid binders, lubricants monostearate, polyvinyl alcohol ) , carbomers (e.g. carboxy
and the like, as suited to the particular dosage form desired . 60 polymethylene, polyacrylic acid , acrylic acid polymer , and
Remington's The Science and Practice of Pharmacy, 21st carboxyvinyl polymer ), carrageenan , cellulosic derivatives
Edition, A. R. Gennaro ( Lippincott, Williams & Wilkins, (e.g. carboxymethylcellulose sodium , powdered cellulose ,
Baltimore, Md . , 2006 ; incorporated herein by reference ) hydroxymethyl cellulose , hydroxypropyl cellulose ,
discloses various excipients used in formulating pharmaceu- hydroxypropyl methylcellulose , methylcellulose ), sorbitan
tical compositions and known techniques for the preparation 65 fatty acid esters ( e.g. polyoxyethylene sorbitan monolaurate
thereof. Except insofar as any conventional excipient [ TWEEN?20 ), polyoxyethylene sorbitan [ TWEEN?60 ],
medium is incompatible with a substance or its derivatives , polyoxyethylene sorbitan monooleate [ TWEEN®80] , sorbi

US 10,898,574 B2
31

32

tan monopalmitate [ SPANR40 ), sorbitan monostearate PLUS® , PHENONIP® , methylparaben, GERMALL®115 ,
[ SPAN®60 ), sorbitan tristearate [SPAN®65 ], glyceryl GERMABEN?II, NEOLONETM , KATHONTM , and /or
monooleate , sorbitan monooleate [SPAN®80 ]), polyoxyeth- EUXYL® .
ylene esters (e.g. polyoxyethylene monostearate
Exemplary buffering agents include , but are not limited
[MYRJ®45 ], polyoxyethylene hydrogenated castor oil , 5 to , citrate buffer solutions , acetate buffer solutions, phos
polyethoxylated castor oil , polyoxymethylene stearate, and
buffer solutions , ammonium chloride, calcium carbon
SOLUTOL® ), sucrose fatty acid esters , polyethylene glycol phate
ate
,
calcium
chloride , calcium citrate , calcium glubionate,
fatty acid esters ( e.g. CREMOPHOR® ), polyoxyethylene calcium gluceptate
, calcium gluconate, D - gluconic acid ,
ethers , ( e.g. polyoxyethylene lauryl ether [ BRIJR30 ] ) , poly calcium glycerophosphate
, calcium lactate , propanoic acid ,

(nolamine
vinyl-pyrrolidone
), diethylene glycol monolaurate, trietha. 10 calcium levulinate, pentanoic acid, dibasic calcium phos
oleate , sodium oleate , potassium oleate , ethyl

, phosphoric acid , tribasic calcium phosphate, calcium
oleate, oleic acid, ethyl laurate, sodium lauryl sulfate , phate
hydroxide
phosphate, potassium acetate, potassium chlo
PLURONIC®F 68 , POLOXAMER®188 , cetrimonium bro ride , potassium
gluconate, potassium mixtures, dibasic
mide, cetylpyridinium chloride , benzalkonium chloride, potassium phosphate
, monobasic potassium phosphate ,
15
docusate sodium , etc. and /or combinations thereof.
Exemplary binding agents include, but are not limited to , potassium phosphate mixtures , sodium acetate, sodium
starch ( e.g. cornstarch and starch paste) ; gelatin ; sugars (e.g. bicarbonate , sodium chloride, sodium citrate , sodium lac
sucrose , glucose , dextrose, dextrin , molasses , lactose , lacti- tate , dibasic sodium phosphate , monobasic sodium phos
tol , mannitol ); natural and synthetic gums (e.g. acacia , phate, sodium phosphate mixtures , tromethamine, magne
sodium alginate, extract of Irish moss , panwar gum , ghatti 20 sium hydroxide, aluminum hydroxide, alginic acid ,
gum , mucilage of isapol husks, carboxymethylcellulose , pyrogen - free water, isotonic saline , Ringer's solution , ethyl
methylcellulose, ethylcellulose, hydroxyethylcellulose, alcohol, etc. , and /or combinations thereof.
hydroxypropyl cellulose , hydroxypropyl methylcellulose ,
Exemplary lubricating agents include, but are not limited
microcrystalline cellulose , cellulose acetate, poly ( vinyl - pyr- to , magnesium stearate, calcium stearate , stearic acid, silica ,
rolidone ) , magnesium aluminum silicate ( VEEGUM® ), and 25 talc , malt , glyceryl behanate, hydrogenated vegetable oils ,
larch arabogalactan ); alginates; polyethylene oxide; poly- polyethylene glycol , sodium benzoate , sodium acetate ,
ethylene glycol ; inorganic calcium salts ; silicic acid ;
polymethacrylates; waxes; water; alcohol, etc .; and combinations thereof.

Exemplary preservatives may include , but are not limited
to , antioxidants, chelating agents , antimicrobial preservatives , antifungal preservatives, alcohol preservatives, acidic
preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to , alpha tocopherol,
ascorbic acid, acorbyl palmitate , butylated hydroxyanisole,
butylated hydroxytoluene, monothioglycerol, potassium
metabisulfite , propionic acid , propyl gallate , sodium ascorbate, sodium bisulfite, sodium metabisulfite , and /or sodium
sulfite . Exemplary chelating agents include ethylenediaminetetraacetic acid ( EDTA ), citric acid monohydrate, disodium edetate , dipotassium edetate, edetic acid , fumaric acid ,
malic acid , phosphoric acid, sodium edetate, tartaric acid ,
and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to , benzalkonium chloride , benzethonium chloride, benzyl alcohol , bronopol, cetrimide , cetylpyridinium chloride, chlorhexidine,
chlorobutanol, chlorocresol, chloroxylenol, cresol , ethyl
alcohol , glycerin, hexetidine, imidurea , phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol , and / or thimerosal. Exemplary antifungal preservatives include , but are not limited to , butyl paraben ,
methyl paraben , ethyl paraben , propyl paraben, benzoic
acid, hydroxybenzoic acid, potassium benzoate , potassium
sorbate, sodium benzoate, sodium propionate, and / or sorbic
acid . Exemplary alcohol preservatives include, but are not
limited to , ethanol, polyethylene glycol, phenol, phenolic
compounds , bisphenol, chlorobutanol, hydroxybenzoate,

sodium chloride, leucine, magnesium lauryl sulfate, sodium
lauryl sulfate, etc. , and combinations thereof.

Exemplary oils include , but are not limited to , almond ,
30 apricot kernel, avocado , babassu, bergamot, black current
seed, borage, cade , camomile , canola, caraway, carnauba ,
castor, cinnamon , cocoa butter, coconut, cod liver, coffee ,
corn , cotton seed, emu, eucalyptus, evening primrose , fish ,
flaxseed , geraniol, gourd , grape seed , hazel nut, hyssop ,
35 isopropyl myristate, jojoba, kukui nut, lavandin , lavender,
lemon, litsea cubeba, macademia nut, mallow , mango seed ,
meadowfoam seed , mink , nutmeg, olive, orange , orange
roughy, palm , palm kernel, peach kernel, peanut, poppy
seed, pumpkin seed , rapeseed, rice bran , rosemary, saf
40 flower, sandalwood, sasquana, savoury, sea buckthorn ,
sesame, shea butter, silicone , soybean , sunflower, tea tree ,
thistle, tsubaki , vetiver, walnut, and wheat germ oils . Exem
plary oils include, but are not limited to , butyl stearate ,
caprylic triglyceride, capric triglyceride, cyclomethicone,
45 diethyl sebacate , dimethicone 360 , isopropyl myristate, min
eral oil , octyldodecanol, oleyl alcohol , silicone oil , and / or
combinations thereof.
Liquid dosage forms for oral and parenteral administra
tion include , but are not limited to , pharmaceutically accept
50 able emulsions, microemulsions, solutions, suspensions,
syrups, and / or elixirs . In addition to active ingredients,
liquid dosage forms may comprise inert diluents commonly
used in the art such as , for example, water or other solvents ,
solubilizing agents and emulsifiers such as ethyl alcohol ,
55 isopropyl alcohol, ethyl carbonate, ethyl acetate , benzyl
alcohol, benzyl benzoate , propylene glycol, 1,3 -butylene
glycol , dimethylformamide, oils (in particular, cottonseed ,

and /or phenylethyl alcohol. Exemplary acidic preservatives groundnut, corn , germ , olive , castor, and sesame oils ) ,
include , but are not limited to , vitamin A , vitamin C , vitamin glycerol, tetrahydrofurfuryl alcohol , polyethylene glycols
E , beta -carotene, citric acid , acetic acid , dehydroacetic acid , 60 and fatty acid esters of sorbitan, and mixtures thereof.
ascorbic acid, sorbic acid , and /or phytic acid . Other preser- Besides inert diluents, oral compositions can include adju
vatives include , but are not limited to , tocopherol, tocoph- vants such as wetting agents, emulsifying and suspending
erol acetate , deteroxime mesylate , cetrimide, butylated agents, sweetening, flavoring, and /or perfuming agents . In
hydroxyanisol ( BHA ) , butylated hydroxytoluened ( BHT ) , certain embodiments for parenteral administration , compo
ethylenediamine, sodium lauryl sulfate ( SLS ) , sodium lauryl 65 sitions are mixed with solubilizing agents such as Cremo
ether sulfate ( SLES ) , sodium bisulfite, sodium metabisulfite , phor® , alcohols , oils , modified oils , glycols , polysorbates,
potassium sulfite, potassium metabisulfite , GLYDANT cyclodextrins, polymers , and / or combinations thereof.

US 10,898,574 B2
33
General considerations in the formulation and / or manu-

facture of pharmaceutical agents may be found, for example,
in Remington : The Science and Practice of Pharmacy 21st
ed . , Lippincott Williams & Wilkins, 2005 ( incorporated
herein by reference ).
In order to prolong the effect of an active ingredient, it is
often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may
be accomplished by the use of a liquid suspension of
crystalline or amorphous material with poor water solubility.
The rate of absorption of the drug then depends upon its rate
of dissolution which , in turn, may depend upon crystal size
and crystalline form . Alternatively, delayed absorption of a
parenterally administered drug form is accomplished by
dissolving or suspending the drug in an oil vehicle . Injectable depot forms are made by forming microencapsule
matrices of the drug in biodegradable polymers such as
polylactide - polyglycolide . Depending upon the ratio of drug
to polymer and the nature of the particular polymer
employed , the rate of drug release can be controlled .
Examples of other biodegradable polymers include poly
( orthoesters ) and poly (anhydrides ). Depot injectable formulations are prepared by entrapping the drug in liposomes or
microemulsions which are compatible with body tissues .
Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing
compositions with suitable non - irritating excipients such as
cocoa butter, polyethylene glycol or a suppository wax
which are solid at ambient temperature but liquid at body
temperature and therefore melt in the rectum or vaginal
cavity and release the active ingredient. Solid dosage forms
for oral administration include capsules , tablets , pills , powders, and granules. In such solid dosage forms, an active
ingredient is mixed with at least one inert, pharmaceutically
acceptable excipient such as sodium citrate or dicalcium
phosphate and/ or fillers or extenders ( e.g. starches, lactose ,
sucrose , glucose , mannitol, and silicic acid ), binders (e.g.
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone , sucrose , and acacia) , humectants (e.g. glycerol),
disintegrating agents (e.g. agar, calcium carbonate, potato or
tapioca starch , alginic acid, certain silicates, and sodium
carbonate ), solution retarding agents ( e.g. paraffin ), absorption accelerators (e.g. quaternary ammonium compounds ),
wetting agents (e.g. cetyl alcohol and glycerol monostearate ) , absorbents ( e.g. kaolin and bentonite clay ) , and lubricants ( e.g. talc , calcium stearate , magnesium stearate , solid
polyethylene glycols , sodium lauryl sulfate ), and mixtures
thereof. In the case of capsules , tablets and pills , the dosage
form may comprise buffering agents .
Solid compositions of a similar type may be employed as
fillers in soft and hard - filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like . Solid dosage
forms of tablets, dragees , capsules , pills , and granules can be
prepared with coatings and shells such as enteric coatings
and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying
agents and can be of a composition that they release the
active ingredient ( s) only, or preferentially, in a certain part
of the intestinal tract, optionally, in a delayed manner .
Examples of embedding compositions which can be used
include polymeric substances and waxes . Solid compositions of a similar type may be employed as fillers in soft and
hard - filled gelatin capsules using such excipients as lactose
or milk sugar as well as high molecular weight polyethylene
glycols and the like.

34
Dosage forms for topical and /or transdermal administra

5

10

15

20

25

30

35
40
45

50

55

60

65

tion of a composition may include ointments , pastes ,
creams, lotions , gels , powders , solutions , sprays, inhalants
and /or patches. Generally, an active ingredient is admixed
under sterile conditions with a pharmaceutically acceptable
excipient and / or any needed preservatives and / or buffers as
may be required. Topically -administrable formulations may,
for example, comprise from about 1 % to about 10 % ( w / w )
active ingredient, although the concentration of active ingre
dient may be as high as the solubility limit of the active
ingredient in the solvent. Formulations for topical adminis
tration may further comprise one or more of the additional
ingredients described herein .
A pharmaceutical composition may be prepared , pack
aged , and / or sold in a formulation suitable for pulmonary
administration via the buccal cavity. Such a formulation may
comprise dry particles which comprise the active ingredient
and which have a diameter in the range from about 0.5 nm
to about 7 nm or from about 1 nm to about 6 nm . Such
compositions are suitably in the form of dry powders for
administration using a device comprising a dry powder
reservoir to which a stream of propellant may be directed to
disperse the powder and / or using a self propelling solvent/
powder dispensing container such as a device comprising
the active ingredient dissolved and / or suspended in a low
boiling propellant in a sealed container. Such powders
comprise particles wherein at least 98 % of the particles by
weight have a diameter greater than 0.5 nm and at least 95 %
of the particles by number have a diameter less than 7 nm .
Alternatively, at least 95 % of the particles by weight have a
diameter greater than 1 nm and at least 90 % of the particles
by number have a diameter less than 6 nm . Dry powder
compositions may include a solid fine powder diluent such
as sugar and are conveniently provided in a unit dose form .
Low boiling propellants generally include liquid propel
lants having a boiling point of below 65 ° F. at atmospheric
pressure . Generally the propellant may constitute 50% to
99.9 % ( w / w ) of the composition, and active ingredient may
constitute 0.1 % to 20 % ( w / w ) of the composition. A pro
pellant may further comprise additional ingredients such as
a liquid non - ionic and / or solid anionic surfactant and / or a
solid diluent ( which may have a particle size of the same
order as particles comprising the active ingredient ).
Pharmaceutical compositions formulated for pulmonary
delivery may provide an active ingredient in the form of
droplets of a solution and / or suspension . Such formulations
may be prepared, packaged , and / or sold as aqueous and /or
dilute alcoholic solutions and / or suspensions, optionally
sterile, comprising active ingredient, and may conveniently
be administered using any nebulization and / or atomization
device . Such formulations may further comprise one or
more additional ingredients including , but not limited to , a
flavoring agent such as saccharin sodium , a volatile oil , a
buffering agent, a surface active agent, and / or a preservative
such as methylhydroxybenzoate. Droplets provided by this
route of administration may have an average diameter in the
range from about 0.1 nm to about 200 nm .
Formulations described herein as being useful for pulmo
nary delivery are useful for intranasal delivery of a phar
maceutical composition . Another formulation suitable for
intranasal administration is a coarse powder comprising the
active ingredient and having an average particle from about
0.2 um to 500 um . Such a formulation is administered in the
manner in which snuff is taken, i.e. by rapid inhalation
through the nasal passage from a container of the powder
held close to the nose .

US 10,898,574 B2
36

35

Formulations suitable for nasal administration may, for
example, comprise from about as little as 0.1 % ( w / w ) and as
much as 100 % ( w / w ) of active ingredient, and may comprise
one or more of the additional ingredients described herein .
A pharmaceutical composition may be prepared , packaged, 5

and / or sold in a formulation suitable for buccal administration . Such formulations may , for example , be in the form of
tablets and / or lozenges made using conventional methods,
and may, for example, 0.1 % to 20% ( w / w ) active ingredient,
the balance comprising an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein . Alternately, formulations suitable for buccal administration may comprise a
powder and / or an aerosolized and / or atomized solution
and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when
dispersed, may have an average particle and / or droplet size
in the range from about 0.1 nm to about 200 nm , and may
further comprise one or more of any additional ingredients
described herein
Properties of the Pharmaceutical Compositions
The pharmaceutical compositions described herein can be
characterized by one or more of the following properties :
Bioavailability
The mmRNA molecules , when formulated into a composition with a delivery agent as described herein , can exhibit
an increase in bioavailability as compared to a composition
lacking a delivery agent as described herein . As used herein ,
the term “ bioavailability ” refers to the systemic availability
of a given amount of a mmRNA molecule administered to a
mammal. Bioavailability can be assessed by measuring the
area under the curve ( AUC ) or the maximum serum or
plasma concentration (Cmar) of the unchanged form of a
compound following administration of the compound to a
mammal. AUC is a determination of the area under the curve
plotting the serum or plasma concentration of a compound
along the ordinate ( Y -axis) against time along the abscissa
(X - axis ) . Generally, the AUC for a particular compound can
be calculated using methods known to those of ordinary skill
in the art and as described in G. S. Banker, Modern Pharmaceutics , Drugs and the Pharmaceutical Sciences , v. 72 ,
Marcel Dekker, New York , Inc. , 1996 , herein incorporated
by reference.
The Cmax value is the maximum concentration of the
compound achieved in the serum or plasma of a mammal
following administration of the compound to the mammal.
The Cmax value of a particular compound can be measured
using methods known to those of ordinary skill in the art.

10

15
20

25

30

35

40

45

composition as compared to the therapeutic window of the
administered mmRNA molecule composition lacking a
delivery agent as described herein . As used herein " thera
peutic window ” refers to the range of plasma concentrations,
or the range of levels of therapeutically active substance at

the site of action , with a high probability of eliciting a
therapeutic effect. In some embodiments, the therapeutic
window of the mmRNA molecule when co - administered
with a delivery agent as described herein can increase by at
least about 2 % , at least about 5 % , at least about 10 % , at least
about 15 % , at least about 20% , at least about 25 % , at least
about 30 % , at least about 35 % , at least about 40 % , at least
about 45 % , at least about 50% , at least about 55 % , at least
about 60 % , at least about 65 % , at least about 70 % , at least
about 75 % , at least about 80% , at least about 85 % , at least
about 90 % , at least about 95 % , or about 100 % .
Volume of Distribution
The mmRNA molecules , when formulated into a compo
sition as described herein , can exhibit an improved volume
of distribution (V dist ). The volume of distribution (V dist)
relates the amount of the drug in the body to the concen
tration of the drug in the blood or plasma . As used herein , the
term “ volume of distribution ” refers to the fluid volume that
would be required to contain the total amount of the drug in
the body at the same concentration as in the blood or plasma :
Vdist equals the amount of drug in the body /concentration of
drug in blood or plasma . For example, for a 10 mg dose and
a plasma concentration of 10 mg / L , the volume of distribu
tion would be 1 liter. The volume of distribution reflects the
extent to which the drug is present in the extravascular
tissue . A large volume of distribution reflects the tendency of
a compound to bind to the tissue components compared with
plasma protein binding . In a clinical setting, Vadist can be
used to determine a loading dose to achieve a steady state
concentration . In some embodiments , the volume of distri
bution of the mmRNA molecule when co -administered with
a delivery agent as described herein can decrease at least
about 2 % , at least about 5 % , at least about 10 % , at least
about 15 % , at least about 20% , at least about 25 % , at least
about 30 % , at least about 35 % , at least about 40 % , at least
about 45 % , at least about 50% , at least about 55 % , at least
about 60 % , at least about 65 % , at least about 70 % .
Devices and Methods for Multi - Administration
Methods and devices for multi -administration may be
employed to deliver the mmRNA of the present invention
according to the split dosing regimens taught herein . Such
methods and devices are described below.
Method and devices known in the art for multi-adminis

The phrases " increasing bioavailability " or " improving the tration to cells , organs and tissues are contemplated for use
pharmacokinetics , " as used herein mean that the systemic 50 in conjunction with the methods and compositions disclosed
availability of a first mmRNA molecule , measured as AUC , herein as embodiments of the present invention. These
Cmax , or Cmin in a mammal is greater, when co - administered include, for example, those methods and devices having
with a delivery agent as described herein , than when such multiple needles , hybrid devices employing for example
co - administration does not take place . In some embodi- lumens or catheters as well as devices utilizing heat, electric
ments
, the bioavailability of the mmRNA molecule can 55 current
or radiation driven mechanisms.
increase by at least about 2 % , at least about 5 % , at least
According to the present invention , these multi -adminis
about 10% , at least about 15 % , at least about 20 % , at least
about 25 % , at least about 30 % , at least about 35 % , at least
about 40% , at least about 45 % , at least about 50 % , at least

tration devices may be utilized to deliver the split doses
contemplated herein .
Suitable devices for use in delivering intradermal phar

about 70% , at least about 75 % , at least about 80 % , at least

needle devices such as those described in U.S. Pat . Nos .

about 55 % , at least about 60 % , at least about 65 % , at least 60 maceutical compositions described herein include short
about 85 % , at least about 90 % , at least about 95 % , or about

4,886,499 ; 5,190,521 ; 5,328,483 ; 5,527,288 ; 4,270,537 ;
5,015,235 ; 5,141,496 ; and 5,417,662 . Intradermal composi
Therapeutic Window
tions may be administered by devices which limit the
The mmRNA molecules , when formulated into a compo- 65 effective penetration length of a needle into the skin , such as
sition as described herein , can exhibit an increase in the those described in PCT publication WO 99/34850 and
therapeutic window of the administered mmRNA molecule functional equivalents thereof. Jet injection devices which
100% .

US 10,898,574 B2
38

37

deliver liquid compositions to the dermis via a liquid jet
injector and /or via a needle which pierces the stratum
corneum and produces a jet which reaches the dermis are
suitable . Jet injection devices are described , for example, in
U.S. Pat . Nos . 5,480,381 ; 5,599,302 ; 5,334,144 ; 5,993,412 ; 5
5,649,912 ; 5,569,189 ; 5,704,911 ; 5,383,851 ; 5,893,397 ;
5,466,220 ; 5,339,163 ; 5,312,335 ; 5,503,627 ; 5,064,413 ;

5,520,639 ; 4,596,556 ; 4,790,824 ; 4,941,880 ; 4,940,460 ; and
PCT publications WO 97/37705 and WO 97/13537 . Ballistic
powder / particle delivery devices which use compressed gas
to accelerate vaccine in powder form through the outer
layers of the skin to the dermis are suitable . Alternatively or
additionally, conventional syringes may be used in the
classical mantoux method of intradermal administration .
A method for delivering therapeutic agents to a solid
tissue has been described by Bahrami et al and is taught for
example in US Patent Publication 20110230839 , the contents of which are incorporated herein by reference in their
entirety. According to Bahrami, an array of needles is
incorporated into a device which delivers a substantially
equal amount of fluid at any location in said solid tissue
along each needle's length .
A device for delivery of biological material across the
biological tissue has been described by Kodgule et al and is
taught for example in US Patent Publication 20110172610 ,
the contents of which are incorporated herein by reference in
their entirety. According to Kodgule, multiple hollow microneedles made of one or more metals and having outer
diameters from about 200 microns to about 350 microns and
lengths of at least 100 microns are incorporated into the
device which delivers peptides , proteins , carbohydrates,
nucleic acid molecules , lipids and other pharmaceutically
active ingredients or combinations thereof.
A delivery probe for delivering a therapeutic agent to a
tissue has been described by Gunday et al and is taught for
example in US Patent Publication 20110270184 , the contents of which are incorporated herein by reference in their
entirety. According to Gunday, multiple needles are incorporated into the device which moves the attached capsules
between an activated position and an inactivated position to
force the agent out of the capsules through the needles .
A multiple -injection medical apparatus has been
described by Assaf and is taught for example in US Patent
Publication 20110218497 , the contents of which are incorporated herein by reference in their entirety. According to
Assaf, multiple needles are incorporated into the device
which has a chamber connected to one or more of said
needles and a means for continuously refilling the chamber
with the medical fluid after each injection .
An at least partially implantable system for injecting a
substance into a patient's body, in particular a penis erection
stimulation system has been described by Forsell and is
taught for example in US Patent Publication 20110196198 ,
the contents of which are incorporated herein by reference in
their entirety . According to Forsell, multiple needles are
incorporated into the device which is implanted along with
one or more housings adjacent the patient's left and right
corpora cavernosa . A reservoir and a pump are also
implanted to supply drugs through the needles.
A method for the transdermal delivery of a therapeutic
effective amount of iron has been described by Berenson and
is taught for example in US Patent Publication
20100130910 , the contents of which are incorporated herein
by reference in their entirety. According to Berenson, multiple needles may be used to create multiple micro channels
in stratum corneum to enhance transdermal delivery of the
ionic iron on an iontophoretic patch .

A method for delivery of biological material across the

biological tissue has been described by Kodgule et al and is
taught for example in US Patent Publication 20110196308 ,

the contents of which are incorporated herein by reference in
their entirety. According to Kodgule , multiple biodegradable

microneedles containing a therapeutic active ingredient are
incorporated in a device which delivers proteins, carbohy
drates, nucleic acid molecules , lipids and other pharmaceu

tically active ingredients or combinations thereof.
10 A transdermal patch comprising a botulinum toxin com
position has been described by Donovan and is taught for
example in US Patent Publication 20080220020 , the con
tents of which are incorporated herein by reference in their
entirety. According to Donovan , multiple needles are incor
15 porated into the patch which delivers botulinum toxin under
stratum corneum through said needles which project through
the stratum corneum of the skin without rupturing a blood
vessel .
A cryoprobe for administration of an active agent to a

20 location of cryogenic treatment has been described by

Toubia and is taught for example in US Patent Publication
20080140061 , the contents ofwhich are incorporated herein

by reference in their entirety . According to Toubia , multiple
needles are incorporated into the probe which receives the

25 active agent into a chamber and administers the agent to the
tissue .
A method for treating or preventing inflammation or
promoting healthy joints has been described by Stock et al
and is taught for example in US Patent Publication
30 20090155186 , the contents of which are incorporated herein
by reference in their entirety. According to Stock , multiple
needles are incorporated in a device which administers
compositions containing signal transduction modulator
compounds.
35 A multi - site injection system has been described by
Kimmell et al and is taught for example in US Patent
Publication 20100256594 , the contents of which are incor
porated herein by reference in their entirety. According to
Kimmell, multiple needles are incorporated into a device

40 which delivers a medication into a stratum corneum through
the needles .
A method for delivering interferons to the intradermal
compartment has been described by Dekker et al and is

taught for example in US Patent Publication 20050181033 ,
45 the contents of which are incorporated herein by reference in

50

55

60

65

their entirety. According to Dekker, multiple needles having
an outlet with an exposed height between 0 and 1 mm are
incorporated into a device which improves pharmacokinet
ics and bioavailability by delivering the substance at a depth
between 0.3 mm and 2 mm .
A method for delivering genes , enzymes and biological
agents to tissue cells has described by Desai and is taught for
example in US Patent Publication 20030073908 , the con
tents of which are incorporated herein by reference in their
entirety. According to Desai, multiple needles are incorpo
rated into a device which is inserted into a body and delivers
a medication fluid through said needles .
A method for treating cardiac arrhythmias with fibroblast
cells has been described by Lee et al and is taught for
example in US Patent Publication 20040005295 , the con
tents of which are incorporated herein by reference in their
entirety. According to Lee , multiple needles are incorporated
into the device which delivers fibroblast cells into the local
region of the tissue .
A method using a magnetically controlled pump for
treating a brain tumor has been described by Shachar et al
and is taught for example in U.S. Pat . No. 7,799,012

US 10,898,574 B2
39

40

(method ) and U.S. Pat . No. 7,799,016 (device ) , the contents uses a stretching assembly to enhance the contact of the
of which are incorporated herein by reference in their needles with the skin and provides a more uniform delivery
entirety. According Shachar, multiple needles were incorpo- of the substance .
rated into the pump which pushes a medicating agent
A perfusion device for localized drug delivery has been
5 described by Zamoyski and is taught for example in U.S.
through the needles at a controlled rate .
Methods of treating functional disorders of the bladder in Pat. No. 6,468,247 , the contents of which are incorporated
mammalian females have been described by Versi et al and herein by reference in their entirety. According to Zamoyski,
are taught for example in U.S. Pat . No. 8,029,496 , the multiple hypodermic needles are incorporated into the
contents of which are incorporated herein by reference in device which injects the contents of the hypodermics into a
their entirety. According to Versi, an array of micro - needles 10 tissue as said hypodermics are being retracted.
is incorporated into a device which delivers a therapeutic
A method for enhanced transport of drugs and biological
agent through the needles directly into the trigone of the molecules across tissue by improving the interaction
bladder.
between micro -needles and human skin has been described
A micro - needle transdermal transport device has been 15 by Prausnitz et al and is taught for example in U.S. Pat . No.
described by Angel et al and is taught for example in U.S. 6,743,211 , the contents of which are incorporated herein by
Pat . No. 7,364,568 , the contents of which are incorporated reference in their entirety. According to Prausnitz , multiple
herein by reference in their entirety. According to Angel, micro -needles are incorporated into a device which is able to
multiple needles are incorporated into the device which present a more rigid and less deformable surface to which
transports a substance into a body surface through the 20 the micro - needles are applied.
needles which are inserted into the surface from different
directions .

A device for intraorgan administration of medicinal

agents has been described by Ting et al and is taught for
A device for subcutaneous infusion has been described by example in U.S. Pat . No. 6,077,251 , the contents of which
Dalton et al and is taught for example in U.S. Pat . No. are incorporated herein by reference in their entirety.
7,150,726 , the contents of which are incorporated herein by 25 According to Ting, multiple needles having side openings
reference in their entirety. According to Dalton, multiple for enhanced administration are incorporated into a device
needles are incorporated into the device which delivers fluid which by extending and retracting said needles from and
into the needle chamber forces a medicinal agent from a
through the needles into a subcutaneous tissue .
reservoir into said needles and injects said medicinal agent
A device and a method for intradermal delivery of vac 30 into
a target organ .
cines and gene therapeutic agents through microcannula
A
multiple needle holder and a subcutaneous multiple
have been described by Mikszta et al and are taught for channel
port has been described by Brown and is
example in U.S. Pat . No. 7,473,247 , the contents of which taught forinfusion
example
in U.S. Pat . No. 4,695,273 , the contents
are incorporated herein by reference in their entirety. of which are incorporated
by reference in their
According to Mitszta , at least one hollow micro - needle is 35 entirety . According to Brown, herein
multiple
on the needle
incorporated into the device which delivers the vaccines to holder are inserted through the septumneedles
of
the
infusion port
the subject's skin to a depth ofbetween 0.025 mm and 2 mm . and communicate with isolated chambers in said
infusion
A method of delivering insulin has been described by port.
Pettis et al and is taught for example in U.S. Pat. No.
A dual hypodermic syringe has been described by Horn

7,722,595 , the contents of which are incorporated herein by 40 and is taught for example in U.S. Pat . No. 3,552,394 , the

reference in their entirety . According to Pettis, two needles contents of which are incorporated herein by reference in
are incorporated into a device wherein both needles insert their entirety. According to Horn , two needles incorporated
essentially simultaneously into the skin with the first at a into the device are spaced apart less than 68 mm and may be
depth of less than 2.5 mm to deliver insulin to intradermal of different styles and lengths, thus enabling injections to be
compartment and the second at a depth of greater than 2.5 45 made to different depths.
mm and less than 5.0 mm to deliver insulin to subcutaneous
A syringe with multiple needles and multiple fluid com
compartment.
partments has been described by Hershberg and is taught for
Cutaneous injection delivery under suction has been example in U.S. Pat . No. 3,572,336 , the contents of which
described by Kochamba et al and is taught for example in are incorporated herein by reference in their entirety.
U.S. Pat . No. 6,896,666 , the contents of which are incorpo- 50 According to Hershberg, multiple needles are incorporated
rated herein by reference in their entirety. According to into the syringe which has multiple fluid compartments and
Kochamba, multiple needles in relative adjacency with each is capable of simultaneously administering incompatible
other are incorporated into a device which injects a fluid drugs which are not able to be mixed for one injection.
below the cutaneous layer.
A surgical instrument for intradermal injection of fluids
A device for withdrawing or delivering a substance 55 has been described by Eliscu et al and is taught for example
through the skin has been described by Down et al and is in U.S. Pat . No. 2,588,623 , the contents of which are
taught for example in U.S. Pat . No. 6,607,513 , the contents incorporated herein by reference in their entirety. According
of which are incorporated herein by reference in their to Eliscu , multiple needles are incorporated into the instru
entirety. According to Down , multiple skin penetrating ment which injects fluids intradermally with a wider dis
members which are incorporated into the device have 60 perse .
lengths of about 100 microns to about 2000 microns and are
An apparatus for simultaneous delivery of a substance to
about 30 to 50 gauge.
multiple breast milk ducts has been described by Hung and
A device for delivering a substance to the skin has been is taught for example in EP 1818017 , the contents of which
described by Palmer et al and is taught for example in U.S. are incorporated herein by reference in their entirety.
Pat . No. 6,537,242 , the contents of which are incorporated 65 According to Hung, multiple lumens are incorporated into
herein by reference in their entirety. According to Palmer, an the device which inserts though the orifices of the ductal
array of micro -needles is incorporated into the device which

networks and delivers a fluid to the ductal networks .

US 10,898,574 B2
41

A catheter for introduction of medications to the tissue of

a heart or other organs has been described by Tkebuchava

and is taught for example in WO2006138109 , the contents
of which are incorporated herein by reference in their
entirety. According to Tkebuchava , two curved needles are
incorporated which enter the organ wall in a flattened
trajectory

42

A surgical device for ablating a channel and delivering at

least one therapeutic agent into a desired region of the tissue

has been described by McIntyre et al and is taught for
example in U.S. Pat . No. 8,012,096 , the contents of which
5
are incorporated herein by reference in their entirety .
According to McIntyre, multiple needles are incorporated
into the device which dispenses a therapeutic agent into a
Devices for delivering medical agents have been region of tissue surrounding the channel and is particularly
described by Mckay et al and are taught for example in well suited for transmyocardial revascularization operations.
WO2006118804 , the content of which are incorporated 10 Methods of treating functional disorders of the bladder in
herein by reference in their entirety . According to Mckay, mammalian
females have been described by Versi et al and
multiple needles with multiple orifices on each needle are are taught for
example in U.S. Pat . No. 8,029,496 , the
incorporated into the devices to facilitate regional delivery contents of which
are incorporated herein by reference in
to a tissue , such as the interior disc space of a spinal disc .
A method for directly delivering an immunomodulatory is their entirety. According to Versi, an array of micro -needles
substance into an intradermal space within a mammalian is incorporated into a device which delivers a therapeutic
skin has been described by Pettis and is taught for example agent through the needles directly into the trigone of the
in WO2004020014 , the contents of which are incorporated bladder.

herein by reference in their entirety. According to Pettis,
A device and a method for delivering fluid into a flexible
multiple needles are incorporated into a device which deliv- 20 biological barrier have been described by Yeshurun et al and
ers the substance through the needles to a depth between 0.3 are taught for example in U.S. Pat . No. 7,998,119 (device)
mm and 2 mm .
and U.S. Pat . No. 8,007,466 (method ), the contents of which
Methods and devices for administration of substances into are incorporated herein by reference in their entirety.
at least two compartments in skin for systemic absorption According to Yeshurun , the micro -needles on the device
and improved pharmacokinetics have been described by 25 penetrate and extend into the flexible biological barrier and
Pettis et al and are taught for example in WO2003094995 , fluid is injected through the bore of the hollow micro

the contents of which are incorporated herein by reference in needles .
their entirety. According to Pettis , multiple needles having
A method for epicardially injecting a substance into an
lengths between about 300 um and about 5 mm are incor- area of tissue of a heart having an epicardial surface and
porated into a device which delivers to intradermal and 30 disposed within a torso has been described by Bonner et al
subcutaneous tissue compartments simultaneously.
and is taught for example in U.S. Pat . No. 7,628,780 , the
A drug delivery device with needles and a roller has been contents of which are incorporated herein by reference in
described by Zimmerman et al and is taught for example in their entirety. According to Bonner, the devices have elon

WO2012006259 , the contents of which are incorporated gate shafts and distal injection heads for driving needles into
herein by reference in their entirety. According to Zimmer- 35 tissue and injecting medical agents into the tissue through

man , multiple hollow needles positioned in a roller are the needles .
incorporated into the device which delivers the content in a
A device for sealing a puncture has been described by
Nielsen et al and is taught for example in U.S. Pat . No.
reservoir through the needles as the roller rotates .
Methods and Devices Utilizing Catheters and/ or Lumens
7,972,358 , the contents of which are incorporated herein by
Methods and devices using catheters and lumens may be 40 reference in their entirety. According to Nielsen, multiple
employed to administer the mmRNA of the present inven- needles are incorporated into the device which delivers a
tion on a split dosing schedule . Such methods and devices closure agent into the tissue surrounding the puncture tract.
are described below.
A method for myogenesis and angiogenesis has been
A catheter -based delivery of skeletal myoblasts to the described by Chiu et al and is taught for example in U.S. Pat.
myocardium of damaged hearts has been described by 45 No. 6,551,338 , the contents of which are incorporated herein
Jacoby et al and is taught for example in US Patent Publi- by reference in their entirety. According to Chiu , 5 to 15
cation 20060263338 , the contents of which are incorporated needles having a maximum diameter of at least 1.25 mm and
herein by reference in their entirety . According to Jacoby, a length effective to provide a puncture depth of 6 to 20 mm
multiple needles are incorporated into the device at least part are incorporated into a device which inserts into proximity

of which is inserted into a blood vessel and delivers the cell 50 with a myocardium and supplies an exogeneous angiogenic

composition through the needles into the localized region of

the subject's heart.
An apparatus for treating asthma using neurotoxin has

been described by Deem et al and is taught for example in

or myogenic factor to said myocardium through the conduits

which are in at least some of said needles.

A method for the treatment of prostate tissue has been
described by Bolmsj et al and is taught for example in U.S.

US Patent Publication 20060225742 , the contents of which 55 Pat. No. 6,524,270 , the contents of which are incorporated
are incorporated herein by reference in their entirety . herein by reference in their entirety. According to Bolmsj, a
According to Deem , multiple needles are incorporated into device comprising a catheter which is inserted through the
the device which delivers neurotoxin through the needles urethra has at least one hollow tip extendible into the
into the bronchial tissue .
surrounding prostate tissue . An astringent and analgesic
A method for administering multiple -component thera- 60 medicine is administered through said tip into said prostate
pies has been described by Nayak and is taught for example tissue .
in U.S. Pat . No. 7,699,803 , the contents of which are
A method for infusing fluids to an intraosseous site has
incorporated herein by reference in their entirety. According been described by Findlay et al and is taught for example in
to Nayak, multiple injection cannulas may be incorporated U.S. Pat. No. 6,761,726 , the contents of which are incorpo
into a device wherein depth slots may be included for 65 rated herein by reference in their entirety. According to
controlling the depth at which the therapeutic substance is Findlay, multiple needles are incorporated into a device
delivered within the tissue .
which is capable of penetrating a hard shell of material

US 10,898,574 B2
44

43

covered by a layer of soft material and delivers a fluid at a

electrodes to penetrate into the tissue surrounding said

predetermined distance below said hard shell of material.
A device for injecting medications into a vessel wall has

lumen . Then the device introduces an agent through at least
one of said needle electrodes and applies electric field by

rated herein by reference in their entirety. According to
Vigil, multiple injectors are mounted on each of the flexible
tubes in the device which introduces a medication fluid
through a multi - lumen catheter, into said flexible tubes and
out of said injectors for infusion into the vessel wall .

site .
A delivery system for transdermal immunization has been
described by Levin et al and is taught for example in

been described by Vigil et al and is taught for example in said pair of needle electrodes to allow said agent pass
U.S. Pat . No. 5,713,863 , the contents of which are incorpo- 5 through the cell membranes into the cells at the treatment

10

A catheter for delivering therapeutic and / or diagnostic

agents to the tissue surrounding a bodily passageway has

WO2006003659 , the contents of which are incorporated
multiple electrodes are incorporated into the device which
applies electrical energy between the electrodes to generate
herein by reference in their entirety. According to Levin ,

been described by Faxon et al and is taught for example in micro channels in the skin to facilitate transdermal delivery.
U.S. Pat . No. 5,464,395 , the contents of which are incorpo
A method for delivering RF energy into skin has been
rated herein by reference in their entirety. According to 15 described
by Schomacker and is taught for example in
Faxon , at least one needle cannula is incorporated into the WO2011163264
, the contents of which are incorporated
catheter which delivers the desired agents to the tissue
through said needles which project outboard of the catheter. herein by reference in their entirety. According to Schoma
Balloon catheters for delivering therapeutic agents have cker, multiple needles are incorporated into a device which
been described by Orr and are taught for example in 20 applies vacuum to draw skin into contact with a plate so that
WO2010024871 , the contents of which are incorporated needles insert into skin through the holes on the plate and
herein by reference in their entirety. According to Orr, deliver RF energy.
multiple needles are incorporated into the devices which Devices and Kits
deliver the therapeutic agents to different depths within the
Devices may also be used in conjunction with the present
tissue .
25 invention. In one embodiment, a device is used to assess
levels of a protein which has been administered in the form
Methods and Devices Utilizing Electrical Current
Methods and devices utilizing electric current may be of a modified mRNA . The device may comprise a blood ,
employed to deliver the mmRNA of the present invention urine or other biofluidic test . It may be as large as to include
according to the split dosing regimens taught herein . Such an automated central lab platform or a small decentralized
methods and devices are described below.
30 bench top device . It may be point of care or a handheld
An electro collagen induction therapy device has been device. The device may be useful in drug discovery efforts
described by Marquez and is taught for example in US as a companion diagnostic.
Patent Publication 20090137945 , the contents of which are

incorporated herein by reference in their entirety. According
to Marquez, multiple needles are incorporated into the
device which repeatedly pierce the skin and draw in the skin
a portion of the substance which is applied to the skin first.
An electrokinetic system has been described by Etheredge
et al and is taught for example in US Patent Publication
20070185432 , the contents ofwhich are incorporated herein
by reference in their entirety. According to Etheredge,
micro -needles are incorporated into a device which drives
by an electrical current the medication through the needles
into the targeted treatment site .
An iontophoresis device has been described by Matsumura et al and is taught for example in U.S. Pat . No.
7,437,189 , the contents of which are incorporated herein by
reference in their entirety. According to Matsumura, multiple needles are incorporated into the device which is
capable of delivering ionizable drug into a living body at
higher speed or with higher efficiency.
Intradermal delivery of biologically active agents by
needle - free injection and electroporation has been described
by Hoffmann et al and is taught for example in U.S. Pat . No.
7,171,264 , the contents of which are incorporated herein by
reference in their entirety. According to Hoffmann, one or
more needle - free injectors are incorporated into an electroporation device and the combination of needle - free injec-

In some embodiments the device is self -contained , and is

35

40

45

50
55

tion and electroporation is sufficient to introduce the agent
into cells in skin , muscle or mucosa .
60

A method for electropermeabilization -mediated intracel-

optionally capable of wireless remote access to obtain
instructions for synthesis and / or analysis of the generated
nucleic acid . The device is capable of mobile synthesis of at
least one nucleic acid , and preferably an unlimited number
of different nucleic acid sequences. In certain embodiments ,
the device is capable of being transported by one or a small
number of individuals. In other embodiments, the device is
scaled to fit on a benchtop or desk . In other embodiments ,
the device is scaled to fit into a suitcase , backpack or
similarly sized object. In further embodiments, the device is
scaled to fit into a vehicle, such as a car , truck or ambulance,
or a military vehicle such as a tank or personnel carrier. The
information necessary to generate a modified mRNA encod
ing protein of interest is present within a computer readable
medium present in the device .
In some embodiments, the device is capable of commu
nication ( e.g. , wireless communication ) with a database of
nucleic acid and polypeptide sequences. The device contains
at least one sample block for insertion of one or more sample
vessels . Such sample vessels are capable of accepting in
liquid or other form any number of materials such as
template DNA , nucleotides , enzymes, buffers, and other
reagents. The sample vessels are also capable of being
heated and cooled by contact with the sample block . The
sample block is generally in communication with a device
base with one or more electronic control units for the at least
one sample block . The sample block preferably contains a
heating module, such heating molecule capable of heating
and / or cooling the sample vessels and contents thereof to
temperatures between about -20 C and above +100 C. The
device base is in communication with a voltage supply such
as a battery or external voltage supply. The device also
contains means for storing and distributing the materials for

lular delivery has been described by Lundkvist et al and is
taught for example in U.S. Pat . No. 6,625,486 , the contents
of which are incorporated herein by reference in their
entirety. According to Lundkvist, a pair of needle electrodes 65
is incorporated into a catheter. Said catheter is positioned
into a body lumen followed by extending said needle RNA synthesis.

US 10,898,574 B2
46

45

Optionally, the sample block contains a module for separating the synthesized nucleic acids . Alternatively, the
device contains a separation module operably linked to the
sample block . Preferably the device contains a means for
analysis of the synthesized nucleic acid . Such analysis 5

example , the term “ C1-6 alkyl ” is specifically intended to
individually disclose methyl, ethyl, C , alkyl, C4 alkyl, Cg
alkyl, and Co alkyl.
Animal: As used herein , the term “ animal ” refers to any
member of the animal kingdom . In some embodiments,
includes sequence identity ( demonstrated such as by hybrid “ animal ” refers to humans at any stage of development. In
ization) , absence of non -desired sequences, measurement of some embodiments, " animal” refers to non - human animals
integrity of synthesized mRNA ( such has by microfluidic at any stage of development. In certain embodiments, the
- human animal is a mammal (e.g. , a rodent, a mouse , a
viscometry combined with spectrophotometry ), and concen 10 non
, a rabbit, a monkey, a dog , a cat , a sheep, cattle, a primate ,
tration and /orpotency of modified RNA ( such as by spec rat
or a pig ) . In some embodiments, animals include , but are not
trophotometry ).
limited to , mammals, birds , reptiles, amphibians, fish , and
In certain embodiments, the device is combined with a worms
In some embodiments, the animal is a transgenic
means for detection of pathogens present in a biological animal,. genetically
- engineered animal, or a clone .
material obtained from a subject, e.g. , the IBIS PLEX - ID 15 Approximately : As used herein , the term “ approximately ”
system ( Abbott) for microbial identification .
or “ about, ” as applied to one or more values of interest,
The present invention provides for devices which incor refers
to a value that is similar to a stated reference value . In
porate mmRNA that encode proteins of interest. These certain embodiments, the term “ approximately ” or “ about”
devices may be implantable in an animal subject or may refers to a range of values that fall within 25 % , 20 % , 19 % ,
supply mmRNA formulations via a catheter or lumen . The 20 18 % , 17 % , 16 % , 15 % , 14 % , 13 % , 12 % , 11 % , 10 % , 9 % , 8 % ,
device may be connected to or incorporate a pump. Such 7 % , 6 % , 5 % , 4 % , 3 % , 2 % , 1 % , or less in either direction
devices include those which can deliver therapeutics to areas (greater than or less than ) of the stated reference value unless
of the body not readily accessible such the CNS or across otherwise stated or otherwise evident from the context
the blood brain barrier. In this embodiment the split dosing (except where such number would exceed 100 % of a pos
25 sible value) .
regimen can be implemented using a regulated pump.
Associated with : As used herein , the terms " associated
Kits
,” “ conjugated " " linked ," " attached , ” and “ tethered , "
The invention provides a variety of kits for conveniently with
and / or effectively carrying out methods of the present inven when used with respect to two or more moieties , means that
moieties are physically associated or connected with one
tion . Typically kits will comprise sufficient amounts and / or 30 the
another, either directly or via one or more additional moi
numbers of components to allow a user to perform multiple eties
that serves as a linking agent, to form a structure that
treatments of a subject( s ) and /or to perform multiple experi is sufficiently
stable so that the moieties remain physically
ments.
associated under the conditions in which the ructure is
In one aspect , the present invention provides kits for
, e.g. , physiological conditions . An “ association ” need
protein production , comprising a first isolated nucleic acid 35 used
not
be
through direct covalent chemical bonding . It
comprising a translatable region and a nucleic acid modifi may alsostrictly
suggest
ionic or hydrogen bonding or a hybrid
cation , wherein the nucleic acid may be capable of evading ization based connectivity sufficiently stable such that the
an innate immune response of a cell into which the first “ associated ” entities remain physically associated .
isolated nucleic acid may be introduced , and packaging and
Bifunctional: As used herein , the term “ bifunctional”
instructions. The kit may further comprise a delivery agent 40 refers to any substance , molecule or moiety which is capable
to form a formulation composition. The delivery composi- of or maintains at least two functions. The functions may
tion may comprise a lipidoid. The lipoid may be selected
from , but is not limited to , C12-200 , 98N12-5 , MD1 , DLinDMA , DLin - K -DMA, DLin -KC2 -DMA , DLin -MC3 - DMA
45
and analogs thereof.

effect the same outcome or a different outcome. The struc
ture that produces the function may be the same or different.
For example , bifunctional modified RNAs of the present
invention may encode a cytotoxic peptide (a first function )
while those nucleosides which comprise the encoding RNA
are, in and of themselves, cytotoxic ( second function ). In
this example , delivery of the bifunctional modified RNA to
a cancer cell would produce not only a peptide or protein

In one aspect , the present invention provides kits for
protein production , comprising a first isolated nucleic acid
comprising a translatable region and a nucleoside modification , wherein the nucleic acid exhibits reduced degradation by a cellular nuclease , and packaging and instructions. 50 molecule which may ameliorate or treat the cancer but
In one aspect , the present invention provides kits for would also deliver a cytotoxic payload of nucleosides to the
protein production , comprising a first isolated nucleic acid cell should degradation, instead of translation of the modi
comprising a translatable region and at least two different fied RNA , occur.
nucleoside modifications , wherein the nucleic acid exhibits
Biologically active : As used herein , the phrase " biologi

reduced degradation by a cellular nuclease, and packaging 55 cally active ” refers to a characteristic of any substance that
has activity in a biological system and /or organism . For
and instructions.
In some embodiments, kits would provide split doses or
instructions for the administration of split dosages of the

mmRNA of the kit .

instance, a substance that, when administered to an organ
ism , has a biological affect on that organism , is considered
to be biologically active . In particular embodiments, a
60 nucleic acid molecule of the present invention may be

considered biologically active if even a portion of the
nucleic acid molecule is biologically active or mimics an
At various places in the present specification, substituents activity considered biologically relevant.
of compounds of the present disclosure are disclosed in
Chemical terms: As used herein, the term “ alkyl” is meant
groups or in ranges. It is specifically intended that the 65 to refer to a saturated hydrocarbon group which is straight
present disclosure include each and every individual sub- chained or branched . Example alkyl groups include methyl
combination of the members of such groups and ranges . For (Me) , ethyl (Et) , propyl (e.g. , n -propyl and isopropyl ), butyl
Definitions

US 10,898,574 B2
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48

( e.g. , n -butyl, isobutyl, t -butyl ), pentyl ( e.g. , n -pentyl, isopentyl, neopentyl), and the like . An alkyl group can contain

different number of neutrons in the nuclei. For example,
isotopes of hydrogen include tritium and deuterium .

from 1 to about 20 , from 2 to about 20 , from 1 to about 12 ,
from 1 to about 8 , from 1 to about 6 , from 1 to about 4 , or

The compounds and salts of the present disclosure can be
prepared in combination with solvent or water molecules to
5 form solvates and hydrates by routine methods .
from 1 to about 3 carbon atoms.
Conserved : As used herein , the term " conserved ” refers to
As used herein , " alkenyl” refers to an alkyl group having
or amino acid residues of a polynucleotide
one or more double carbon - carbon bonds . Example alkenyl nucleotides
sequence or polypeptide sequence, respectively, that are

groups include ethenyl, propenyl, and the like .
occur unaltered in the same position of two or
As used herein , “ alkoxy ” refers to an O -alkyl group. those that
sequences being compared. Nucleotides or amino
Example alkoxy groups include methoxy, ethoxy, propoxy 10 more
acids that are relatively conserved are those that are con
( e.g. , n - propoxy and isopropoxy ), t - butoxy, and the like .
served amongst more related sequences than nucleotides or
As used herein , “ alkenyl ” refers to an alkyl, as defined amino
acids appearing elsewhere in the sequences .
above , containing at least one double bond between adjacent
In some embodiments, two or more sequences are said to

carbon
atoms. Alkenyls include both cis and trans isomers. 15 be “completely conserved” if they are 100% identical to one
Representative straight chain and branched alkenyls include another. In some embodiments, two or more sequences are

ethylenyl, propylenyl, 1 - butenyl, 2 -butenyl, isobutylenyl,
1 -pentenyl, 2 -pentenyl, 3 -methyl - 1 -butenyl, 2-methyl-2butenyl, 2,3 - dimethyl - 2 - butenyl , and the like .

said to be “ highly conserved ” if they are at least 70 %
identical, at least 80% identical, at least 90 % identical, or at
least 95 % identical to one another. In some embodiments,
As used herein, “ alkynyl” refers to an alkyl group having 20 two or more sequences are said to be “ highly conserved ” if
one or more triple carbon - carbon bonds . Example alkynyl they are about 70 % identical, about 80% identical, about
groups include ethynyl, propynyl, and the like .
90% identical, about 95 % , about 98 % , or about 99 % iden
As used herein , " aryl” refers to monocyclic or polycyclic tical to one another. In some embodiments, two or more
( e.g. , having 2 , 3 or 4 fused rings ) aromatic hydrocarbons sequences are said to be “ conserved ” if they are at least 30 %
such as , for example, phenyl, naphthyl, anthracenyl, 25 identical, at least 40 % identical, at least 50% identical, at
phenanthrenyl, indanyl, indenyl, and the like. In some least 60% identical, at least 70 % identical, at least 80 %
embodiments, aryl groups have from 6 to about 20 carbon identical, at least 90% identical, or at least 95 % identical to
atoms .
one another. In some embodiments , two or more sequences
As used herein , “ halo ” or “ halogen ” includes fluoro , are said to be " conserved ” if they are about 30 % identical,
chloro , bromo , and iodo .
30 about 40 % identical, about 50% identical, about 60 % iden
Compound: As used herein , the term “ compound,” is tical , about 70% identical, about 80% identical, about 90 %
meant to include all stereoisomers , geometric isomers, tau- identical, about 95 % identical, about 98 % identical, or about
99 % identical to one another. Conservation of sequence may
tomers , and isotopes of the structures depicted .

The compounds described herein can be asymmetric ( e.g. , apply to the entire length of an oligonucleotide or polypep
having one or more stereocenters ). All stereoisomers, such 35 tide or may apply to a portion, region or feature thereof.

as enantiomers and diastereomers , are intended unless otherwise indicated . Compounds of the present disclosure that
contain asymmetrically substituted carbon atoms can be
isolated in optically active or racemic forms. Methods on
how to prepare optically active forms from optically active
starting materials are known in the art, such as by resolution
of racemic mixtures or by stereoselective synthesis. Many
geometric isomers of olefins, C=N double bonds, and the
like can also be present in the compounds described herein ,
and all such stable isomers are contemplated in the present
disclosure . Cis and trans geometric isomers of the compounds of the present disclosure are described and may be
isolated as a mixture of isomers or as separated isomeric
forms.
Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of
a single bond with an adjacent double bond and the concomitant migration of a proton . Tautomeric forms include
prototropic tautomers which are isomeric protonation states
having the same empirical formula and total charge.
Examples prototropic tautomers include ketone - enol pairs ,
amide - imidic acid pairs , lactam - lactim pairs , amide - imidic
acid pairs , enamine - imine pairs , and annular forms where a
proton can occupy two or more positions of a heterocyclic
system , such as , 1H- and 3H - imidazole , 1H- , 2H- and
4H - 1,2,4 -triazole, 1H- and 2H - isoindole, and 1H- and
2H -pyrazole. Tautomeric forms can be in equilibrium or
sterically locked into one form by appropriate substitution .
Compounds of the present disclosure also include all of
the isotopes of the atoms occurring in the intermediate or
final compounds. “ Isotopes ” refers to atoms having the same
atomic number but different mass numbers resulting from a

40

45

50

55

60

65

Delivery : As used herein , “ delivery ” refers to the act or
manner of delivering a compound, substance, entity , moiety,
cargo or payload .
Delivery Agent: As used herein , “ delivery agent ” refers to
any substance which facilitates, at least in part, the in vivo
delivery of a nucleic acid molecule to targeted cells .
Detectable label : As used herein , " detectable label” refers
to one or more markers , signals, or moieties which are
attached , incorporated or associated with another entity that
is readily detected by methods known in the art including
radiography , fluorescence, chemiluminescence, enzymatic
activity, absorbance and the like . Detectable labels include
radioisotopes, fluorophores, chromophores, enzymes , dyes ,
metal ions , ligands such as biotin, avidin , strepavidin and
haptens, quantum dots , and the like . Detectable labels may
be located at any position in the peptides or proteins dis
closed herein . They may be within the amino acids , the
peptides , or proteins, or located at the N- or C - termini.
Distal : As used herein " distal ” means farther from center
mass or line of symmetry of subject or reference point. For
limbs, it is farther from body.
Dosing regimen : As used herein , a “ dosing regimen ” is a
schedule of administration or physician determined regimen
of treatment, prophylaxis, or palliative care .
Dose splitting factor (DSF )-ratio of PUD of dose split
treatment divided by PUD of total daily dose or single unit
dose . The value is derived from comparison of dosing
regimens groups.
Expression: As used herein , “ expression ” of a nucleic acid
sequence refers to one or more of the following events : ( 1 )
production of an RNA template from a DNA sequence ( e.g. ,
by transcription ); (2 ) processing of an RNA transcript ( e.g. ,

US 10,898,574 B2
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50

by splicing , editing , 5 ' cap formation , and / or 3 ' end processing ) ; ( 3 ) translation of an RNA into a polypeptide or protein ;
and (4 ) post - translational modification of a polypeptide or

account the number of gaps, and the length of each gap ,
which needs to be introduced for optimal alignment of the
two sequences. The comparison of sequences and determi
protein .
nation of percent identity between two sequences can be
Formulation : As used herein , a “ formulation ” includes at 5 accomplished using a mathematical algorithm . For example,
least a modified nucleic acid molecule and a delivery agent. the percent identity between two nucleotide sequences can
Functional : As used herein , a “ functional ” biological be determined using methods such as those described in

molecule is a biological molecule in a form in which it
exhibits a property and / or activity by which it is characterized.

Homology : As used herein , the term “ homology ” refers to
the overall relatedness between polymeric molecules , e.g.
between nucleic acid molecules ( e.g. DNA molecules and /or
RNA molecules ) and / or between polypeptide molecules . In
some embodiments, polymeric molecules are considered to
be “ homologous” to one another if their sequences are at
least 25 % , at least 30 % , at least 35 % , at least 40 % , at least
45 % , at least 50 % , at least 55 % , at least 60 % , at least 65 % ,
at least 70 % , at least 75 % , at least 80 % , at least 85 % , at least
90 % , at least 95 % , or at least 99 % identical. In some
embodiments, polymeric molecules are considered to be
“ homologous ” to one another if their sequences are at least
25 % , at least 30 % , at least 35 % , at least 40 % , at least 45 % ,
at least 50 % , at least 55 % , at least 60 % , at least 65 % , at least
70 % , at least 75 % , at least 80 % , at least 85 % , at least 90 % ,
at least 95 % , or at least 99 % similar. The term “ homologous” necessarily refers to a comparison between at least
two sequences ( polynucleotide or polypeptide sequences ).
In accordance with the invention, two polynucleotide
sequences are considered to be homologous if the polypeptides they encode are at least about 50% identical, at least
about 60 % identical, at least about 70 % identical, at least
about 80 % identical, or at least about 90 % identical for
least one stretch of at least about 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 . In accordance
with the invention , two protein sequences are considered to
be homologous if the proteins are at least about 50 %
identical, at least about 60 % identical, at least about 70 %
identical, at least about 80 % identical, or at least about 90%
identical for at least one stretch of at least about 20 amino
acids .

Identity: As used herein , the term “ identity ” refers to the

overall relatedness between polymeric molecules , e.g. ,
between oligonucleotide molecules ( e.g. DNA molecules
and / or RNA molecules ) and / or between polypeptide molecules . Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning
the two sequences for optimal comparison purposes ( e.g. ,

gaps can be introduced in one or both of a first and a second
nucleic acid sequences for optimal alignment and non-

identical sequences can be disregarded for comparison purposes ) . In certain embodiments, the length of a sequence
aligned for comparison purposes is at least 30% , at least

Computational Molecular Biology, Lesk , A. M. , ed . , Oxford
University Press, New York, 1988 ; Biocomputing: Informat

10 ics and Genome Projects, Smith, D. W., ed . , Academic Press,
New York , 1993 ; Sequence Analysis in Molecular Biology,
von Heinje , G. , Academic Press , 1987 ; Computer Analysis
of Sequence Data, Part I , Griffin , A. M. , and Griffin , H. G. ,
eds . , Humana Press, New Jersey, 1994 ; and Sequence
15 Analysis Primer, Gribskov, M. and Devereux , J. , eds . , M
Stockton Press , New York , 1991 ; each of which is incorpo
rated herein by reference. For example, the percent identity
between two nucleotide sequences can be determined using
the algorithm of Meyers and Miller (CABIOS , 1989 , 4 :11
20 17 ) , which has been incorporated into the ALIGN program
( version 2.0 ) using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4. The percent
identity between two nucleotide sequences can , alterna
tively, be determined using the GAP program in the GCG
25 software package using an NWSgapdna.CMP matrix . Meth
ods commonly employed to determine percent identity
between sequences include , but are not limited to those
disclosed in Carillo , H. , and Lipman , D. , SIAM J Applied
Math ., 48 : 1073 ( 1988 ) ; incorporated herein by reference .
30 Techniques for determining identity are codified in publicly
available computer programs. Exemplary computer soft
ware to determine homology between two sequences
include, but are not limited to , GCG program package ,
Devereux, J. , et al . , Nucleic Acids Research , 12 ( 1 ) , 387
35 ( 1984 ) ) , BLASTP, BLASTN , and FASTA Atschul, S. F. et
al . , J. Molec. Biol., 215 , 403 ( 1990 ) ) .
Inhibit expression of a gene: As used herein , the phrase
“ inhibit expression of a gene” means to cause a reduction in
the amount of an expression product of the gene. The
40 expression product can be an RNA transcribed from the gene
(e.g. , an mRNA ) or a polypeptide translated from an mRNA
transcribed from the gene. Typically a reduction in the level
of an mRNA results in a reduction in the level of a
polypeptide translated therefrom . The level of expression
45 may be determined using standard techniques for measuring
mRNA or protein .
In vitro : As used herein , the term " in vitro ” refers to
events that occur in an artificial environment, e.g. , in a test
tube or reaction vessel , in cell culture, in a Petri dish, etc.,
50 rather than within an organism ( e.g. , animal, plant, or
microbe ) .
In vivo : As used herein , the term " in vivo ” refers to events
that occur within an organism (e.g. , animal, plant, or
microbe or cell or tissue thereof).
55 Isolated : As used herein , the term “ isolated ” refers to a
substance or entity that has been separated from at least

some of the components with which it was associated
( whether in nature or in an experimental setting ). Isolated

40 % , at least 50 % , at least 60 % , at least 70 % , at least 80 % , substances may have varying levels of purity in reference to
at least 90 % , at least 95 % , or 100 % of the length of the 60 the substances from which they have been associated . Iso
reference sequence. The nucleotides at corresponding lated substances and / or entities may be separated from at
nucleotide positions are then compared . When a position in least about 10 % , about 20 % , about 30% , about 40 % , about
the first sequence is occupied by the same nucleotide as the 50% , about 60 % , about 70 % , about 80 % , about 90% , or
corresponding position in the second sequence, then the more of the other components with which they were initially
molecules are identical at that position . The percent identity 65 associated . In some embodiments, isolated agents are more
between the two sequences is a function of the number of than about 80 % , about 85 % , about 90 % , about 91 % , about
identical positions shared by the sequences , taking into 92 % , about 93 % , about 94 % , about 95 % , about 96 % , about

US 10,898,574 B2
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52

97 % , about 98 % , about 99 % , or more than about 99 % pure .
Pharmaceutically acceptable salts : The present disclosure
As used herein, a substance is “ pure ” if it is substantially free also includes pharmaceutically acceptable salts of the com
of other components. Substantially isolated : By “ substan- pounds described herein . As used herein , “ pharmaceutically
tially isolated” is meant that the compound is substantially acceptable salts ” refers to derivatives of the disclosed com
separated from the environment in which it was formed or 5 pounds wherein the parent compound is modified by con
detected . Partial separation can include, for example, a verting an existing acid or base moiety to its salt form .
composition enriched in the compound of the present dis- Examples of pharmaceutically acceptable salts include, but
closure . Substantial separation can include compositions are not limited to , mineral or organic acid salts of basic
containing at least about 50 % , at least about 60 % , at least residues such as amines ; alkali or organic salts of acidic

about 70% , at least about 80 % , at least about 90 % , at least 10 residues such as carboxylic acids ; and the like . The phar
about 95 % , at least about 97 % , or at least about 99 % by maceutically acceptable salts of the present disclosure
weight of the compound of the present disclosure, or salt include the conventional non - toxic salts of the parent com
thereof. Methods for isolating compounds and their salts are pound formed , for example, from non - toxic inorganic or
routine in the art.
organic acids . The pharmaceutically acceptable salts of the
Modified: As used herein “ modified ” refers to a changed 15 present disclosure can be synthesized from the parent com
state or structure of a molecule of the invention . Molecules pound which contains a basic or acidic moiety by conven
may be modified in many ways including chemically, struc- tional chemical methods. Generally, such salts can be pre
turally, and functionally. In one embodiment, the mRNA pared by reacting the free acid or base forms of these
molecules of the present invention are modified by the compounds with a stoichiometric amount of the appropriate
introduction of non - natural nucleosides and / or nucleotides. 20 base or acid in water or in an organic solvent, or in a mixture
Modified , as it pertains to a modified mRNA may also mean of the two; generally, nonaqueous media like ether, ethyl
acetate , ethanol, isopropanol , or acetonitrile are preferred .
any alteration which is different from the wild type.
Naturally occurring: As used herein , “ naturally occurring " Lists of suitable salts are found in Remington's Pharmaceu
means existing in nature without artificial aid .
tical Sciences, 17th ed . , Mack Publishing Company, Easton ,
Patient: As used herein , “ patient ” refers to a subject who 25 Pa . , 1985 , p . 1418 and Journal of Pharmaceutical Science,
may seek or be in need of treatment, requires treatment, is 66 , 2 ( 1977 ) , each of which is incorporated herein by
receiving treatment, will receive treatment, or a subject who reference in its entirety .
is under care by a trained professional for a particular
Polypeptide: As used herein , “ polypeptide” means a poly
disease or condition .
mer of amino acid residues linked together by peptide bonds.

Peptide : As used herein, “ peptide ” is less than or equal to 30 The term , as used herein, refers to proteins, polypeptides ,

50 amino acids long , e.g. , about 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , and peptides of any size , structure, or function . Typically,
45 , or 50 amino acids long .
however, a polypeptide will be at least 50 amino acids long .
Prodrug: The present disclosure also includes prodrugs of In some instances the polypeptide encoded is smaller than
the compounds described herein . As used herein , “ prodrugs ” about 50 amino acids and the polypeptide is termed a
refer to any substance , molecule or entity which is in a form 35 peptide. If the polypeptide is a peptide, it will be at least
predicate for that substance, molecule or entity to act as a about 5 amino acid residues long. Thus, polypeptides
therapeutic upon chemical or physical alteration . Prodrugs include gene products, naturally occurring polypeptides,
may by covalently bonded or sequestested in some way and synthetic polypeptides, homologs , orthologs, paralogs, frag
which release or are converted into the active drug moiety ments and other equivalents, variants, and analogs of the
prior to , upon or after administered to a mammalian subject. 40 foregoing. A polypeptide may be a single molecule or may
Prodrugs can be prepared by modifying functional groups be a multi-molecular complex such as a dimer, trimer or
present in the compounds in such a way that the modifica- tetramer . The term polypeptide may also apply to amino acid
tions are cleaved , either in routine manipulation or in vivo , polymers in which one or more amino acid residues are an
to the parent compounds. Prodrugs include compounds artificial chemical analogue of a corresponding naturally

wherein hydroxyl, amino , sulfhydryl, or carboxyl groups are 45 occurring amino acid .
bonded to any group that, when administered to a mammaPolypeptide per unit drug (PUD ) : As used herein , a PUD
lian subject, cleaves to form a free hydroxyl, amino , sulf- or product per unit drug, is defined as a subdivided portion
hydryl, or carboxyl group respectively. Preparation and use of total daily dose , usually 1 mg , pg , kg , etc. , of a product
of prodrugs is discussed in T. Higuchi and V. Stella , “ Pro- ( such as a polypeptide ) as measured in body fluid or tissue ,
drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. 50 usually defined in concentration such as pmol/mL , mmol /
Symposium Series, and in Bioreversible Carriers in Drug mL , etc divided by the measure in the body fluid .
Design, ed . Edward B. Roche , American Pharmaceutical
Proximal : As used herein , “ proximal” means closer to
Association and Pergamon Press , 1987 , both of which are center mass or line of symmetry of subject or reference
hereby incorporated by reference in their entirety.
point. For limbs, it is closer to body.
Proliferate: As used herein , the term “ proliferate ” means 55 Sample: As used herein , the term “ sample ” refers to a
to grow , expand or increase or cause to grow , expand or subset of its tissues , cells or component parts ( e.g. body
increase rapidly. “ Proliferative ” means having the ability to fluids, including but not limited to peripheral blood , serum ,
proliferate. “ Anti -proliferative ” means having properties plasma, ascites, urine , cerebrospinal fluid ( CSF ) , sputum ,
counter to or inapposite to proliferative properties.
saliva , bone marrow , synovial fluid , aqueous humor, amni

Pharmaceutically acceptable : The phrase “ pharmaceuti- 60 otic fluid , cerumen , breast milk , broncheoalveolar lavage
cally acceptable” is employed herein to refer to those fluid, semen , prostatic fluid , cowper's fluid or pre- ejacula
compounds, materials, compositions , and / or dosage forms tory fluid , sweat, fecal matter, hair, tears , cyst fluid , pleural
which are , within the scope of sound medical judgment, and peritoneal fluid, pericardial fluid , lymph , chyme, chyle,
suitable for use in contact with the tissues of human beings bile , interstitial fluid , menses , pus , sebum , vomit , vaginal
and animals without excessive toxicity, irritation , allergic 65 secretions, mucosal secretion , stool water, pancreatic juice ,
response , or other problem or complication , commensurate lavage fluids from sinus cavities , bronchopulmonary aspi
with a reasonable benefit/ risk ratio .
rates, blastocyl cavity fluid , and umbilical cord blood ). A

US 10,898,574 B2
53

sample further may include a homogenate, lysate or extract
prepared from a whole organism or a subset of its tissues ,
cells or component parts, or a fraction or portion thereof,
including but not limited to , for example , plasma , serum ,
spinal fluid , lymph fluid , the external sections of the skin ,
respiratory, intestinal, and genitourinary tracts, tears, saliva ,
milk , blood cells , tumors, organs. A sample further refers to
a medium , such as a nutrient broth or gel , which may contain
cellular components, such as proteins or nucleic acid molecule .
Similarity: As used herein , the term " similarity ” refers to
the overall relatedness between polymeric molecules , e.g.
between polynucleotide molecules ( e.g. DNA molecules
and / or RNA molecules ) and / or between polypeptide molecules . Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner
as a calculation of percent identity, except that calculation of
percent similarity takes into account conservative substitutions as is understood in the art.
Stable : As used herein “ stable ” refers to a compound that
is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture , and preferably capable of
formulation into an efficacious therapeutic agent.
Subject: As used herein , the term “ subject ” or “ patient ”
refers to any organism to which a composition in accordance
with the invention may be administered , e.g. , for experimental, diagnostic, prophylactic, and / or therapeutic purposes . Typical subjects include animals (e.g. , mammals such
as mice , rats, rabbits , non -human primates, and humans)
and / or plants .
Substantially: As used herein , the term " substantially "
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely,
if ever, go to completion and /or proceed to completeness or
achieve or avoid an absolute result . The term “ substantially ”
is therefore used herein to capture the potential lack of

completeness inherent in many biological and chemical
Substantially equal: As used herein as it relates to time
differences between doses, the term means plus/minus 2 % .
Substantially simultaneously : As used herein and as it

phenomena.

relates to plurality of doses , the term means within 2
seconds.
Simultaneously: As used herein , “ simultaneously ” means

within scientific reproducibility, at same time .

Suffering from : An individual who is “ suffering from ” a
or displays one or more symptoms of a disease, disorder,

disease , disorder, and / or condition has been diagnosed with
and / or condition .

5

10

15
20

25

30

54
( 6 ) exposure to and /or infection with a microbe associated
with development of the disease , disorder, and / or condition .
In some embodiments, an individual who is susceptible to a
disease , disorder, and / or condition will develop the disease ,
disorder, and / or condition . In some embodiments , an indi
vidual who is susceptible to a disease , disorder, and /or
condition will not develop the disease, disorder, and / or
condition .
Synthetic: The term “ synthetic ” means produced, pre
pared, and / or manufactured by the hand ofman . Synthesis of
polynucleotides or polypeptides or other molecules of the
present invention may be chemical or enzymatic .
Single unit dose : As used herein , a “ single unit dose ” is a
dose of any therapeutic administered in one dose/at one
time/ single route/ single point of contact, i.e. , single admin
istration event.
Total daily dose: As used herein , a “ total daily dose” is an
amount given or prescribed in 24 hr period. It may be
administered as a single unit dose .
Split dose: As used herein , a “ split dose ” is the division of
single unit dose or total daily dose into two or more doses.
Targeted Cells : As used herein , “ targeted cells ” refers to
any one or more cells of interest. The cells may be found in
vitro, in vivo , in situ or in the tissue or organ of an organism .
The organism may be an animal , preferably a mammal, more
preferably a human and most preferably a patient.
Therapeutic Agent: The term “ therapeutic agent ” refers to
any agent that, when administered to a subject, has a
therapeutic, diagnostic , and / or prophylactic effect and / or
elicits a desired biological and / or pharmacological effect.
Therapeutically effective amount: As used herein , the
term “ therapeutically effective amount ” means an amount of
an agent to be delivered (e.g. , nucleic acid, drug, therapeutic

agent, diagnostic agent, prophylactic agent, etc.) that is
35 sufficient, when administered to a subject suffering from or
susceptible to a disease , disorder, and / or condition , to treat,
improve symptoms of, diagnose , prevent, and / or delay the
onset of the disease , disorder, and / or condition .
Transcription factor: As used herein, the term “ transcrip
40 tion factor” refers to a DNA -binding protein that regulates
transcription of DNA into RNA, for example , by activation
or repression of transcription . Some transcription factors
effect regulation of transcription alone , while others act in
concert with other proteins. Some transcription factor can
45 both activate and repress transcription under certain condi
tions . In general, transcription factors bind a specific target
sequence or sequences highly similar to a specific consensus
sequence in a regulatory region of a target gene. Transcrip
tion factors may regulate transcription of a target gene alone
50 or in a complex with other molecules .
Treating: As used herein , the term “ treating” refers to
partially or completely alleviating, ameliorating, improving ,
relieving, delaying onset of, inhibiting progression of,
reducing severity of, and / or reducing incidence of one or
55 more symptoms or features of a particular disease , disorder,
and / or condition . For example , " treating " cancer may refer
to inhibiting survival, growth , and / or spread of a tumor.

Susceptible to : An individual who is " susceptible to ” a
disease , disorder, and / or condition has not been diagnosed
with and / or may not exhibit symptoms of the disease ,
disorder, and /or condition but harbors a propensity to
develop a disease or its symptoms. In some embodiments, an
individual who is susceptible to a disease , disorder, and / or
condition ( for example, cancer) may be characterized by one Treatment may be administered to a subject who does not
or more of the following: ( 1 ) a genetic mutation associated exhibit signs of a disease , disorder, and / or condition and /or
with development of the disease, disorder, and / or condition ; 60 to a subject who exhibits only early signs of a disease,
( 2 ) a genetic polymorphism associated with development of disorder, and / or condition for the purpose of decreasing the
the disease , disorder, and / or condition ; (3 ) increased and / or risk of developing pathology associated with the disease ,
decreased expression and / or activity of a protein and / or disorder, and / or condition .
nucleic acid associated with the disease , disorder, and /or
Unmodified : As used herein , “ unmodified ” refers to any
condition ; (4 ) habits and /or lifestyles associated with devel- 65 substance, compound or molecule prior to being changed in
opment of the disease , disorder, and / or condition ; ( 5 ) a any way. Unmodified may , but does not always , refer to the
family history of the disease , disorder, and / or condition ; and wild type or native form of a biomolecule . Molecules may

US 10,898,574 B2
55

56

undergo a series of modifications whereby each modified

information available to the applicants and does not consti

molecule may serve as the “ unmodified ” starting molecule tute any admission as to the correctness of the dates or
for a subsequent modification.
contents of these documents.
All cited sources, for example, references, publications,
Equivalents and Scope
5 databases, database entries, and art cited herein , are incor
porated into this application by reference , even if not
Those skilled in the art will recognize , or be able to expressly stated in the citation . In case of conflicting state
ascertain using no more than routine experimentation, many
of a cited source and the instant application, the
equivalents to the specific embodiments in accordance with ments
statement
in the instant application shall control.
the invention described herein . The scope of the present 10
invention is not intended to be limited to the above Descrip
EXAMPLES
tion, but rather is as set forth in the appended claims .
In the claims , articles such as “ a , ” “ an ,” and “ the ” may
Example 1. Modified mRNA Production
mean one or more than one unless indicated to the contrary
or otherwise evident from the context. Claims or descrip- 15 Modified mRNAs (mmRNA ) according to the invention
tions that include “ or” between one or more members of a
using standard laboratory methods and mate
group are considered satisfied if one , more than one , or all may. beThemade
open reading frame (ORF ) of the gene of interest
of the group members are present in, employed in , or rials
otherwise relevant to a given product or process unless may be flanked by a 5 ' untranslated region (UTR ) which may

indicated to the contrary or otherwise evident from the 20 contain a strong Kozak translational initiation signal and/or
context. The invention includes embodiments in which

an alpha - globin 3 ' UTR which may include an oligo (dT )

exactly one member of the group is present in , employed in , sequence for templated addition of a poly - A tail . The modi
or otherwise relevant to a given product or process. The fied mRNAs may be modified to reduce the cellular innate
invention includes embodiments in which more than one , or immune response . The modifications to reduce the cellular
all of the group members are present in , employed in , or 25 response may include pseudouridine (4 ) and 5-methyl
otherwise relevant to a given product or process .
cytidine ( 5meC or mC ). ( see , Kariko K et al . Immunity
It is also noted that the term “ comprising " is intended to 23 : 165-75 (2005 ) , Kariko K et al . Mol Ther 16 : 1833-40
be open and permits the inclusion of additional elements or
(2008 ) , Anderson B R et al . NAR ( 2010 ) ; herein incorpo
steps .
rated
reference ).
Where ranges are given, endpoints are included . Further- 30 ThebyORF
may also include various upstream or down
more , it is to be understood that unless otherwise indicated stream additions ( such as , but not limited to , ß - globin , tags ,
or otherwise evident from the context and understanding of etc. ) may be ordered from an optimization service such as ,
one of ordinary skill in the art, values that are expressed as but
limited to , DNA2.0 ( Menlo Park , Calif.) and may
ranges can assume any specific value or subrange within the contain
multiple cloning sites which may have Xbal recog
stated ranges in different embodiments of the invention , to 35 nition . Upon
receipt of the construct, it may be reconstituted
the tenth of the unit of the lower limit of the range, unless and transformed
into chemically competent E. coli.
the context clearly dictates otherwise .
For
the
present
invention , NEB DH5 - alpha Competent E.
In addition , it is to be understood that any particular
coli
are
used
.
Transformations
are performed according to
embodiment of the present invention that falls within the
prior
art may be explicitly excluded from any one or more 40 NEB
instructions
using
100
ng
of plasmid . The protocol is
:
of the claims . Since such embodiments are deemed to be as 1.follows
Thaw a tube ofNEB 5 - alpha Competent E. coli cells on
known to one of ordinary skill in the art, they may be
ice for 10 minutes.
excluded even if the exclusion is not set forth explicitly
herein . Any particular embodiment of the compositions of
2. Add 1-5 ul containing 1 pg - 100 ng of plasmid DNA to
the invention ( e.g. , any nucleic acid or protein encoded 45
the cell mixture . Carefully flick the tube 4-5 times to
mix cells and DNA . Do not vortex .
thereby ; any method of production; any method of use ; etc.)
can be excluded from any one or more claims, for any
3. Place the mixture on ice for 30 minutes. Do not mix .
reason , whether or not related to the existence of prior art.
4. Heat shock at 42 ° C. for exactly 30 seconds . Do not
As used herein and in the claims , the singular forms
mix .
include the plural reference and vice versa unless the context 50 5. Place on ice for 5 minutes . Do not mix .
clearly indicates otherwise . Other than in the operating
6. Pipette 950 ul of room temperature SOC into the
examples, or where otherwise indicated , all numbers
mixture.
expressing quantities of ingredients or reaction conditions
7. Place at 37 ° C. for 60 minutes. Shake vigorously ( 250
used herein should be understood as modified in all

instances by the term “ about.”

55

rpm ) or rotate .
8. Warm selection plates to 37 ° C.
9. Mix the cells thoroughly by flicking the tube and
inverting

All patents, oligonucleotide sequences identified by gene
identification numbers, and other publications identified
herein are expressly incorporated by reference for the pur
10. Spread 50-100 ul of each dilution onto a selection
pose of describing and disclosing , for example, the meth
plate and incubate overnight at 37 ° C. Alternatively,
odologies described in such publications that might be used 60
incubate at 30 ° C. for 24-36 hours or 25 ° C. for 48
in connection with the present invention . These publications
hours
.
are provided solely for their disclosure prior to the filing date
of the present application. Nothing in this regard should be
A single colony is then used to inoculate 5 ml of LB
construed as an admission that the inventors are not entitled growth media using the appropriate antibiotic and then
to antedate such disclosure by virtue of prior invention or for 65 allowed to grow ( 250 RPM , 37 ° C. ) for 5 hours . This is then
any other reason . All statements as to the date or represen used to inoculate a 200 ml culture medium and allowed to
tation as to the contents of these documents is based on the

grow overnight under the same conditions .

US 10,898,574 B2
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57

To isolate the plasmid ( up to 850 ug ), a maxi prep is

Calif.) per manufacturer's instructions. Larger scale purifi

performed using the Invitrogen PURELINKTM HiPure cations may need to be done with a product that has a larger
Maxiprep Kit (Carlsbad, Calif. ), following the manufactur- load capacity such as Invitrogen's standard PURELINKTM
er's instructions .
PCR Kit ( Carlsbad , Calif .). Following the cleanup, the
In order to generate cDNA for In Vitro Transcription 5 linearized vector is quantified using the NanoDrop and
( IVT ) , the plasmid (an Example of which is shown in FIG . analyzed to confirm linearization using agarose gel electro
2 ) is first linearized using a restriction enzyme such as Xbal . phoresis.

A typical restriction digest with Xbal will comprise the
As a non -limiting example, G -CSF may represent the
following: Plasmid 1.0 ug ; 10x Buffer 1.0 ul; Xbal 1.5 ul; polypeptide of interest . Sequences used in the steps outlined
dH20 up to 10 ul ; incubated at 37 ° C. for 1 hr. If performing 10 in Examples 1-5 are shown in Table 2. It should be noted that
at lab scale ( < 5 ug) , the reaction is cleaned up using
Invitrogen's PURELINKTM PCR Micro Kit (Carlsbad,

the start codon (ATG ) has been underlined in each sequence
of Table 2 .

TABLE 2
G - CSF Sequences

SEQ
ID

NO
1

Description
cDNAsequence :
ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCT
GCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTG
CCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAG
ATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGGTGAGTGAGTGTGCCAC
CTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCA
TCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGC
TGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCC
CTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGA
CGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGG
CCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCC
AGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAG
GTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA

2

CDNA having T7 polymerase site and Xba restriction site :
TTGGACCCTCGTACAGAAGCTAATACGACTCACTATA

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC

ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCT
GCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTG
CCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAG
ATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGGTGAGTGAGTGTGCCAC
CTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCA
TCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGC
TGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCC
CTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGA
CGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGG
CCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCC

AGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAG
GTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGC

GGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGG
TCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA
3

Optimized sequence ; containing T7 polymerase site and Xba restriction site
TTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAA
AAGAAGAGTAAGAAGAAATATAAGAGCCACC

ATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCG
ACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGG
AGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTG
CGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCT
TGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGG
CAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGC
AAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAG
CTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGG
GATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCG
CGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTT
TGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAGCCTTCTGCG
GGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGT
CTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCA
4

mRNA sequence ( transcribed )
CUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAG
CCACCA

AUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAG
CGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUG

UCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAA
GCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGG

CACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUU
UGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCA

US 10,898,574 B2
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60
TABLE 2 - continued
G - CSF Sequences

SEQ
ID
NO

Description
GGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUG
GACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGA
UGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCC
GGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGC

CACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCG
CAGCCGUGAGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUC
CCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGGCGG
CCGCUCGAGCAUGCAU
15

to confirm the RNA is the proper size and that no degrada

Example 2 : PCR for cDNA Production

PCR procedures for the preparation of cDNA are per
formed using 2xKAPA HIFITM HotStart ReadyMix by Kapa
Biosystems (Woburn, Mass . ) . This system includes

tion of the RNA has occurred .
20

2xKAPA ReadyMix12.5 ul ; Forward Primer ( 10 uM) 0.75
ul ; Reverse Primer ( 10 uM ) 0.75 ul ; Template cDNA 100 ng;
and dH2O diluted to 25.0 ul. The reaction conditions are at
95 ° C. for 5 min . and 25 cycles of 98 ° C. for 20 sec , then 58 ° 25
C. for 15 sec , then 72 ° C. for 45 sec , then 72 ° C. for 5 min .
then 4 ° C. to termination .
The reverse primer of the instant invention incorporates a
poly - T120 for a poly - A120 in the mRNA . Other reverse
primers with longer or shorter poly ( T ) tracts can be used to 30
adjust the length of the poly (A) tail in the mRNA .
The reaction is cleaned up using Invitrogen’s PURE
LINKTM PCR Micro Kit ( Carlsbad , Calif.) per manufactur
er's instructions ( up to 5 ug ) . Larger reactions will require a
cleanup using a product with a larger capacity. Following the 35

cleanup , the cDNA is quantified using the Nano Drop and
analyzed by agarose gel electrophoresis to confirm the
cDNA is the expected size . The cDNA is then submitted for
sequencing analysis before proceeding to the in vitro tran
scription reaction.
Example 3. In Vitro Transcription (IVT)
The in vitro transcription reaction generates mRNA con
taining modified nucleotides or modified RNA . The input
nucleotide triphosphate (NTP ) mix is made in -house using
natural and un - natural NTPs .
A typical in vitro transcription reaction includes the
following:
1. Template cDNA
10x transcription buffer (400 mM Tris -HCl pH
8.0 , 190 mM MgCl2 , 50 mM DTT, 10 mm
Spermidine)

2.

1.0 ug

2.0 ul

40

Example 4. Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the
mixture includes : IVT RNA 60 ug - 180 ug and dH2O up to
72 ul . The mixture is incubated at 65 ° C. for 5 minutes to
denature RNA, and then is transferred immediately to ice .
The protocol then involves the mixing of 10x Capping
Buffer ( 0.5 M Tris -HCl (pH 8.0) , 60 mM KC1 , 12.5 mM

MgCl2 ) ( 10.0 ul); 20 mM GTP ( 5.0 ul ) ; 20 mM S -Adenosyl

Methionine ( 2.5 ul ) ; RNase Inhibitor ( 100 U) ; 2'-O -Meth
yltransferase ( 400 U) ; Vaccinia capping enzyme (Guanylyl
transferase) (40 U) ; dH2O (Up to 28 ul ) ; and incubation at
37 ° C. for 30 minutes for 60 ug RNA or up to 2 hours for
180 ug of RNA .

The mRNA is then purified using Ambion’s MEGA
CLEARTM Kit ( Austin, Tex .) following the manufacturer's
instructions. Following the cleanup, the RNA is quantified
using the NANODROPTM ( ThermoFisher, Waltham , Mass . )
and analyzed by agarose gel electrophoresis to confirm the
RNA is the proper size and that no degradation of the RNA
has occurred . The RNA product may also be sequenced by
running a reverse - transcription -PCR to generate the cDNA
for sequencing

45

Example 5. PolyA Tailing Reaction
Without a poly - T in the cDNA, a poly -A tailing reaction
50 must be performed before cleaning the final product. This is
done by mixing Capped IVTRNA ( 100 ul ) ; RNase Inhibitor
(20 U) ; 10x Tailing Buffer (0.5 M Tris - HCl ( pH 8.0 ) , 2.5 M

NaCl , 100 mM MgCl2 ) ( 12.0 ul ) ; 20 mM ATP ( 6.0 ul ) ;
Poly -A Polymerase ( 20 U) ; dH20 up to 123.5 ul and
RNase Inhibitor
20 U
55
incubation
at 37 ° C. for 30 min . If the poly -A tail is already
3000 U
5. T7 RNA polymerase
in
the
transcript
, then the tailing reaction may be skipped and
6 . dH20
Up to 20.0 ul . and
proceed directly to cleanup with Ambion's MEGA
7 . Incubation at 37 ° C. for 3 hr- 5 hrs .
CLEARTM kit ( Austin , Tex .) ( up to 500 ug ) . Poly - A Poly
merase
The crude IVT mix may be stored at 4 ° C. overnight for 60 yeast . is preferably a recombinant enzyme expressed in
cleanup the next day. 1 U of RNase - free DNase is then used
to digest the original template. After 15 minutes of incubaFor studies performed and described herein , the poly -A
tion at 37 ° C. , the mRNA is purified using Ambion's tail is encoded in the IVT template to comprise 160 nucleo
MEGACLEARTM Kit ( Austin , Tex .) following the manu- tides in length . However, it should be understood that the
3.
4.

Custom NTPs (25 mM each )

7.2 ul

facturer's instructions. This kit can purify up to 500 ug of 65 processivity or integrity of the Poly-A tailing reaction may

RNA . Following the cleanup , the RNA is quantified using
the NanoDrop and analyzed by agarose gel electrophoresis

not always result in exactly 160 nucleotides. Hence Poly -A
tails of approximately 160 nucleotides, e.g , about 150-165 ,

US 10,898,574 B2
61

62

155 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 or 165 are
within the scope of the invention .

Example 6. Formulation of Modified mRNA Using
Lipidoids

D. Capping Reaction Efficiency
Synthetic mRNAs encoding human G - CSF ( cDNA shown

5

in SEQ ID NO : 1 ) containing the ARCA cap analog or the
Capl structure can be analyzed for capping reaction effi
ciency by LC - MS after capped mRNA nuclease treatment.
Nuclease treatment of capped mRNAs would yield a mix
ture of free nucleotides and the capped 5 ' - 5 - triphosphate cap
structure detectable by LC - MS . The amount of capped
product on the LC - MS spectra can be expressed as a percent
of total mRNA from the reaction and would correspond to
capping reaction efficiency. The cap structure with a higher
capping reaction efficiency would have a higher amount of

5 ' - capping of modified RNA may be completed concomitantly during the in vitro -transcription reaction using the
following chemical RNA cap analogs to generate the
5 - guanosine cap structure according to manufacturer pro- 10
tocols : 3 ' - O - Me - m7G ( 5 ' ) ppp ( 5 ' ) G [ the ARCA cap ]; G ( 5 ' )
ppp ( 5 ' ) A ; G ( 5'ppp ( 5 ' ) G ; m7G ( 5 ') ppp ( 5 ') A ; m76 ( 5 ')ppp
( 5 ' ) G (New England BioLabs , Ipswich , Mass . ) . 5 ' - capping
of modified RNA may be completed post -transcriptionally capped product by LC -MS .
using a Vaccinia Virus Capping Enzyme to generate the 15 Example 8. Formulation of Modified mRNA Using
“ Cap 0 ” structure: m7G ( 5 ' ) ppp ( 5 ' ) G (New England Bio
Lipidoids
Labs , Ipswich , Mass . ) . Cap 1 structure may be generated
using both Vaccinia Virus Capping Enzyme and a 2-0
Modified mRNAs (mmRNA ) are formulated for in vitro
methyl -transferase to generate: m7G (54)ppp (5 ')G -2-0
methyl. Cap 2 structure may be generated from the Cap 1 20 experiments
by mixing the mmRNA with the lipidoid at a set
structure followed by the 2 - O -methylation of the 5'-ante ratio prior to addition to cells . In vivo formulation may
penultimate nucleotide using a 2-0 methyl - transferase . Cap require the addition of extra ingredients to facilitate circu
3 structure may be generated from the Cap 2 structure lation throughout the body. To test the ability of these
followed by the 2 - O -methylation of the 5'-preantepenulti- lipidoids to form particles suitable for in vivo work , a
mate nucleotide using a 2 ' -0 methyl -transferase. Enzymes 25 standard formulation process used for siRNA - lipidoid for
mulations was used as a starting point. Initial mmRNA
are preferably derived from a recombinant source .
When transfected into mammalian cells , the modified lipidoid formulations may consist of particles composed of
mRNAs have a stability of between 12-18 hours or more 42 % lipidoid, 48 % cholesterol and 10 % PEG , with further
than 18 hours , e.g. , 24 , 36 , 48 , 60 , 72 or greater than 72 optimization of ratios possible . After formation of the par
hours.
30 ticle , mmRNA is added and allowed to integrate with the
complex. The encapsulation efficiency is determined using a
Example 7. Capping
standard dye exclusion assays .
Materials and Methods for Examples 9-13
A. Lipid Synthesis
A. Protein Expression Assay
Synthetic mRNAs encoding human G - CSF ( cDNA shown 35 Six lipids , DLin - DMA , DLin- K - DMA , DLin -KC2 -DMA ,
in SEQ ID NO : 1 ) containing the ARCA (3 ' O - Me -m7G ( 5 ' ) 98N12-5 , C12-200 and DLin -MC3 -DMA, were synthesized
ppp ( 5 ' ) G ) cap analog or the Capl structure can be trans- by methods outlined in the art in order to be formulated with
fected into human primary keratinocytes at equal concen modified RNA . DLin - DMA and precursors were synthe
trations. 6 , 12 , 24 and 36 hours post - transfection the amount sized as described in Heyes et. al , J. Control Release , 2005 ,
of G - CSF secreted into the culture medium can be assayed 40 107 , 276-287 . DLin - K - DMA and DLin - KC2 - DMA and pre
by ELISA . Synthetic mRNAs that secrete higher levels of cursors were synthesized as described in Semple et . al ,
G - CSF into the medium would correspond to a synthetic Nature Biotechnology, 2010 , 28 , 172-176 . 98N12-5 and
mRNA with a higher translationally -competent Cap struc- precursor were synthesized as described in Akinc et . al ,
ture .
Nature Biotechnology, 2008 , 26 , 561-569 .
B. Purity Analysis Synthesis
45
C12-200 and precursors were synthesized according to
mRNAs encoding human G - CSF ( cDNA shown in SEQ

ID NO : 1 ) containing the ARCA cap analog or the Cap1
structure crude synthesis products can be compared for
purity using denaturing Agarose -Urea gel electrophoresis or

the method outlined in Love et . al , PNAS , 2010 , 107 ,

1864-1869 . 2 -epoxydodecane ( 5.10 g , 27.7 mmol, 8.2 eq)

was added to a vial containing Amine 200 (0.723 g , 3.36
mmol, 1 eq) and a stirring bar. The vial was sealed and
HPLC analysis. Synthetic mRNAs with a single, consoli- 50 warmed to 80 ° C. The reaction was stirred for 4 days at 80 °
dated band by electrophoresis correspond to the higher C. Then the mixture was purified by silica gel chromatog
purity product compared to a synthetic mRNA with multiple raphy using a gradient from pure dichloromethane ( DCM ) to
bands or streaking bands . Synthetic mRNAs with a single DCM : MeOH 98 : 2 . The target compound was further puri
HPLC peak would also correspond to a higher purity prod- fied by RP - HPLC to afford the desired compound.
uct . The capping reaction with a higher efficiency would 55 DLin -MC3 -DMA and precursors were synthesized
provide a more pure mRNA population .
according to procedures described in WO 2010054401
C. Cytokine Analysis
herein incorporated by reference in its entirety . A mixture of
Synthetic mRNAs encoding human G - CSF (cDNA shown dilinoleyl methanol ( 1.5 g , 2.8 mmol , 1 eq) , N , N -dimethyl
in SEQ ID NO : 1 ) containing the ARCA cap analog or the aminobutyric acid ( 1.5 g , 2.8 mmol, 1 eq ), DIPEA (0.73 mL ,
Capl structure can be transfected into human primary kera- 60 4.2 mmol, 1.5 eq ) and TBTU ( 1.35 g , 4.2 mmol, 1.5 eq) in

tinocytes at multiple concentrations. 6 , 12 , 24 and 36 hours 10 mL of DMF was stirred for 10 h at room temperature.
post - transfection the amount of pro -inflammatory cytokines Then the reaction mixture was diluted in ether and washed
such as TNF - alpha and IFN -beta secreted into the culture with water . The organic layer was dried over anhydrous
medium can be assayed by ELISA . Synthetic mRNAs that sodium sulfate, filtrated and concentrated under reduced
secrete higher levels of pro - inflammatory cytokines into the 65 pressure. The crude product was purified by silica gel
medium would correspond to a synthetic mRNA containing chromatography using a gradient DCM to DCM : MeOH
98 : 2 . Subsequently the target compound was subjected to an
an immune -activating cap structure .

US 10,898,574 B2
64

63

additional RP - HPLC purification which was done using a
YMC - Pack C4 column to afford the target compound.
B. Formulation of Modified RNA Nanoparticles
Solutions of synthesized lipid, 1,2- distearoyl-3 -phosphatidylcholine (DSPC) ( Avanti Polar Lipids , Alabaster, Ala . ) , 5
cholesterol ( Sigma - Aldrich, Taufkirchen , Germany ), and
a-[ 3'- (1,2 -dimyristoyl- 3 -propanoxy)-carboxamide-propyl]0 -methoxy -polyoxyethylene ( PEG - C - DOMG) (NOF,
Bouwelven , Belgium) were prepared at concentrations of 50
mM in ethanol and stored at -20 ° C. The lipids were 10
combined to yield molar ratio of 50 : 10 : 38.5 : 1.5 ( Lipid :
DSPC : Cholesterol: PEG - c - DOMG) and diluted with ethanol to a final lipid concentration of 25 mM . Solutions of
modified mRNA at a concentration of 1-2 mg /mL in water
were diluted in 50 mM sodium citrate buffer at a pH of 3 to 15
form a stock modified mRNA solution . Formulations of the
lipid and modified mRNA were prepared by combining the
synthesized lipid solution with the modified mRNA solution
at total lipid to modified mRNA weight ratio of 10 : 1 , 15 : 1 ,
20 : 1 and 30 : 1 . The lipid ethanolic solution was rapidly 20
injected into aqueous modified mRNA solution to afford a
suspension containing 33 % ethanol. The solutions were
injected either manually (MI ) or by the aid of a syringe
pump ( SP) (Harvard Pump 33 Dual Syringe Pump Harvard
Apparatus Holliston , Mass . ) .
25
To remove the ethanol and to achieve the buffer exchange,
the formulations were dialyzed twice against phosphate
buffered saline (PBS ) , pH 7.4 at volumes 200 -times of the
primary product using a Slide - A - Lyzer cassettes ( Thermo
Fisher Scientific Inc. Rockford , Ill . ) with a molecular weight 30
cutoff ( MWCO ) of 10 kD . The first dialysis was carried at
room temperature for 3 hours and then the formulations
were dialyzed overnight at 4 ° C. The resulting nanoparticle
suspension was filtered through 0.2 um sterile filter
( Sarstedt, Niimbrecht, Germany) into glass vials and sealed 35
with a crimp closure .
C. Characterization of Formulations
A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern ,
Worcestershire , UK) was used to determine the particle size ,
the polydispersity index (PDI ) and the zeta potential of the 40
modified mRNA nanoparticles in 1xPBS in determining
particle size and 15 mM PBS in determining zeta potential.
Ultraviolet - visible spectroscopy was used to determine
the concentration of modified mRNA nanoparticle formulation. 100 uL of the diluted formulation in 1xPBS was added 45
to 900 uL of a 4 : 1 ( v / v ) mixture of methanol and chloroform .
After mixing , the absorbance spectrum of the solution was

recorded between 230 nm and 330 nm on a DU 800

spectrophotometer ( Beckman Coulter, Beckman Coulter,
nanoparticle formulation was calculated based on the extinc

Inc., Brea, Calif .). The modified RNA concentration in the 50

tion coefficient of the modified RNA used in the formulation
and on the difference between the absorbance at a wave-

length of 260 nm and the baseline value at a wavelength of

330 nm .

QUANT-ITTM RIBOGREEN® RNA assay ( Invitrogen

Corporation Carlsbad, Calif .) was used to evaluate the
encapsulation of modified RNA by the nanoparticle. The
samples were diluted to a concentration of approximately 5
ug /mL in TE buffer ( 10 mM Tris - HCl, 1 mM EDTA , pH
7.5 ) . 50 uL of the diluted samples were transferred to a
polystyrene 96 well plate , then either 50 uL of TE buffer or
50 uL of a 2 % Triton X - 100 solution was added . The plate
was incubated at a temperature of 37 ° C. for 15 minutes. The
RIBOGREEN® reagent was diluted 1 : 100 in TE buffer, 100
uL of this solution was added to each well . The fluorescence
intensity was measured using a fluorescence plate reader

( Wallac Victor 1420 Multilablel Counter; Perkin Elmer,
Waltham , Mass . ) at an excitation wavelength of ~ 480 nm
and an emission wavelength of ~ 520 nm . The fluorescence
values of the reagent blank were subtracted from that of each
of the samples and the percentage of free modified RNA was
determined by dividing the fluorescence intensity of the
intact sample (without addition of Triton X - 100 ) by the
fluorescence value of the disrupted sample ( caused by the
addition of Triton X - 100 ).
D. In Vitro Incubation
Human embryonic kidney epithelial (HEK293 ) and hepa
tocellular carcinoma epithelial (HepG2 ) cells (LGC stan
dards GmbH , Wesel, Germany ) were seeded on 96 - well
plates (Greiner Bio - one GmbH , Frickenhausen , Germany )
and plates for HEK293 cells were precoated with collagen
typel. HEK293 were seeded at a density of 30,000 and
HepG2 were seeded at a density of 35,000 cells per well in
100 ul cell culture medium . For HEK293 the cell culture
medium was DMEM , 10 % FCS , adding 2 mM L - Glutamine ,
1 mM Sodiumpyruvate and 1x non - essential amino acids
( Biochrom AG , Berlin , Germany) and 1.2 mg/ml Sodium
bicarbonate ( Sigma - Aldrich, Munich, Germany ) and for
HepG2 the culture medium was MEM (Gibco Life Tech
nologies , Darmstadt, Germany ), 10% FCS adding 2 mM
L -Glutamine, 1 mM Sodiumpyruvate and 1x non -essential
amino acids (Biochrom AG , Berlin , Germany. Formulations
containing mCherry mRNA (mRNA sequence shown in
SEQ ID NO : 5 ; poly -A tail of approximately 160 nucleotides
not shown in sequence ; 5 ' cap , Capl) were added in qua
druplicates directly after seeding the cells and incubated .
The mCherry cDNA with the T7 promoter, 5'untranslated
region (UTR ) and 3 ' UTR used in in vitro transcription (IVT )
is given in SEQ ID NO : 6 .
Cells were harvested by transferring the culture media
supernatants to a 96 -well Pro -Bind U - bottom plate (Beckton
Dickinson GmbH , Heidelberg , Germany ). Cells were
trypsinized with 1/2 volume Trypsin /EDTA ( Biochrom AG ,
Berlin, Germany), pooled with respective supernatants and
fixed by adding one volume PBS /2 % FCS (both Biochrom
AG, Berlin, Germany) /0.5% formaldehyde (Merck, Darm
stadt, Germany ). Samples then were submitted to a flow
cytometer measurement with a 532 nm excitation laser and
the 610/20 filter for PE - Texas Red in a LSRII cytometer
(Beckton Dickinson GmbH , Heidelberg , Germany ). The
mean fluorescence intensity (MFI ) of all events and the
standard deviation of four independent wells are presented
in for samples analyzed .
Example 9. Purification on Nanoparticle

Formulations
Nanoparticle formulations of DLin -KC2 -DMA and
98N12-5 in HEK293 and HepG2 were tested to determine if

the mean fluorescent intensity (MFI ) was dependent on the
55 lipid to modified RNA ratio and / or purification. Three for
mulations of DLin -KC2 - DMA and two formulations of
98N12-5 were produced using a syringe pump to the speci
fications described in Table 3. Purified samples were purified
by SEPHADEXTM G - 25 DNA grade (GE Healthcare , Swe
60 den ). Each formulation before and after purification (aP )
were tested at concentration of 250 ng modified RNA per
well in a 24 well plate . The percentage of cells that are
positive for the marker for FL4 channel (% FL4 -positive )
when analyzed by the flow cytometer for each formulation
65 and the background sample are shown in FIGS . 3A and 3B ,
and the MFI of the marker for the FL4 channel for each
formulation and the background sample are shown in FIGS .

US 10,898,574 B2
65

66

4A and 4B . The formulations which had been purified had a
purification.

TABLE 4

slightly higher MFI than those formulations tested before

Formulations
Formulation #

5

TABLE 3
Formulations
Formulation # Lipid

10

NPA -001-1 aP DLin -KC2 - DMA

10

NPA -002-1

DLin -KC2 -DMA

15

NPA -002-1 aP

DLin -KC2- DMA

15

125 nm

NPA -003-1

DLin - KC2 - DMA

20

PDI : 0.12
114 nm
PDI : 0.08

NPA -003-1 aP

DLin -KC2 -DMA

20

155 nm

NPA - 005

DLin -KC2

98N12-5

Lipid / RNA

20

15

114 nm
PDI : 0.08

106 nm
PDI : 0.12

DMA

Lipid / RNA wt/ wt Mean size (nm)

DLin -KC2 - DMA

NPA -001-1

NPA - 003

Lipid
wt/wt
Mean size

10

PDI : 0.08
141 nm
PDI : 0.14
140 nm
PDI : 0.11

TABLE 5

15

Concentration and MFI

104 nm

MFI mCherry

PDI : 0.06

NPA - 005-1

98N12-5

15

127 nm
PDI : 0.12

NPA - 005-1 aP

98N12-5

15

98N12

20

NPA -006-1 aP 98N12

20

NPA - 006-1

Formulation

20

134 nm
PDI : 0.17
126 nm
PDI : 0.08
118 nm

NPA - 003

NPA - 005

25 ngwell

11963.25

0.25 ng /well
0.025 ng /well

1349.75
459.50
310.75

12256.75
2572.75
534.75
471.75

0.0025 ng/well
25

PDI : 0.13

Example 11. Manual Injection and Syringe Pump
Formulations

Example 10. Concentration Response Curve

Nanoparticle formulations of 98N12-5 (NPA -005 ) and
trations to determine the MFI of FL4 or mCherry (mRNA
sequence shown in SEQ ID NO : 5 ; poly -A tail of approxi

30

DLin - KC2 - DMA (NPA - 003 ) were tested at varying concen

Two formulations of DLin -KC2 -DMA and 98N12-5 were

prepared by manual injection ( MI ) and syringe pump injec
tion ( SP ) and analyzed along with a background sample to
compare the MFI of mCherry (mRNA shown in SEQ ID

NO : 5 ; poly -A tail of approximately 160 nucleotides not

35

mately 160 nucleotides not shown in sequence; 5 ' cap , Capl)
over a range of doses. The formulations tested are outlined
in Table 4. To determine the optimal concentration of
nanoparticle formulations of 98N12-5 , varying concentra 40
tions of formulated modified RNA ( 100 ng , 10 ng , 1.0 ng ,
0.1 ng and 0.01 ng per well ) were tested in a 24 -well plate
of HEK293 , and the results of the FL4 MFI of each dose are
shown in FIG . 5A . Likewise, to determine the optimal
concentration of nanoparticle formulations of DLin -KC2 45
DMA, varying concentrations of formulated modified RNA
(250 ng 100 ng, 10 ng , 1.0 ng , 0.1 ng and 0.01 ng per well)
were tested in a 24 -well plate of HEK293 , and the results of
the FL4 MFI of each dose are shown in FIG . 5B . Nanopar

shown in sequence ; 5 ' cap , Capl) of the different formula
tions . Table 5 shows that the syringe pump formulations had
a higher MFI as compared to the manual injection formu
lations of the same lipid and lipid / RNA ratio .
TABLE 5
Formulations and MFI

Lipid /
Formulation
#

Lipid

wt/wt (nm )

Untreated
Control
NPA -002

N/A

N /A

DLin -KC2

15

DLin -KC2

varying concentrations of formulated modified RNA (250

NPA - 003

DMA
DLin -KC2
DMA

20

and the results of the FL4 MFI of each dose are shown in

NPA - 003-2

DLin -KC2

20

FIG . 5C . A dose of 1 ng/well for 98N12-5 and a dose of 10
ng /well for DLin - K2 - DMA were found to resemble the FL4 55

NPA - 005

DMA
98N12-5

NPA - 005-2

98N12-5

15

To determine how close the concentrations resembled the

NPA - 006

98N12-5

20

ng, 100 ng and 30 ng per well ) in a 24 well plate of HEK293 ,

MFI of the background.

background, we utilized a flow cytometer with optimized
filter sets for detection of mCherry expression, and were able 60
to obtain results with increased sensitivity relative to back
ground levels . Doses of 25 ng/well, 0.25 ng /well, 0.025
ng /well and 0.0025 ng/well were analyzed for 98N12-5
(NPA -005 ) and DLin -K2 - DMA ( NPA - 003) to determine the
MFI of mCherry. As shown in Table 5 , the concentration of 65
0.025 ng/well and lesser concentrations are similar to the

background MFI level of mCherry which is about 386.125 .

for
mulation

MFI

674.67

N/A

N /A

140 nm

MI

10318.25

SP

37054.75

MI

22037.5

SP

37868.75

MI

11504.75

SP

9343.75

MI

11182.25

SP

5167

PDI : 0.11

DMA
NPA - 002-2

ticle formulations of DLin -KC2 -DMA were also tested at 50

Method of

RNA Mean size

15

105 nm
PDI : 0.04

114 nm
PDI : 0.08
95 nm
PDI : 0.02

15

127 nm
PDI : 0.12
106 nm
PDI : 0.07
126 nm
PDI : 0.08

NPA -006-2

98N12-5

20

93 nm
PDI : 0.08

Example 12. mCherry Fluorescence of
Formulations

Formulations of DLin - DMA , DLin - K - DMA , DLin -KC2

DMA , 98N12-5 , C12-200 and DLin -MC3 -DMA were incu

US 10,898,574 B2
67

68

bated at a concentration of 60 ng /well or 62.5 ng /well in a

administered the formulation . Serum from the mice admin

plate of HEK293 and 62.5 ng /well in a plate of HepG2 cells
for 24 hours to determine the MFI of mCherry (mRNA

istered formulations containing human G - CSF modified
mRNA are measured by specific G - CSF ELISA and serum

shown in SEQ ID NO : 5 ; poly -A tail of approximately 160 from mice administered human Factor IX modified RNA is
nucleotides not shown in sequence ; 5 ' cap , Capl ) for each 5 analyzed by specific Factor IX ELISA or chromogenic assay.

The liver and spleen from the mice administered with

formulation . The formulations tested are outlined in Table 6

mCherry modified mRNA are analyzed by immunohisto
chemistry ( IHC ) or fluorescence - activated cell sorting
( FACS ). As a control, a group of mice are not injected with

below. As shown in FIG . 6A for the 60 ng /well and FIGS .
6B , 6C , 6D , and 6E for the 62.5 ng /well, the formulation of

NPA -003 and NPA -018 have the highest mCherry MFI and 10 any formulation and their serum and tissue are collected
the formulations of NPA -008, NPA -010 and NPA -013 are
most the similar to the background sample mCherry MFI analyzed by ELISA , FACS and / or IHC .
value .

Example 14. In Vitro and In Vivo Expression
TABLE 6

Formulation # Lipid
DLin - KC2 - DMA

NPA -001

Formulations

Lipid / RNA wt/wt Mean size (nm)
10

155 nm
PDI : 0.08

NPA - 002

DLin -KC2 - DMA

15

NPA - 002-2

DLin -KC2 - DMA

15

105 nm
PDI : 0.04
114 nm
PDI : 0.08

NPA - 003

DLin -KC2 -DMA

20

NPA -003-2

DLin -KC2 - DMA

20

140 nm
PDI : 0.11

95 nm
PDI : 0.02

NPA - 005

98N12-5

15

127 nm
PDI : 0.12

NPA - 006

98N12-5

A. A. In Vitro Expression in Human Cells Using Lipidoid
The ratio of mmRNA to lipidoid used to test for in vitro
transfection is tested empirically at different lipidoid :
mmRNA ratios. Previous work using siRNA and lipidoids
20 have utilized 2.5 : 1 , 5 : 1 , 10 : 1 , and 15 : 1 lipidoid : siRNA wt:wt
ratios. Given the longer length of mmRNA relative to
siRNA , a lower wt : wt ratio of lipidoid to mmRNA may be
effective. In addition, for comparison mmRNA were also
formulated using RNAIMAXTM ( Invitrogen, Carlsbad ,
Calif.) or TRANSIT -mRNA (Mirus Bio , Madison , Wis .)
25 cationic lipid delivery vehicles . The ability of lipidoid
formulated Luciferase (IVT cDNA sequence as shown in
15

Formulations

20

126 nm
PDI : 0.08

NPA -007

DLin - DMA

15

NPA - 008

DLin - K - DMA

15

NPA -009

C12-200

15

NPA -010

DLin - MC3 - DMA

15

NPA -012

DLin - DMA

20

NPA -013

DLin- K- DMA

20

104 nm
PDI : 0.03
101 nm
PDI : 0.06

148 nm
PDI : 0.09
121 nm
PDI : 0.08

NPA -014

C12-200

20

NPA -015

DLin- MC3 - DMA

20

138 nm
PDI : 0.15
126 nm
PDI : 0.09
86 nm
PDI : 0.08

109 nm
PDI : 0.07

SEQ ID NO : 10) , green fluorescent protein (GFP ) ( IVT
cDNA sequence as shown in SEQ ID NO : 11 ) , G- CSF
(mRNA sequence shown in SEQ ID NO : 4 ; poly -A tail of
30 approximately 160 nucleotides not shown in sequence ; 5 '
cap , Capl ) , and EPO mmRNA (mRNA sequence shown in
SEQ ID NO : 7 ; poly -A tail of approximately 160 nucleotides
not shown in sequence ; 5 ' cap , Capl ) to express the desired
protein product can be confirmed by luminescence for
expression,
35 luciferase expression, flow cytometry for GFP
and by ELISA for G - CSF and Erythropoietin ( EPO ) secre
tion .
B. In Vivo Expression Following Intravenous Injection

Systemic intravenous administration of the formulations
are created using various different lipidoids including, but

40 not limited to , 98N12-5 , C12-200 , and MD1 .
Lipidoid formulations containing mmRNA are injected
45

Example 13. In Vivo Formulation Studies

intravenously into animals . The expression of the modified
mRNA (mmRNA ) -encoded proteins are assessed in blood
and / or other organs samples such as , but not limited to , the
liver and spleen collected from the animal . Conducting
single dose intravenous studies will also allow an assess
ment of the magnitude, dose responsiveness, and longevity
of expression of the desired product.
In one embodiment, lipidoid based formulations of

Mice ( n = 5 ) are administered intravenously a single dose
of a formulation containing a modified mRNA and a lipid . 98N12-5 , C12-200 , MD1 and other lipidoids, are used to
The modified mRNA administered to the mice is selected 50 deliver luciferase, green fluorescent protein (GFP ), mCherry
from G - CSF (mRNA shown in SEQ ID NO : 4 ; poly - A tail fluorescent protein , secreted alkaline phosphatase ( SAP) ,
of approximately 160 nucleotides not shown in sequence ; 5 ' human G - CSF, human Factor IX , or human Erythropoietin
cap , Capl ) , erythropoietin (EPO ) (mRNA shown in SEQ ID (EPO ) mmRNA into the animal. After formulating mmRNA
NO : 7 ; poly -A tail of approximately 160 nucleotides not with a lipid , as described previously, animals are divided
shown in sequence ; 5 ' cap , Capl ) , Factor IX (mRNA shown 55 into groups to receive either a saline formulation, or a
in SEQ ID NO : 8 ; poly - A tail of approximately 160 nucleo- lipidoid - formulation which contains one of a different
selected from luciferase, GFP , mCherry, SAP ,
tides not shown in sequence ; 5 ' cap , Capl ) or mCherry mmRNA
G - CSF, human Factor IX , and human EPO . Prior to
(mRNA sequence shown in SEQ ID NO : 5 ; poly - A tail of human
injection into the animal , mmRNA -containing lipidoid for
approximately 160 nucleotides not shown in sequence ; 5 ' mulations
are diluted in PBS . Animals are then administered
cap , Capl ) . The erythropoietin cDNA with the T7 promoter, 60 a single dose
of formulated mmRNA ranging from a dose of
5'untranslated region (UTR ) and 3 ' UTR used in in vitro 10 mg/kg to doses
as low as 1 ng/kg, with a preferred range
transcription (IVT) is given in SEQ ID NO : 9 .
to
be
10
mg
/
kg
to
100 ng /kg, where the dose of mmRNA
Each formulation also contains a lipid which is selected depends on the animal
body weight such as a 20 gram mouse
from one of DLin - DMA , DLin - K - DMA, DLin -KC2 -DMA , receiving a maximum formulation of 0.2 ml ( dosing is based
98N12-5, C12-200 or DLin-MC3- DMA. The mice are 65 no mmRNA per kg body weight).After the administration of
injected with 100 ug , 10 ug or 1 ug of the formulated the mmRNA - lipidoid formulation, serum , tissues , and / or
modified mRNA and are sacrificed 8 hours after they are

tissue lysates are obtained and the level of the mmRNA

US 10,898,574 B2
69

70

encoded product is determined at a single and /or a range of mCherry fluorescent protein, secreted alkaline phosphatase
time intervals . The ability of lipidoid - formulated Luciferase , ( SAP ) , human G - CSF, human factor IX , or human Erythro
GFP, mCherry, SAP, G - CSF, Factor IX , and EPO mmRNA poietin ( EPO ) mmRNA to express the desired protein prod
to express the desired protein product is confirmed by uct is confirmed by luminescence for luciferase expression,
luminescence
for the expression of Luciferase, flow cytom- 5 flow cytometry for GFP and mCherry expression , by enzy
etry for the expression of GFP and mCherry expression , by matic activity for SAP, and by ELISA for G - CSF, Factor IX
enzymatic activity for SAP, or by ELISA for the section of and Erythropoietin ( EPO ) secretion .
G - CSF, Factor IX and / or EPO .
Additional studies for a multi-dose regimen are also
Further studies for a multi -dose regimen are also per
formed to determine the maximal expression of mmRNA, to performed to determine the maximal expression using
evaluate the saturability of the mmRNA - driven expression 10 mmRNA, to evaluate the saturability of the mmRNA - driven
(by giving a control and active mmRNA formulation in expression (achieved by giving a control and active
parallel or in sequence ), and to determine the feasibility of mmRNA formulation in parallel or in sequence ), and to
repeat drug administration (by giving mmRNA in doses determine the feasibility of repeat drug administration (by

separated by weeks ormonths and then determining whether

giving mmRNA in doses separated by weeks or months and

expression
level is affected by factors such as immunoge- 15 then determining whether expression level is affected by
nicity) . An assessment of the physiological function of factors such as immunogenicity ) . Studies utilizing multiple

proteins such as G -CSF and EPO are also determined subcutaneous or intramuscular injection sites at one time
through analyzing samples from the animal tested and point
, are also utilized to further increase mmRNA drug
detecting increases in granulocyte and red blood cell counts, exposure
and improve protein production. An assessment of
respectively. Activity of an expressed protein product such 20 the physiological
function of proteins , such as GFP,
as Factor IX , in animals can also be assessed through mCherry , SAP, human G - CSF, human factor IX , and human
analysis of Factor IX enzymatic activity ( such as an acti EPO , are determined through analyzing samples from the
vated partial thromboplastin time assay ) and effect of clot tested
animals
andcounts
detecting
a change
in granulocyte
and /or
ting times .
red
blood
cell
.
Activity
of
an
expressed
protein
C. In Vitro Expression Following Intramuscular and / or 25 product such as Factor IX , in animals can also be assessed
Subcutaneous Injection
through analysis of Factor IX enzymatic activity ( such as an
The use of lipidoid formulations to deliver oligonucle activated partial thromboplastin time assay) and effect of
otides, including mRNA , via an intramuscular route or a clotting times .
subcutaneous route of injection needs to be evaluated as it
has not been previously reported. Intramuscular and / or sub
Example 15. Split Dose Studies
cutaneous injection of mmRNA are evaluated to determine 30
if mmRNA -containing lipidoid formulations are capable to
produce both localized and systemic expression of a desired
Studies utilizing multiple subcutaneous or intramuscular
portions.
injection
at one time point were designed and per
Lipidoid formulations of 98N12-5 , C12-200 , and MD1 formed to sites
investigate ways to increase mmRNA drug expo
containing
mmRNA
selected
from
luciferase
,
green
fluores
cent protein (GFP) , mCherry fluorescent protein, secreted 35 sure
andtheimprove
protein
production
to detec
of
expressed
protein
product., In
an addition
assessment
of the
alkaline phosphatase (SAP ), human G - CSF, human factor tion
physiological function of proteins was also determined
IX , or human Erythropoietin ( EPO ) mmRNA are injected through
analyzing samples from the animal tested .
intramuscularly and / or subcutaneously into animals. The
Surprisingly
, it has been determined that split dosing of
expression of mmRNA - encoded proteins are assessed both
within the muscle or subcutaneous tissue and systemically in 40 mmRNA produces greater protein production and pheno
blood and other organs such as the liver and spleen . Single multi
typic -responses
than those produced by single unit dosing or
dosing schemes .
dose studies allow an assessment of the magnitude, dose
responsiveness, and longevity of expression of the desired
The design of a single unit dose , multi -dose and split dose
product.
experiment involved using human erythropoietin (EPO )
Animals are divided into groups to receive either a saline 45 mmRNA (mRNA sequence shown in SEQ ID NO : 7 ; poly -A
formulation or a formulation containing modified mRNA . tail of approximately 160 nucleotides not shown in
Prior to injection mmRNA -containing lipidoid formulations sequence; 5 ' cap , Capl ) administered in buffer alone . The
are diluted in PBS . Animals are administered a single

intramuscular dose of formulated mmRNA ranging from 50
mg/kg to doses as low as 1 ng /kg with a preferred range to

dosing vehicle (F. buffer) consisted of 150 mM NaCl , 2 mM

CaCl2, 2 mM Nat -phosphate ( 1.4 mM monobasic sodium
phosphate; 0.6 mM dibasic sodium phosphate ), and 0.5 mm

be
10 mg/kg to 100 ng /kg. A maximum dose for intramus- 50 EDTA, pH 6.5. The pH was adjusted using sodium hydrox
cular administration , for a mouse , is roughly 1 mg mmRNA ide and the final solution was filter sterilized . The mmRNA

or as low as 0.02 ng mmRNA for an intramuscular injection was modified with 5meC at each cytosine and pseudouridine
into the hind limb of the mouse . For subcutaneous admin
replacement at each uridine site .
istration , the animals are administered a single subcutaneous
Animals ( n = 5 ) were injected IM ( intramuscular ) for the
dose
fromrange
400 mg
kg to80 55 single
unitdose of 100 ug. For multi-dosing, two schedules
doses ofasformulated
low as 1 ngmmRNA
/kg withranging
a preferred
to /be
were
used
doses of 100 ug and 6 doses of 100 ug . For the
mg/kg to 100 ng/kg. A maximum dose for subcutaneous split dosing, 3 scheme
schedules were used , 3 doses at
administration, for a mouse , is roughly 8 mg mmRNA or as 33.3 ug and 6 doses, two
of
16.5
ug mmRNA . Control dosing
low as 0.02 ng mmRNA .
involved
use
of
buffer
only
at
6 doses. Control mmRNA
For a 20 gram mouse the volume of a single intramuscular
the use of luciferase mmRNA ( IVT cDNA
injection is maximally 0.025 ml and a single subcutaneous 60 involved
sequence shown in SEQ ID NO : 10) dosed 6 times at 100 ug .
injection is maximally 0.2 ml . The optimal dose of mmRNA Blood
administered is calculated from the body weight of the tion . and muscle tissue were evaluated 13 hrs post injec
animal. At various points in time points following the
Human EPO protein was measured in mouse serum 13 h
administration of the mmRNA - lipidoid, serum , tissues , and
tissue lysates is obtained and the level of the mmRNA- 65 post I.M. single , multi- or split dosing of the EPO mmRNA
encoded product is determined. The ability of lipidoid- in buffer. Seven groups of mice ( n = 5 mice per group ) were
formulated luciferase, green fluorescent protein ( GFP ) ,

treated and evaluated . The results are shown in Table 7 .

US 10,898,574 B2
72

71
TABLE 7
Split dose study

Group Treatment
1

Human EPO mmRNA

2
3
4
5
6
7

Human EPO mmRNA
Human EPO mmRNA
Human EPO mmRNA

Human EPO mmRNA

Luciferase mmRNA
Buffer Alone

Avg.

Polypeptide

pmol/mL

per unit

(pmol /ug )

Dose of
mmRNA

Total
Dose

human
EPO

1 x 100 ug
3 x 100 ug
6 x 100 ug
3 x 33.3 ug
6 x 16.5 ug
6 x 100 ug

100 ug
300 ug
600 ug
100 ug
100 ug
600 ug

14.3
82.5
273.0
104.7
127.9
0

drug
.14
.28
.46
1.1
1.3

Dose

Splitting
Factor
1
2

3.3
7.9
9.3

-

0

15

The splitting factor is defined as the product per unit drug

following the protocol outlined in Example 15. In these

divided by the single dose product per unit drug (PUD ) . For studies, varied doses of 1 ug , 5 ug , 10 ug , 25 ug , 50 ug , and
example for treatment group 2 the value 0.28 or product values in between are used to determine dose response
( EPO ) per unit drug (mmRNA ) is divided by the single dose outcomes. Split dosing for a 100 ug total dose includes three
product per unit drug of 0.14 . The result is 2. Likewise , for 20 or six doses of 1.6 ug , 4.2 ug , 8.3 ug , 16.6 ug , or values and
treatment group 4 , the value 1.1 or product ( EPO ) per unit total doses equal to administration of the total dose selected .
drug (mmRNA) is divided by the single dose product per
unit drug of 0.14 . The result is 7.9 . Consequently , the dose
Injection sites are chosen from the limbs or any body
splitting factor ( DSF ) may be used as an indicator of the surface presenting enough area suitable for injection. This

efficacy
of a split dose regimen . For any single administra- 25 may also include a selection of injection depth to target the
tion of a total daily dose, the DSF should be equal to 1 .
Therefore any DSF greater than this value in a split dose
regimen is an indication of increased efficacy.
To determine the dose response trends, impact of injection
site and impact of injection timing , studies are performed. In 30
these studies, varied doses of 1 ug , 5 ug , 10 ug , 25 ug , 50 ug ,
and values in between are used to determine dose response
outcomes . Split dosing for a 100 ug total dose includes three
or six doses of 1.6 ug , 4.2 ug , 8.3 ug , 16.6 ug , or values and
total doses equal to administration of the total dose selected . 35
Injection sites are chosen from the limbs or any body

dermis ( Intradermal), epidermis ( Epidermal ), subcutaneous

tissue ( SC ) or muscle (IM) . Injection angle will vary based
on targeted delivery site with injections targeting the intra
dermal site to be 10-15 degree angles from the plane of the
surface of the skin , between 20-45 degrees from the plane of
the surface of the skin for subcutaneous injections and
angles of between 60-90 degrees for injections substantially
into the muscle. RNAIMAXTM

surface presenting enough area suitable for injection. This
Example 17. Routes of Administration
may also include a selection of injection depth to target the
dermis ( Intradermal ), epidermis (Epidermal ), subcutaneous
tissue ( SC ) or muscle ( IM) . Injection angle will vary based
Further studies were performed to investigate dosing
on targeted delivery site with injections targeting the intra- 40 using different routes of administration. Following the pro
dermal site to be 10-15 degree angles from the plane of the tocol outlined in Example 15 , 4 mice per group were dosed
surface of the skin , between 20-45 degrees from the plane of
the surface of the skin for subcutaneous injections and intramuscularly ( I.M. ) , intravenously ( IV) or subcutane
angles of between 60-90 degrees for injections substantially

into the muscle .

Example 16 : Dose Response and Injection Site
Selection and Timing
To determine the dose response trends, impact of injection
site and impact of injection timing, studies are performed

ously ( S.C. ) by the dosing chart outlined in Table 8. Serum
45 was collected 13 hours post injection from all mice , tissue
was collected from the site of injection from the intramus

cular and subcutaneous group and the spleen , liver and
kidneys were collected from the intravenous group . The
results from the intramuscular group are show in FIG . 7A
and the subcutaneous group results are shown in FIG . 7B .
TABLE 8
Dosing Chart

Group Treatment
1
2
3

4
5
6

Route Dose of mmRNA

Total
Dose

Dosing
Vehicle

Lipoplex - human EPO mmRNA

I.M. 4 x 100 ug + 30 %

4 x 70 ul Lipoplex

Lipoplex - human EPO mmRNA

I.M. 4 x 100 ug

4 x 70 ul Buffer

Lipoplex

Lipoplex -human EPO mmRNA

S.C. 4 x 100 ug + 30 %

4 x 70 ul Lipoplex

Lipoplex - human EPO mmRNA
Lipoplex - human EPO mmRNA

S.C. 4 x 100 ug

4 x 70 ul Buffer

I.V. 200 ug + 30 %

140 ul Lipoplex

Lipoplexed -Luciferase mmRNA

I.M. 100 ug + 30 %

4 x 70 ul Lipoplex

Lipoplex

Lipoplex

Lipoplex

US 10,898,574 B2
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TABLE 8 - continued
Dosing Chart

Group Treatment
7
8
9

10
11

Lipoplexed -Luciferase mmRNA
Lipoplexed - Luciferase mmRNA
Lipoplexed - Luciferase mmRNA
Lipoplexed - human EPO mmRNA
Formulation Buffer

Total
Dose

Route Dose of mmRNA

Dosing
Vehicle

I.M. 100 ug
S.C. 100 ug + 30 %

4 x 70 ul Buffer

S.C. 100 ug
I.V. 200 ug + 30 %

4 x 70 ul Buffer

Lipoplex

Lipoplex
I.M. 4x multi dosing

Example 18 : In Vivo Delivery of Modified mRNA

4 x 70 ul Lipoplex

140 ul Lipoplex
4 x 70 ul Buffer

15

Modified RNA was delivered to C57 /BL6 mice intramus-

cularly, subcutaneously, or intravenously to evaluate the
bio - distribution of modified RNA using luciferase. A formulation buffer used with all delivery methods contained
150 mM sodium chloride, 2 mM calcium chloride, 2 mM
Na + -phosphate which included 1.4 mM monobasic sodium
phosphate and 0.6 mM of dibasic sodium phosphate , and 0.5
mM ethylenediaminetetraacetic acid (EDTA ) was adjusted
using sodium hydroxide to reach a final pH of 6.5 before
being filtered and sterilized . A 1x concentration was used as
the delivery buffer. To create the lipoplexed solution deliv
ered to the mice , in one vial 50 ug of RNA was equilibrated
for 10 minutes at room temperature in the delivery buffer
and in a second vial 10ul RNAiMAXTM was equilibrated for
10 minutes at room temperature in the delivery buffer. After
equilibrium , the vials were combined and delivery buffer
was added to reach a final volume of 100 ul which was then
incubated for 20 minutes at room temperature . Luciferin was
administered by intraperitoneal injection ( IP ) at 150 mg/kg
to each mouse prior to imaging during the plateau phase of
the luciferin exposure curve which was between 15 and 30
minutes . To create luciferin , 1 g of D - luciferin potassium or
sodium salt was dissolved in 66.6 ml of distilled phosphate
buffer solution (DPBS ) , not containing Mg2 + or Ca2 + , to
make a 15 mg/ml solution . The solution was gently mixed
and passed through a 0.2 um syringe filter, before being
purged with nitrogen , aliquoted and frozen at -80 ° C. while
being protected from light as much as possible . The solution
was thawed using a waterbath if luciferin was not dissolved ,
gently mixed and kept on ice on the day of dosing.
Whole body images were taken of each mouse 2 , 8 and 24
hours after dosing . Tissue images and serum was collected

the left hind limb . The bioluminescence average for the
luciferase expression signals for each group at 2 , 8 and 24
hours after dosing are shown in FIG . 8A for the left hind
limb and FIG . 8B for the right hind limb . The biolumines

20 ug
cence showed a positive signal at the injection site of the 5

and 50 ug modified RNA formulations containing and not
containing lipoplex .
B. Subcutaneous Administration
Mice were subcutaneously ( S.C. ) administered either

25 modified luciferase mRNA (Naked -Luc ), lipoplexed modi
fied luciferase mRNA (Lipoplex - luc ) , lipoplexed modified
G - CSF mRNA ( Lipoplex - G - CSF ) or the formation buffer at
a single dose of 50 ug of modified mRNA in an injection

of 100 ul for each formulation. The bioluminescence
30 volume
average for the luciferase expression signals for each group

at 2 , 8 and 24 hours after dosing are shown in FIG . 8C . The

bioluminescence showed a positive signal at the injection
site of the 50 ug modified mRNA formulations containing
35 and not containing lipoplex .
C. Intravenous Administration

40
45

from each mouse 24 hours after dosing. Mice administered
doses intravenously had their liver, spleen , kidneys, lungs , 50
heart, peri -renal adipose tissue and thymus imaged . Mice
administered doses intramuscularly or subcutaneously had

their liver, spleen, kidneys , lungs, peri - renal adipose tissue ,
and muscle at the injection site . From the whole body
images the bioluminescence was measured in photon per 55
second for each route of administration and dosing regimen .
A. Intramuscular Administration
Mice were intramuscularly ( I.M. ) administered either
modified luciferase mRNA (IVT cDNA sequence shown in
SEQ ID NO : 10) (Naked -Luc ), lipoplexed modified lucifer- 60
ase mRNA (Lipoplex -luc ), lipoplexed modified granulocyte
colony -stimulating factor (G - CSF) mRNA (mRNA
sequence shown in SEQ ID NO : 4 ; poly -A tail of approximately 160 nucleotides not shown in sequence ; 5 ' cap , Capl)
( Lipoplex - Cytokine) or the formation buffer at a single dose 65
of 50 ug of modified RNA in an injection volume of 50 ul
for each formulation in the right hind limb and a single dose

of 5 ug of modified RNA in an injection volume of 50 ul in

Mice were intravenously ( I.V.) administered either modi
fied luciferase mRNA ( Naked -Luc ), lipoplexed modified
luciferase mRNA (Lipoplex - luc ), lipoplexed modified
G - CSF mRNA ( Lipoplex - G - CSF ) or the formation buffer at
a single dose of 50 ug of modified mRNA in an injection
volume of 100 ul for each formulation . The bioluminescence
average for the luciferase expression signal in the spleen
from each group at 2 hours after dosing is shown in FIG . 8D .
The bioluminescence showed a positive signal in the spleen
of the 50 ug modified mRNA formulations containing
lipoplex .
Example 19 : In Vivo Delivery Using Lipoplexes
A. Human EPO Modified RNA Lipoplex
A formulation containing 100 ug of modified human
erythropoietin mRNA (mRNA sequence shown in SEQ ID
NO : 7 ; poly -A tail of approximately 160 nucleotides not
shown in sequence ; 5 ' cap , Capl) (EPO ; fully modified
5 -methylcytosine; N1-methylpseudouridine) was lipoplexed
with 30 % by volume of RNAIMAXTM (Lipoplex -h - Epo - 46 ;
Generation 2 or Gen2) in 50-70 uL delivered intramuscu
larly to four C57 / BL6 mice . Other groups consisted of mice
receiving an injection of the lipoplexed modified luciferase
mRNA (Lipoplex - luc ) ( IVT cDNA sequence shown in SEQ
ID NO : 10 ) which served as a control containing 100 ug of
modified luciferase mRNA was lipoplexed with 30 % by
volume of RNAIMAXTM or mice receiving an injection of
the formulation buffer as negative control at a dose volume
of 65 ul. 13 hours after the intramuscular injection, serum

was collected from each mouse to measure the amount of

US 10,898,574 B2
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75

human EPO protein in the mouse serum by human EPO ul/eye . A formulation buffer is also administered by IVT to
ELISA and the results are shown in FIG . 9 .
a control group of rats in a dose volume of 5 ul/eye . Eyes
B. Human G - CSF Modified RNA Lipoplex
from treated rats can be collected after 18 hours post
A formulation containing 100 ug of one of the two types injection for sectioning and lysating to determine whether
of modified human G - CSF mRNA (mRNA sequence shown 5 mmRNA can be effectively delivered in vivo to the eye and
in SEQ ID NO : 4 ; poly - A tail of approximately 160 nucleo- result in protein production, and to also determine the cell
tides not shown in sequence ; 5 ' cap , Capl ) (G - CSF fully type( s) responsible for producing protein in vivo .
modified with 5 -methylcytosine and pseudouridine ( G - CSF )
Intranasal Administration
or G - CSF fully modified with 5 -methylcytosine and
N1-methyl-pseudouridine ( G - CSF -N1) lipoplexed with 10 A formulation containing 100 ug of either modified
30 % by volume of RNAIMAXTM and delivered in 150 uL mCherry mRNA lipoplexed with 30 % by volume of
intramuscularly (I.M) , in 150 L subcutaneously ( S.C ) and RNAIMAXTM , modified mCherry mRNA in saline, modi
in 225 uL intravenously ( I.V) to C57 / BL6 mice . Three fied luciferase mRNA lipoplexed with 30% by volume of
control groups were administered either 100 ug ofmodified

RNAIMAXTM or modified luciferase mRNA in saline is

luciferase mRNA ( IVT cDNA sequence shown in SEQ ID 15 delivered intranasally. A formulation buffer is also admin
NO : 10) intramuscularly ( Luc -unsp I.M. ) or 150 ug of istered to a control group intranasally. Lungs may be col
modified luciferase mRNA intravenously (Luc - unsp I.V.) or lected about 13 hours post instillation for sectioning ( for
150 uL of the formulation buffer intramuscularly (Buffer those receiving mCherry mRNA ) or homogenization ( for
I.M. ) . 6 hours after administration of a formulation, serum those receiving luciferase mRNA ). These samples will be
was collected from each mouse to measure the amount of 20 used to determine whether mmRNA can be effectively

human G - CSF protein in the mouse serum by human G - CSF delivered in vivo to the lungs and result in protein produc
ELISA and the results are shown in FIG . 10 .
tion , and to also determine the cell type (s ) responsible for
C. Human G - CSF Modified RNA Lipoplex Comparison producing protein in vivo .
A formulation containing 100 ug of either modified
human G - CSF mRNA lipoplexed with 30 % by volume of 25 Example 20 : In Vivo Delivery Using Varying Lipid
RNAIMAXTM with a 5 -methylcytosine ( 5mc ) and a
Ratios
pseudouridine ( 4 ) modification (G - CSF - Gen1 - Lipoplex ) ,
modified human G - CSF mRNA with a 5mc and y modifi
Modified mRNA was delivered to C57 /BL6 mice to
cation in saline ( G - CSF - Gen1 - Saline) , modified human
G- CSF mRNA with a N1-5 -methylcytosine (N1-5mc) and a 30 evaluate varying lipid ratios and the resulting protein expres
W modification lipoplexed with 30% by volume of sion . Formulations of 100 ug modified human EPO mRNA
RNAIMAXTM (G -CSF -Gen 2 -Lipoplex ), modified human (mRNA sequence shown in SEQ ID NO : 7 ; poly -A tail of
G - CSF mRNA with a N1-5mc and y modification in saline approximately 160 nucleotides not shown in sequence ; 5 '

( G - CSF -Gen2 -Saline ), modified luciferase with a 5mc and y cap , Capl ) lipoplexed with 10 % , 30 % or 50 %
modification lipoplexed with 30 % by volume of 35 RNAIMAXTM , 100 ug modified luciferase mRNA ( IVT
RNAIMAXTM (Luc -Lipoplex ), or modified luciferase cDNA sequence shown in SEQ ID NO : 10 ) lipoplexed with

mRNA with a 5mc and y modification in saline (Luc -Saline ) 10% , 30 % or 50 % RNAIMAXTM or a formulation buffer
was delivered intramuscularly ( I.M. ) or subcutaneously were administered intramuscularly to mice in a single 70 ul
( S.C. ) and a control group for each method of administration dose . Serum was collected 13 hours post injection to
was giving a dose of 80 uL of the formulation buffer (F. 40 undergo a human EPO ELISA to determine the human EPO
Buffer) to C57 / BL6 mice . 13 hours post injection serum and protein level in each mouse . The results of the human EPO
tissue from the site of injection were collected from each ELISA , shown in FIG . 12 , show that modified human EPO

mouse and analyzed by G - CSF ELISA to compare human
G - CSF protein levels . The results of the human G - CSF

expressed in the muscle is secreted into the serum for each
of the different percentage of RNAIMAXTM .

tion are shown in FIG . 11A , and the subcutaneous admin

Example 21 : Intramuscular and Subcutaneous In
Vivo Delivery in Mammals

protein in mouse serum from the intramuscular administra- 45
istration results are shown in FIG . 11B .

D. mCherry Modified RNA Lipoplex Comparison
A formulation containing 100 ug of either modified 50 Modified human EPO mRNA (mRNA sequence shown in
SEQ ID NO : 7 ; poly -A tail of approximately 160 nucleotides
mCherry mRNA (mRNA sequence shown in SEQ ID NO : 5 ; not
in sequence ; 5 ' cap , Capl ) formulated in saline
poly -A tail of approximately 160 nucleotides not shown in was shown
delivered to either C57 /BL6 mice or Sprague -Dawley
sequence; 5 ' cap , Capl ) lipoplexed with 30% by volume of
RNAIMAXTM or modified mCherry mRNA in saline is rats to evaluate the dose dependency on human EPO pro
Intramuscular and Subcutaneous Administration

delivered intramuscularly and subcutaneously to mice . A 55 modified
duction. Rats
were intramuscularly injected with 50 ul of the
human EPO mRNA ( h -EPO ), modified luciferase
formulation buffer is also administered to a control group of

mice either intramuscularly or subcutaneously. The site of
injection on the mice may be collected 17 hours post
injection for sectioning to determine the cell type ( s) respon

sible for producing protein .

60

mRNA ( Luc ) ( IVT DNA sequence shown in SEQ ID NO :
10) or the formulation buffer ( F.Buffer) as described in the
dosing chart Table 9 .
Mice were intramuscularly or subcutaneously injected

with 50 ul of the modified human EPO mRNA ( h - EPO ),
A formulation containing 10 ug of either modified modified luciferase mRNA ( Luc ) or the formulation buffer
mCherry mRNA lipoplexed with RNAIMAXTM , modified ( F.Buffer) as described in the dosing chart Table 10. 13 hours
mCherry mRNA in a formulation buffer, modified luciferase post injection blood was collected and serum was analyzed
mRNA lipoplexed with RNAMAXTM , modified luciferase 65 to determine the amount human EPO for each mouse or rat.
mRNA in a formulation buffer can be administered by The average and geometric mean in pg /ml for the rat study
Intravitreal Administration

intravitreal injection ( IVT ) in rats in a dose volume of 5

are also shown in Table 9 .

US 10,898,574 B2
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77
TABLE 9
Rat Study

Group
h - EPO
h - EPO
h - EPO
h - EPO
h - EPO

Luc
F. Buffer

G
G
G
G
G
G
G

Dose

R # 2

R #1

R #3

R #4

18.5

14.7

21.2

mean

R # 6 pg/ml

pg /ml

74.2 67.7
161.5 79.4
23.9 101.5
150.3 46.3
34.1 28.7

66.9
85.4
31.2

R#5

150 ug 61.8 86.3 69.9 55.2 59
100 ug 69.4 77.8 48.2 17.6 101.9
50 ug 143.6 60.9 173.4 145.9 61.5
10 ug 7.8 11.8 30.9 36.2 40.6
46.2 18.1
1 ug 9.1 35.8
100 ug 34.1 36.5 13.5 13.7

#1
# 2
# 3
#4
#5
#6
#7

Geometric

Avg.

24.5

18.7

20.3

67.1

25.4
22.4
18.5

15

Example 23. In Vitro Transfection of VEGF - A

TABLE 10
Mouse Study

Average
Level in
serum

Route

Treatment

Group

Dose

pg/ml

IM
IM
IM
IM
IM

h- EPO
h- EPO
h - EPO
h - EPO
h- EPO
Luc
F. Buffer
h - EPO

1
2
3
4
5
6

100 ug

96.2
63.5
18.7
25.9
2.6

1
2
3

100 ug
100 ug

IM
IM
SC
SC
SC

Luc

F. Buffer

50 ug
25 ug
10 ug
ug

100 ug

0
1.0
72.0
26.7
17.4

Human vascular endothelial growth factor - isoform A
(VEGF- A ) modified mRNA (mRNA sequence shown in
20 SEQ ID NO : 12 ; poly -A tail of approximately 160 nucleo
tides not shown in sequence ; 5 ' cap , Capl ) was transfected
via reverse transfection in Human Keratinocyte cells in 24
multi -well plates. Human Keratinocytes cells were grown in
EPILIFE® medium with Supplement S7 from Invitrogen
25 ( Carlsbad, Calif .) until they reached a confluence of
50-70% . The cells were transfected with 0 , 46.875 , 93.75 ,
187.5 , 375 , 750 , and 1500 ng ofmodified mRNA (mmRNA )
encoding VEGF - A which had been complexed with
RNAIMAXTM from Invitrogen ( Carlsbad, Calif . ). The RNA :
30
RNAIMAXTM complex was formed by first incubating the
RNA with Supplement - free EPILIFE® media in a 5x volu
metric dilution for 10 minutes at room temperature. In a

Example 22 : Duration of Activity after
Intramuscular In Vivo Delivery in Rats

35

Modified human EPO mRNA (mRNA sequence shown in
SEQ ID NO : 7 ; poly -A tail of approximately 160 nucleotides
not shown in sequence ; 5 ' cap , Capl ) formulated in saline

was delivered to Sprague -Dawley rats to determine the
duration of the dose response . Rats were intramuscularly
injected with 50 ul of the modified human EPO mRNA
(h- EPO ) , modified luciferase mRNA (IVT cDNA sequence
shown in SEQ ID NO : 10 ) (Luc) or the formulation buffer
( F.Buffer) as described in the dosing chart Table 11. The rats
were bled 2 , 6 , 12 , 24 , 48 and 72 hours after the intramus

cular injection to determine the concentration of human EPO
in serum at a given time . The average and geometric mean
in pg /ml for this study are also shown in Table 11 .

second vial , RNAIMAXTM reagent was incubated with
Supplement- free EPILIFE® Media in a 10x volumetric
dilution for 10 minutes at room temperature. The RNA vial

was then mixed with the RNAIMAXTM vial and incubated
for 20-30 minutes at room temperature before being added
to the cells in a drop -wise fashion .
The fully optimized mRNA encoding VEGF -A trans
40 fected
with the Human Keratinocyte cells included modifi
cations during translation such as natural nucleoside triphos
phates (NTP ), pseudouridine at each uridine site and
5 -methylcytosine at each cytosine site (pseudo - U /5mC ), and
45 N1 -methyl-pseudouridine at each uridine site and 5 -meth
ylcytosine at each cytosine site (N1-methyl- Pseudo - U /
5mC ) . Cells were transfected with the mmRNA encoding
VEGF -A and secreted VEGF -A concentration ( pg /ml) in the
culture medium was measured at 6 , 12 , 24 , and 48 hours
post - transfection for each of the concentrations using an

ELISA kit from Invitrogen ( Carlsbad , Calif.) following the

TABLE 11

Dosing Chart
Group

Dose

R #1

R #2

R #3

Luc

100 ug 60.0 62.4 53.6
100 ug 66.4 102.5 45.6
100 ug 132.9 55.1 89.0
100 g 51.1 76.3 264.3
100 ug 96.3 59.0 85.7
100 ug 46.3 66.9 73.5
24 , 48 and 72 hour 100 ug 60.2 38.5 48.8

F. Buffer

24 , 48 and 72 hour

h - EPO

h - EPO
h - EPO
h - EPO
h - EPO

h - EPO

2
6
12
24
48
72

hour
hour
hour
hour
hour
hour

50.0

10.0

R #4

R# 5

R #6

R#7

33.2 68.6 66.4 72.8
78.1 56.8 122.5 8.1
80.1 85.6 105.6 63.3
142.4 77.6 73.5 75.0
82.6 63.5 80.3
69.7
57.3 136.7 110
46.1
3.6 26.1
80.9 54.7

Avg.
pg

Geometric

ml

mean pg /ml

59.6
68.6
87.4
108.6
77.9

58.2
55.8
84.5
95.3
77.0

80.1
37.2
48.9

29.2

75.8
10.4

US 10,898,574 B2
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80

manufacturers recommended instructions. These data ,
shown in Table 12 , show that modified mRNA encoding
VEGF -A is capable of being translated in Human Keratinocyte cells and that VEGF - A is transported out of the cells and

bled at 13 hours after the intramuscular injection to deter
mine the concentration of human the polypeptide in serum
in pg /mL . The results revealed that administration of Factor

released into the extracellular environment.

lation buffer ( F.Buffer) at 2x100 ug /mouse. The mice were

5 IX mmRNA resulted in levels of 1600 pg mL
/ at 13 hours as

TABLE 12
VEGF- A Dosing and Protein Secretion

Dose (ng)

6 hours

12 hours

24 hours

48 hours

(pg /ml)

(pg /ml)

(pg ml
/ )

(pg /ml)

10

VEGF- A Dose Containing Natural NTPs
46.875

93.75
187.5
375
750
1500

10.37
9.79
14.07
19.16

21.51
36.11

18.07
20.54
24.56

37.53
38.90
61.90

33.90
41.95
45.25
53.61

88.28

51.44

61.79

76.70

86.54

67.02
65.75
64.39

VEGF -A Dose Containing Pseudo - U / 5mC
46.875
93.75

187.5
375
750
1500

10.13

16.67

33.99

72.88

11.00
16.04
69.15
133.95
198.96

20.00

46.47
83.00

145.61

448.50
524.02

392.44
526.58

426.97

505.41

34.07
188.10
304.30
345.65

120.77

VEGF - A Dose Containing N1 -methyl- Pseudo - U /5mC
46.875
93.75
187.5
375
750

1500

0.03
12.37
104.55

605.89
445.41
261.61

6.02
46.38
365.71
1201.23

27.65

100.42
167.56
1056.91

121.23

1036.45

1025.41
1653.63
1522.86

714.68

1053.12

1889.23
1954.81
1513.39

Example 24. In Vivo Studies of Factor IX

15

compared to less than 100 pg/mL of Factor IX for either
Luciferase or buffer control administration .
Example 25. Multi -Site Administration :
Intramuscular and Subcutaneous

Human G - CSF mmRNA (mRNA sequence shown in SEQ
ID NO : 4 ; poly - A tail of approximately 160 nucleotides not
shown in sequence ; 5 ' cap , Capl ) modified as either Genl or
Gen2 ( 5 -methylcytosine ( 5mc ) and a pseudouridine ( 4 )

modification , G - CSF - Genl ; or N1-5 -methylcytosine (N1

5mc ) and a y modification , G - CSF -Gen2 ) and formulated in
saline were delivered to mice via intramuscular ( IM ) or
20 subcutaneous ( SC ) injection. Injection of four doses or 2x50
ug ( two sites ) daily for three days ( 24 hrs interval) was
performed. The fourth dose was administered 6 hrs before
blood collection and CBC analysis. Controls included
Luciferase ( cDNA sequence for IVT shown in SEQ ID NO :
25 10) or the formulation buffer ( F.Buffer ). The mice were bled
at 72 hours after the first mmRNA injection ( 6 hours after the
last mmRNA dose ) to determine the effect of mmRNA
encoded human G - CSF on the neutrophil count. The dosing
regimen
is shown in Table 13 as are the resulting neutrophil
30
counts (thousands/uL ). Asterisks indicate statistical signifi
cance at p <0.05 .
For intramuscular administration , the data reveal a four
fold increase in neutrophil count above control at day 3 for
35

Human Factor IX mmRNA (mRNA shown in SEQ ID

NO : 8 ; poly -A tail of approximately 160 nucleotides not

the Gen1 G - CSF mmRNA and a two fold increase for the

Gen2 G - CSF mmRNA . For subcutaneous administration,
the data reveal a two fold increase in neutrophil count above
control at day 3 for the Gen2 CT -CSF mmRNA
TABLE 13
Dosing Regimen
Dose

Vol.
Gr. Treatment

1
2
3
4
5
6
7

G- CSF ( Gen1 )
G- CSF ( Gen1 )
G- CSF ( Gen2 )
G- CSF ( Gen2 )
Luc (Gen1 )
Luc (Gen1)
Luc ( Gen2 )

8 Luc (Gen2)

9 F. Buffer
10 F. Buffer

11 Untreated

shown in sequence ; 5 ' cap , Capl ) (Gen1; fully modified

5 -methylcytosine and pseudouridine ) formulated in saline
was delivered to mice via intramuscular injection. The

Route

I.M
S.C
I.M
S.C
I.M.
S.C.
I.M

S.C

I.M

S.C.

Dosing

N = Dose (ug /mouse )
(ul/mouse) Vehicle
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
F. buffer
50
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
50
F. buffer
5 2 x 50 ug ( four doses )
50
F. buffer
4 0 ( four doses )
50
F. buffer
4
4

O ( four doses )

50

F. buffer

Neutrophil
Thous/ uL
840 *
430

746 *
683
201

307
336
357

245
509
312

Example 26. Intravenous Administration

Human G - CSF mmRNA (mRNA sequence shown in SEQ

results demonstrate that Factor IX protein was elevated in 60 ID NO : 4 ; poly - A tail of approximately 160 nucleotides not
serum as measured 13 hours after administration .
shown in sequence ; 5 ' cap , Capl) modified with 5 -methyl
In this study, mice (N= 5 for Factor IX , N=3 for Luciferase cytosine ( 5mc ) and a pseudouridine (W ) modification; or
or Buffer controls) were intramuscularly injected with 50 ul having no modifications and formulated in 10 % lipoplex
of the Factor IX mmRNA (mRNA sequence shown in SEQ (RNAIMAXTM ) were delivered to mice at a dose of 50 ug
ID NO : 8 ; poly - A tail of approximately 160 nucleotides not 65 RNA and in a volume of 100 ul via intravenous (IV)
shown in sequence ; 5 ' cap , Capl ) , Luciferase ( cDNA injection at days 0 , 2 and 4. Neutrophils were measured at
sequence for IVT shown in SEQ ID NO : 10) or the formu- days 1 , 5 and 8. Controls included non -specific mammalian

US 10,898,574 B2
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82

RNA or the formulation buffer alone ( F.Buffer ). The mice
were bled at days 1 , 5 and 8 to determine the effect of
mmRNA - encoded human G - CSF to increase neutrophil
count. The dosing regimen is shown in Table 14 as are the

TABLE 15

resulting neutrophil counts ( thousands /uL ; K /uL ).

Dosing Regimen
5

Dose

For intravenous administration, the data reveal a four to
five fold increase in neutrophil count above control at day 5

Group

Treatment

with G - CSF mmRNA but not with unmodified G - CSF

G - CSF

G - CSF

mRNA or non - specific controls . Blood count returned to

baseline four days after the final injection. No other changes

in leukocyte populations were observed .
An asterisk indicates statistical significance at p <0.001
compared to buffer.

10 G -CSF
G - CSF
EPO
EPO
EPO

Vol.
1
2

3

G-CSF (Gen1)
Day 1
G- CSF (Gen1 )
Day 5
G- CSF (Gen1)
Day 8

4 G-CSF (no

5

5
5
5

Dosing

(ul/mouse ) Vehicle
100
10% lipoplex
100
10% lipoplex
100
10% lipoplex
100
10% lipoplex

Neutrophil

20

K /uL
2.91
5.32 *

25
2.06

1.88

modification )

Day 1

5

100

10% lipoplex

1.95

Day 5
G- CSF (no
modification )

5

100

10% lipoplex

2.09

RNA Control

5

100

10% lipoplex

2.90

5

100

1.68

4

100

4

100

4

100

4

100

10% lipoplex
10% lipoplex
10% lipoplex
10% lipoplex
10% lipoplex

5 G- CSF (no

modification )

6

Day 8
7
8
9

10

11
12

Day 1
RNA Control
Day 5
RNA Control
Day 8
F. Buffer
Day 1
F. Buffer
Day 5
F. Buffer
Day 8

EPO

Luciferase

50
50
50
50
50

5
5
5
5
5

Saline
Saline
F. buffer
Saline

Saline
F. buffer

F. buffer

serum

19.8
0.5
0.5

191.5
15.0
4.8

Example 28. EPO Multi - Dose /Multi - Administration

Dose
N=

Luciferase
F. buffer

Dosing

(ul/mouse) Vehicle

15

TABLE 14
Dosing Regimen
Gr. Treatment

Vol.
N=

Average
Protein
Product
pg /mL ,

30

35

Control dosing involved use of buffer at a single dose .

Human EPO blood levels were evaluated 13 hours post
injection .

1.72
2.51

Studies utilizing multiple intramuscular injection sites at
one time point were designed and performed .
The design of a single multi -dose experiment involved
using human erythropoietin ( EPO ) mmRNA (mRNA
sequence shown in SEQ ID NO : 7 ; poly -A tail of approxi
mately 160 nucleotides not shown in sequence ; 5 cap , Capl)
or G - CSF (mRNA sequence shown in SEQ ID NO : 4 ;
poly - A tail of approximately 160 nucleotides not shown in
sequence ; 5 ' cap , Capl ) administered in saline . The dosing
vehicle (F. buffer ) was used as a control. The EPO and
G - CSF mmRNA were modified with 5 -methylcytosine at
each cytosine and pseudouridine replacement at each uridine
site .
Animals ( n = 5 ) , Sprague- Dawley rats, were injected IM
( intramuscular) for the single unit dose of 100 ug ( delivered
to one thigh ). For multi -dosing 6 doses of 100 ug ( delivered
to two thighs ) were used for both EPO and G - CSF mmRNA .

40

1.31

Human EPO protein was measured in rat serum 13 hours
post I.M. Five groups of rats were treated and evaluated . The
results are shown in Table 16 .
TABLE 16

1.92

Multi- dose study
45

Avg.

Pg /mL

Example 27. Saline Formulation : Intramuscular

human

Administration

Human G - CSF mmRNA (mRNA sequence shown in SEQ
ID NO : 4 ; poly - A tail of approximately 160 nucleotides not
shown in sequence ; 5 ' cap , Capl) and human EPO mmRNA

Group Treatment
50

(mRNA sequence shown in SEQ ID NO : 7 ; poly - A tail of
approximately 160 nucleotides not shown in sequence; 5 ' 55
cap , Capl ) ; G - CSF mmRNA (modified with 5 -methylcyto
sine ( 5mc ) and pseudouridine ( V )) and EPO mmRNA (modi
fied with N1-5 -methylcytosine (N1-5mc ) and y modifica

1
2

3
4
5

Human EPO mmRNA
Human EPO mmRNA
G- CSF mmRNA
G- CSF mmRNA
Buffer Alone

Dose of
mmRNA

1 x 100 ug
6 x 100 mg

1 x 100 ug
6 x 100 ug

Total
Dose

100 ug

600 ug
100 ug
600 ug

EPO ,
serum

143
256
43
58
20

Example 29. Signal Sequence Exchange Study
tion ), were formulated in saline and delivered to mice via
Several variants of mmRNAs encoding human Granulo
intramuscular ( IM) injection at a dose of 100 ug .
60 cyte colony stimulating factor ( G -CSF) (mRNA sequence
Controls included Luciferase ( IVT cDNA sequence shown in SEQ ID NO : 4 ; poly - A tail of approximately 160
shown in SEQ ID NO : 10 ) or the formulation buffer ( F.Buf- nucleotides not shown in sequence; 5 ' cap , Capl) were
fer ). The mice were bled at 13 hours after the injection to synthesized using modified nucleotides pseudouridine and
determine the concentration of the human polypeptide in 5 -methylcytosine (pseudo - U /5mC ). These variants included
serum in pg /mL (G - CSF groups measured human G - CSF in 65 the G - CSF constructs encoding either the wild - type N
mouse serum and EPO groups measured human EPO in terminal secretory signal peptide sequence
mouse serum ). The data are shown in Table 15 .
(MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ

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83

ID NO : 13 ) , no secretory signal peptide sequence , or secretory signal peptide sequences taken from other mRNAs.
These included sequences where the wild type GCSF signal
peptide sequence was replaced with the signal peptide
of

sequence

either:

human

a - 1 -anti

trypsin

Transfection Preparation:
5

(MMPSSVSWGILLLAGLCCLVPVSLA ; SEQ ID NO :
14 ) , human Factor IX (MQRVNMIMAESPSLITI

CLLGYLLSAECTVFLDHENANKILNRPKR ; SEQ ID
NO :

15 ) ,

human

Prolactin

(MKGSLLLLL-

VSNLLLCQSVAP; SEQ ID NO : 16 ) , or human Albumin
( MKWVTFISLLFLFSSAYSRGVFRR ; SEQ ID NO : 17 ) .
250 ng of modified mRNA encoding each G - CSF variant
was transfected into HEK293A ( 293A in the table) , mouse
myoblast ( MM in the table ) ( C2C12 , CRL - 1772, ATCC ) and
rat myoblast ( RM in the table) ( L6 line , CRL - 1458 , ATCC )

mmRNA encoding human G - CSF (mRNA sequence
shown in SEQ ID NO : 4 ; poly - A tail of approximately 160
nucleotides not shown in sequence ; 5 ' cap , Capl ) ( contain
ing either ( 1 ) natural NTPs , ( 2 ) 100 % substitution with
5 -methyl cytidine and pseudouridine, or (3 ) 100 % substitu

tion with 5 -methyl cytidine and N1 -methyl pseudouridine;
10

mmRNA encoding luciferase (IVT cDNA sequence shown

in SEQ ID NO : 10 ) ( containing either ( 1 ) natural NTPs or
(2 ) 100 % substitution with 5 -methyl cytidine and pseudou
ridine) and TLR agonist R848 ( Invivogen tlrl - r848 ) were

diluted to 38.4 ng/uL in a final volume of 2500 uL Optimem
15 1 .

Separately, 432 uL of Lipofectamine 2000 (Invitrogen

cell lines in a 24 well plate using 1 ul of Lipofectamine 2000

( Life Technologies ), each well containing 300,000 cells . The 11668-027 , lot 1070962 ) was diluted with 13.1 mL
supernatants were harvested after 24 hrs and the secreted Optimem I. In a 96 well plate nine aliquots of 135 uL of each
G - CSF protein was analyzed by ELISA using the Human 20 mmRNA , positive control (R - 848 ) or negative control (Op
G - CSF ELISA kit (Life Technologies ). The data shown in timem I ) was added to 135 uL of the diluted Lipofectamine
Table 17 reveal that cells transfected with G - CSF mmRNA 2000. The plate containing the material to be transfected was
encoding the Albumin signal peptide secrete at least 12 fold incubated for 20 minutes. The transfection mixtures were
more G - CSF protein than its wild type counterpart.
25 then transferred to each of the human PBMC plates at 50 uL
per well . The plates were then incubated at 37 C. At 2 , 4 , 8 ,
TABLE 17
20 , and 44 hours each plate was removed from the incubator,
Signal Peptide Exchange
and the supernatants were frozen .
MM
RM
293A
After the last plate was removed, the supernatants were
30
Signal peptides
(pg /ml)

(pg/ml)

( pg /ml)

G- CSF Natural
a - 1 - anti trypsin

9650
9950

3450

6050
8475

Factor IX

11675

Prolactin
Albumin

7875

No Signal peptide

122050

5000
6175
1525
81050

173300

0

0

0

assayed using a human G - CSF ELISA kit ( Invitrogen
KHC2032 ) and human IFN - alpha ELISA kit ( Thermo Sci
entific 41105-2 ) . Each condition was done in duplicate .

11675
9800

Example 30. Cytokine Study: PBMC
PBMC Isolation and Culture :

50 mL of human blood from two donors was received

Results :
35

The ability of unmodified and modified mRNA (mmR
NAs) to produce the encoded protein was assessed (G - CSF
production ) over time as was the ability of the mRNA to
trigger innate immune recognition as measured by inter

40 feron - alpha production . Use of in vitro PBMC cultures is an
accepted way to measure the immunostimulatory potential
of oligonucleotides (Robbins et al . , Oligonucleotides 2009

from Research Blood Components (lots KP30928 and 19 : 89-102).
KP30931 ) in sodium heparin tubes . For each donor, the
blood was pooled and diluted to 70 mL with DPBS ( SAFC 45 Results were interpolated against the standard curve of
Bioscience 59331C , lot 071M8408 ) and split evenly each ELISA plate using a four parameter logistic curve fit.
between two 50 mL conical tubes . 10 mL of Ficoll Paque Shown in Tables 18 and 19 are the average from 2 separate
( GE Healthcare 17-5442-03 , lot 10074400 ) was gently dis- PBMC donors of the G - CSF and IFN - alpha production over
pensed below the blood layer. The tubes were centrifuged at time as measured by specific ELISA .
2000 rpm for 30 minutes with low acceleration and braking . 50
In the G - CSF ELISA , background signal from the Lipo
The tubes were removed and the buffy coat PMBC layers fectamine
untreated condition was subtracted at each
were gently transferred to a fresh 50 mL conical and washed timepoint. 2000
The data demonstrated specific production of
with DPBS . The tubes were centrifuged at 1450 rpm for 10
human G - CSF protein by human peripheral blood mononu
minutes .

The supernatant was aspirated and the PBMC pellets were 55 clear is seen with G - CSF mRNA containing natural NTPs ,
resuspended and washed in 50 mL of DPBS . The tubes were 100 % substitution with 5 -methyl cytidine and pseudouri
centrifuged at 1250 rpm for 10 minutes. This wash step was dine , or 100% substitution with 5 -methyl cytidine and
repeated, and the PBMC pellets were resuspended in 19 mL N1-methyl pseudouridine. Production of G - CSF was sig
of Optimem I (Gibco 11058, lot 1072088 ) and counted . The nificantly increased through the use of modified mRNA

cell
suspensions were adjusted to a concentration of 3.0x 60 relative to unmodified mRNA, with the 5 -methyl cytidine
10 ^ 6 cells /mL live cells .

N1 - methyl pseudouridine containing G - CSF mmRNA
These cells were then plated on five 96 well tissue culture and
showing
highest level of G - CSF production. With
treated round bottom plates (Costar 3799 ) per donor at 50 ML regards totheinnate
immune recognition, unmodified mRNA
per well . Within 30 minutes, transfection mixtures were
added to each well at a volume of 50 uL per well . After 4 65 resulted in substantial IFN - alpha production, while the
hours post transfection , the media was supplemented with modified mRNA largely prevented interferon -alpha produc
10 uL of Fetal Bovine Serum ( Gibco 10082 , lot 1012368 )

tion .

US 10,898,574 B2
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86

TABLE 18

AMBION® , Austin , Tex .; MSCRIPTTM mRNA Production
Kit , EPICENTRE® Biotechnologies, Madison , Wis . ). An in
vitro transcription reaction contains between 1-2 ug of
template DNA in the form of a linearized plasmid , PCR
product, or single-stranded oligonucleotide with a double
stranded polymerase promoter region . The template DNA
encodes a strong translation initiation sequence such as a
strong consensus Kozak sequence or an optimized , high

G- CSF Signal

G - CSF signal - 2 Donor Average

pg /mL

2 Hr 4 Hr

20 Hr

44 Hr

G - CSF ( 5mC /pseudouridine)
G- CSF (5mC /N1-methyl
pseudouridine )

120.3 136.8 421.0 346.1
256.3 273.7 919.3 1603.3

431.8
1843.3

258.3

242.4
58.0

GCSF ( Natural-no modification )

Luciferase ( 5mC /pseudouridine)

8 Hr

63.5 92.6 129.6
4.5 153.7 33.0

186.5

TABLE 19
IFN- alpha signal

10

15

IFN -alpha signal - 2 donor average

pg /mL

2 Hr

4 Hr

8 Hr

G- CSF (5mC /pseudouridine)
G- CSF ( 5mC /N1-methyl
pseudouridine)

21.1

2.9

3.7

0.5

0.4

3.0

G- CSF (Natural)

0.0
0.4
39.1
0.8

2.1
0.4
151.3
17.2

23.3

Luciferase ( 5mC /pseudouridine)
R - 848
Lipofectamine 2000 control

5

4.7
278.4

16.5

20 Hr 44 Hr
22.7
2.3

4.3

74.9
1.0
362.2
0.7

119.7
2.4

2.1

208.1
3.1

expression IRES including the EMCV IRES . Reaction vol
umes are between 20-40 ul and contain 3 ' - O - Me - mP - G ( 5 ' )
ppp ( 5 ')G ARCA cap analog (NEW ENGLAND
BIOLABS® ) in addition to an optimized ribonucleotide
mixture of determined modified adenine, guanine, cytidine
and uridine ribonucleotide analogs . Final reaction concen
trations for nucleotide are 6 mM for the cap analog and
1.5-7.5 mM for each of the other nucleotides. The tempera

ture and duration of the in vitro transcription reaction are
optimized for efficiency, fidelity and yield. Reactions may be
20 incubated from 3-6 hours and up to 16 hours at 37 ° C.
Following the in vitro transcription reaction, the capped
mRNA undergoes polyadenylation using a commercially
available poly -A tailing kit ( EPICENTRE® Biotechnolo
gies , Madison , Wis . ). The resulting capped and polyade

25 nylated synthetic mRNA is then purified by denaturing
agarose gel electrophoresis to confirm production of full
length product and to remove any degradation products
Example 31. Quantification in Exosomes
followed by spin column filtration (RNeasy Kit , Qiagen ,
The quantity and localization of the mmRNA of the Valencia , Calif .; MEGACLEARTM AMBION® , Austin ,

present invention can be determined by measuring the 30 Tex . ). Purified synthetic mRNAs are resuspended in RNase
amounts ( initial , timecourse , or residual basis ) in isolated free water containing an RNase inhibitor (RNASIN® Plus
exosomes . In this study, since the mmRNA are typically RNase Inhibitor, Promega, Madison , Wis . ), quantified by
codon - optimized and distinct in sequence from endogenous NANODROPTM ( Thermo Scientific , Logan, Utah ) and
mRNA , the levels of mmRNA are quantitated as compared 35 stored at -20 ° C.
to endogenous levels of native or wild type mRNA by using
the methods of Gibbings , PCT/IB2009 /005878 , the contents
Example 34 : Bulk Transfection of Modified mRNA
into Cell Culture
of which are incorporated herein by reference in their
entirety.
In these studies, the method is performed by first isolating
A. Cationic Lipid Delivery Vehicles
exosomes or vesicles preferably from a bodily fluid of a 40 RNA transfections are carried out using RNAIMax (Invit
patient previously treated with a polynucleotide, primary rogen , Carlsbad , Calif .) or TRANSIT -mRNA (Mirus Bio ,
construct or mmRNA of the invention, then measuring, in Madison , Wis .) cationic lipid delivery vehicles. RNA and
said exosomes , the polynucleotide, primary construct or reagent are first diluted in Opti - MEM basal media (Invitro
mmRNA levels by one of mRNA microarray, qRT- PCR , or gen , Carlsbad, Calif. ). 100 ng/uL RNA is diluted 5x and 5
other means for measuring RNA in the art including by 45 uL of RNAIMax perm of RNA is diluted 10x . The diluted
components are pooled and incubated 15 minutes at room
suitable antibody or immunohistochemical methods.
temperature before they are dispensed to culture media . For
Example 32 : Bifunctional mmRNA
TRANSIT -mRNA transfections , 100 ng /uL RNA is diluted
10x in Opti - MEM and BOOST reagent is added ( at a
Using the teachings and synthesis methods described 50 concentration of 2 ul perm of RNA ), TRANSIT -mRNA is
herein , modified RNAs are designed and synthesized to be added ( at a concentration of 2 uL perm of RNA ), and then
bifunctional, thereby encoding one or more cytotoxic pro- the RNA - lipid complexes are delivered to the culture media
tein molecules as well as be synthesized using cytotoxic after a 2 -minute incubation at room temperature. RNA
nucleosides.
transfections are performed in Nutristem xenofree hES
Administration of the bifunctional modified mRNAs is 55 media (STEMGENT® , Cambridge, Mass . ) for RiPS deri
effected using either saline or a lipid carrier. Once admin- vations , Dermal Cell Basal Medium plus Keratinocyte
istered , the bifunctional modified mRNA is translated to Growth Kit ( ATCC ) for keratinocyte experiments, and Opti
produce the encoded cytotoxic peptide. Upon degradation of MEM plus 2 % FBS for all other experiments. Successful
the delivered modified mRNA , the cytotoxic nucleosides are introduction of a modified mRNA (mmRNA ) into host cells

released which also effect therapeutic benefit to the subject. 60 can
be monitored using various known methods, such as a
fluorescent marker, such as Green Fluorescent Protein

Example 33. Synthesis of Modified mRNA
(GFP ) . Successful transfection of a modified mRNA can
also be determined by measuring the protein expression
Modified mRNA is generated from a cDNA template level of the target polypeptide by e.g. , Western Blotting or
containing a 17 RNA -polymerase promoter sequence using 65 immunocytochemistry. Similar methods may be followed
a commercially available 17 RNA polymerase transcription for large volume scale -up to multi - liter ( 5-10,000 L ) culture
kit (MEGASCRIPT® High Yield Transcription KIT, format following similar RNA - lipid complex ratios.

US 10,898,574 B2
87
mRNA Transcripts

88
trolled via rotameters. Air was sparged into the reactor via a
submerged frit ( 5 um pore size ) and through the reactor head

cally detect the exogenous transcripts. Discharging a 150 uF
capacitor charged to F into 2.5x10 cells suspended in 50 ul
of Opti - MEM ( Invitrogen , Carlsbad , Calif.) in a standard

used for cell counting was stained with trypan blue ( Sigma ,
St. Louis , Mo. ) . Cell count and cell viability determination
were performed via hemocytometry using a microscope. For

repeated delivery in excess of 10,000 copies of modified
mRNA transcripts per cell , as determined using the standard
curve method, while maintaining high viability (> 70 % ).
Further experiments may reveal that the voltage required to
efficiently transfect cells with mmRNA transcripts can 15
depend on the cell density during electroporation . Cell
density may vary from 1x10 cell / 50 ul to a density of
2.5x10 cells / 50 ul and require from 110V to 145V to
transfect cells with similar efficiencies measured in transcript copies per cell . Large multi- liter ( 5-10,000 L ) elec- 20
troporation may be performed similar to large volume flow
electroporation strategies similar to methods described with

for 20 minutes at 2000 rpm ( 4 ° C. ) for cell separation.
Supernatant was analyzed for the following parameters:
titer, sialic acid , glucose , lactate , glutamine, glutamate, pH ,
p02 , pCO2 , ammonia , and, optionally, lactate dehydroge
nase (LDH ). Additional back-up samples were frozen at
-20 ° C. To measure secreted humanized IgG antibody titers ,
supernatant is taken from seed - stock cultures of all stable
cell pools , the IgG titer is determined by ELISA and divided
by the mean number of cells to calculate the specific
productivity. The highest values are the cell pools with the
Ser132A CERT mutant, followed by wild type CERT. In
both, IgG expression is markedly enhanced compared to

B. Electroporation Delivery of Exogenous Synthetic
Electroporation parameters are optimized by transfecting space for CO2 removal. Molecular oxygen was sparged
MRC - 5 fibroblasts with in vitro synthetic modified mRNA through the same frit for DO control. CO2 was sparged
( mmRNA ) transcripts and measuring transfection efficiency 5 through same frit as used for pH control. Samples of cells
by quantitative RT - PCR with primers designed to specifi- were removed from the reactor on a daily basis . A sample

electroporation cuvette with a 2 mm gap is sufficient for 10 analysis of metabolites, additional samples were centrifuged

the above described constraints (Li et al . , 200 Geng et al . ,

carrier -alone or untransfected cells .

Continuous or Batch -Fed Culture

2010 ) .
25

Example 35. Overexpression of Ceramide Transfer
Protein to Increase Therapeutic Antibody Protein
Production in Established CHO Cell Lines

A. Batch Culture
30
An antibody producing CHO cell line (CHO DG44 )
secreting a humanized therapeutic IgG antibody is transfected a single time with lipid cationic delivery agent alone
( control) or a synthetic mRNA transcript encoding wild type
ceramide transfer protein ( CERT) or a non -phosphorylation 35
competent Ser132A CERT mutant. The sequences are taught
in for example, U.S. Ser. No. 13 /252,049 , the contents of
which are incorporated herein by reference in their entirety.
CERT is an essential cytosolic protein in mammalian cells
that transfers the sphingolipid ceramide from the endoplas- 40
mic reticulum to the Golgi complex where it is converted to
sphingomyelin ( Hanada et al . , 2003 ). Overexpression of
CERT significantly enhances the transport of secreted proteins to the plasma membrane and improves the production
of proteins that are transported via the secretory pathway 45
from eukaryotic cells thereby enhancing secretion of proteins in the culture medium . Synthetic mRNA transcripts are

pre -mixed with a lipid cationic delivery agent at a 2-5 : 1
carrier :RNA ratio . The initial seeding density is about 2x105
viable cells /mL . The synthetic mRNA transcript is delivered 50
after initial culture seeding during the exponential culture
growth phase to achieve a final synthetic mRNA copy
number between 10x102 and 10x109 per cell . The basal cell
culture medium used for all phases of cell inoculum generation and for growth of cultures in bioreactors was modi- 55
fied CD - CHO medium containing glutamine , sodium bicarbonate , insulin and methotrexate . The pH of the medium was
adjusted to 7.0 with 1 N HCl or IN NaOH after addition of
all components. Culture run times ended on days 7 , 14 , 21
or 28+ . Production - level 50 L scale reactors ( stainless steel 60
reactor with two marine impellers ) were used and are
scalable to > 10,000 L stainless steel reactors ( described in
commonly - assigned patent application U.S. Ser. No. 60/436 ,
050 , filed Dec. 23 , 2002 , and U.S. Ser. No. 10/740,645 ) . A
data acquisition system ( Intellution Fix 32 , OSIsoft, LLC , 65
San Leandro , Calif.) recorded temperature, pH , and dissolved oxygen (DO ) throughout runs. Gas flows were con-

An antibody producing CHO cell line (CHO DG44 )
secreting humanized IgG antibody is transfected with lipid
cationic delivery agent alone ( control) or a synthetic mRNA
transcript encoding wild type ceramide transfer protein or a
non -phosphorylation competent Ser132A CERT mutant.
Synthetic mRNA transcripts are pre -mixed with a lipid
cationic delivery agent at a 2-5 : 1 carrier :RNA ratio . The
initial seeding density was about 2x10% viable cells/mL .
Synthetic mRNA transcript is delivered after initial culture
seeding during the exponential culture growth phase to
achieve a final synthetic mRNA copy number between
10x102 and 10x103 per cell . The basal cell culture medium
used for all phases of cell inoculum generation and for
growth of cultures in bioreactors was modified CD - CHO
medium containing glutamine, sodium bicarbonate , insulin
and methotrexate . The pH of the medium was adjusted to 7.0
with 1 N HCl or IN NaOH after addition of all components .
Bioreactors of 5 L scale (glass reactor with one marine
impeller ) were used to obtain maximum CERT protein
production and secreted humanized IgG antibody curves .
For continuous or fed - batch cultures, the culturing run time
is increased by supplementing the culture medium one or
more times daily (or continuously ) with fresh medium
during the run . In the a continuous and fed -batch feeding
regimens, the cultures receive feeding medium as a continu
ously - supplied infusion , or other automated addition to the
culture, in a timed , regulated, and / or programmed fashion so
as to achieve and maintain the appropriate amount of
synthetic mRNA : carrier in the culture. The preferred
method is a feeding regimen of a once per day bolus feed
with feeding medium containing synthetic mRNA : carrier on
each day of the culture run , from the beginning of the culture
run to the day of harvesting the cells . The daily feed amount
was recorded on batch sheets. Production - level 50 L scale
reactors ( stainless steel reactor with two marine impellers )
were used and are scalable to > 10,000 L stainless steel
reactors. A data acquisition system ( Intellution Fix 32 )
recorded temperature , pH , and dissolved oxygen ( DO )
throughout runs. Gas flows were controlled via rotameters.
Air was sparged into the reactor via a submerged frit ( 5 um
pore size) and through the reactor head space for CO2
removal. Molecular oxygen was sparged through the same
frit for DO control. CO2 was sparged through same frit as

US 10,898,574 B2
89

90

used for pH control. Samples of cells were removed from the

reactor on a daily basis . A sample used for cell counting was
stained with trypan blue ( Sigma , St. Louis , Mo. ) . Cell count
and cell viability determination were performed via hemo-

B. Continuous or Batch - Fed Culture

A primary CHO cell line derived and expanded as
described above ( see Example 36a ) is transfected with lipid
cationic delivery agent alone ( control) or a synthetic mRNA

cytometry using a microscope. For analysis of metabolites, 5 transcript encoding human erythropoietin protein . Synthetic
additional samples were centrifuged for 20 minutes at 2000 mRNA transcripts are pre -mixed with a lipid cationic deliv
rpm ( 4 ° C. ) for cell separation. Supernatant was analyzed for ery agent at a 2-5 : 1 carrier :RNA ratio . The initial seeding
the following parameters: titer, sialic acid , glucose , lactate , density was about 2x10 % viable cells/mL . Synthetic mRNA
glutamine , glutamate , pH , pO2 , pCO2 , ammonia, and, transcript is delivered after initial culture seeding during the

optionally, lactate dehydrogenase (LDH ). Additional back- 10 exponential culture growth phase to achieve a final synthetic

up samples were frozen at -20 ° C. To measure secreted mRNA copy number between 10x102 and 10x103 per cell .
humanized IgG antibody titers, supernatant is taken from Culture conditions were as described above ( Example 35a) .
seed - stock cultures of all stable cell pools , the IgG titer is For continuous or fed -batch cultures, the culturing run time
determined by ELISA and divided by the mean number of is increased by supplementing the culture medium one or
cells to calculate the specific productivity. The highest 15 more times daily (or continuously ) with fresh medium
values are the cell pools with the Ser132A CERT mutant, during the run . In the a continuous and fed -batch feeding
followed by wild type CERT. In both , IgG expression is regimens, the cultures receive feeding medium as a continu
markedly enhanced compared to carrier - alone or untrans- ously - supplied infusion , or other automated addition to the
culture, in a timed , regulated, and / or programmed fashion so
fected cells .
20 as to achieve and maintain the appropriate amount of
Example 36. De Novo Generation of a Mammalian
synthetic mRNA : carrier in the culture. The preferred
Cell Line Expressing Human Erythropoietin as a
method is a feeding regimen of a once per day bolus feed
Therapeutic Agent
with feeding medium containing synthetic mRNA : carrier on
each day of the culture run , from the beginning of the culture
A. Batch Culture
25 run to the day of harvesting the cells . The daily feed amount
This Example describes the production of human eryth- was recorded on batch sheets. Production - level 50 L scale
ropoietin protein ( EPO ) from cultured primary CHO cells . reactors ( stainless steel reactor with two marine impellers )
Erythropoietin is a glycoprotein hormone that is required for were used and are scalable to > 10,000 L stainless steel
red blood cell synthesis. EPO protein may be used as a reactors. Culture growth and analysis were performed as
therapeutic agent for anemia from cancer, heart failure, 30 described herein ( see Example 35 ) .
chronic kidney disease and myelodysplasia . Primary CHO
It is to be understood that the words which have been used
cells are isolated and cultured as described ( Tjio and Puck , are words of description rather than limitation , and that
1958 ) . Primary CHO cells were then expanded in modified changes may be made within the purview of the appended
CD - CHO medium containing glutamine , sodium bicarbon- claims without departing from the true scope and spirit of the
ate , insulin, and methotrexate ( see Example 35 ) using T -75 35 invention in its broader aspects .
flasks ( Corning, Corning, N.Y.) and 250 and 500 mL spinWhile the present invention has been described at some
ners (Bellco , Vineland , N.J. ) . T - flasks and spinners were length and with some particularity with respect to the several
incubated at 37 ° C. in 6 % CO2 . After sufficient inoculum described embodiments, it is not intended that it should be
was generated, the culture was transferred into a either a 5 limited to any such particulars or embodiments or any
Lor a 50 L bioreactor as described above ( see Example 35 ) . 40 particular embodiment, but it is to be construed with refer
Synthetic mRNA transcript encoding the human erythropoi- ences to the appended claims so as to provide the broadest
etin protein are pre -mixed with a lipid cationic delivery possible interpretation of such claims in view of the prior art
agent at a 2-5 : 1 carrier:RNA
in a minimum of 1 % total and , therefore, to effectively encompass the intended scope
culture volume . The initial seeding density is about 2x105 of the invention .
viable cells /mL . The synthetic mRNA transcript is delivered 45 All publications , patent applications, patents, and other
after initial culture seeding during the exponential culture references mentioned herein are incorporated by reference in
growth phase to achieve a final synthetic mRNA copy their entirety. In case of conflict, the present specification,
number between 10x102 and 10x103 per cell . Culture including definitions, will control. In addition , section head
growth and analysis were performed as described above ( see ings , the materials, methods, and examples are illustrative
Example 34 ) .
only and not intended to be limiting .
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS :

17

< 210 > SEQ ID NO 1
< 211 > LENGTH : 624
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 1

atggctggac ctgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg

60

cacagtgcac tctggacagt gcaggaagcc acccccctgg gccctgccag ctccctgccc

120

cagagcttcc tgctcaagtg cttagagcaa gtgaggaaga tccagggcga tggcgcagcg

180

US 10,898,574 B2

92

91
- continued
ctccaggaga agctggtgag tgagtgtgcc acctacaagc tgtgccaccc cgaggagctg

240

gtgctgctcg gacactctct gggcatcccc tgggctcccc tgagcagctg ccccagccag

300

gccctgcagc tggcaggctg cttgagccaa ctccatagcg gccttttcct ctaccagggg

360

ctcctgcagg ccctggaagg gatctccccc gagttgggtc ccaccttgga cacactgcag

420

ctggacgtcg ccgactttgc caccaccato tggcagcaga tggaagaact gggaatggcc

480

cctgccctgc agcccaccca gggtgccatg ccggccttcg cctctgcttt ccagcgccgg

540

gcaggagggg tcctggttgc ctcccatctg cagagcttcc tggaggtgtc gtaccgcgtt

600

ctacgccacc ttgcccagcc ctga

624

< 210 > SEQ ID NO 2
< 211 > LENGTH : 829
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 2

ttggaccctc gtacagaagc taatacgact cactataggg aaataagaga gaaaagaaga
gtaagaagaa atataagagc caccatggct ggacctgcca cccagagccc catgaagctg

120

atggccctgc agctgctgct gtggcacagt gcactctgga cagtgcagga agccaccccc

180

ctgggccctg ccagctccct gccccagagc ttcctgctca agtgcttaga gcaagtgagg

240

aagatccagg gcgatggcgc agcgctccag gagaagctgg tgagtgagtg tgccacctac
aagctgtgcc accccgagga gctggtgctg ctcggacact ctctgggcat cccctgggct

300

cccctgagca gctgccccag ccaggccctg cagctggcag gctgcttgag ccaactccat

420

agcggccttt tcctctacca ggggctcctg caggccctgg aagggatctc ccccgagttg

480

ggtcccacct tggacacact gcagctggac gtcgccgact ttgccaccac catctggcag

540

cagatggaag aactgggaat ggcccctgcc ctgcagcoca cccagggtgc catgccggcc

600

ttcgcctctg ctttccagcg ccgggcagga ggggtcctgg ttgcctccca tctgcagage

660

ttcctggagg tgtcgtaccg cgttctacgc caccttgccc agccctgaag cgctgccttc

720

tgcggggctt gccttctggc catgcccttc ttctctccct tgcacctgta cctcttggtc
tttgaataaa gcctgagtag gaaggcggcc gctcgagcat gcatctaga

780

60

360

829

< 210 > SEQ ID NO 3
< 211 > LENGTH : 772
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 3

ttggaccctc gtacagaagc taatacgact cactataggg aaataagaga gaaaagaaga

60

gtaagaagaa atataagagc caccatggcc ctgcagttgc tgctttggca ctcggccctc

120

tggacagtcc aagaagcgac tcctctcgga cctgcctcat cgttgccgca gtcattcctt

180

ttgaagtgtc tggagcaggt gcgaaagatt cagggcgatg gagccgcact ccaagagaag

240

ctctgcgcga catacaaact ttgccatccc gaggagctcg tactgctcgg gcacagcttg
gggattccct gggctcctct ctcgtcctgt ccgtcgcagg ctttgcagtt ggcagggtgc

300

ctttcccagc tccactccgg tttgttcttg tatcagggac tgctgcaagc ccttgaggga

420

CCC gacgctggac ac -tgcagc tcgacg ggc ggatttcgca

480

acaaccatct ggcagcagat ggaggaactg gggatggcac ccgcgctgca gcccacgcag

540

ggggcaatgc cggcctttgc gtccgcgttt cagcgcaggg cgggtggagt cctcgtagcg

600

ata

cag aat

360

US 10,898,574 B2
94

93
- continued

agccaccttc aatcattttt ggaagtctcg taccgggtgc tgagacatct tgcgcagccg

660

tgagccttct gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac

720

ctcttggtct ttgaataaag cctgagtagg aaggcggccg ctcgagcatg ca

772

< 210 > SEQ ID NO 4
< 211 > LENGTH : 746
< 212 > TYPE : RNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE :

4

cucacuauag ggaaauaaga gagaaaagaa gaguaagaag aaauauaaga gccaccaaug

60

goccugcagu ugcugcuuug gcacucggco cucuggacag uccaagaagc gacuccucuc

120

ggaccugccu caucguugcc gcagucauuc cuuuugaagu gucuggagca ggugcgaaag

180

auucagggcg auggagccgc acuccaagag aagcucugcg cgacauacaa acuuugccau

240

cccgaggagc ucguacugcu cgggcacagc uuggggauuc ccugggcucc ucucucgucc

300

uguccgucgc aggcuuugca guuggcaggg ugccuuuccc agcuccacuc cgguuuguuc
uuguaucagg gacugcugca agcccuugag ggaaucucgc cagaauuggg cccgacgcug

360

gacacguugc agcucgacgu ggcggauuuc gcaacaacca ucuggcagca gauggaggaa

480

cuggggaugg cacccgcgcu gcagcccacg cagggggcaa ugccggccuu ugcguccgcg

540

uuucagcgca gggcgggugg aguccucgua gcgagccacc uucaaucauu uuuggaaguc

600

ucguaccggg ugcugagaca ucuugcgcag ccgugagccu ucugcggggc uugccuucug

660

gccaugcccu ucuucucucc cuugcaccug uaccucuugg ucuuugaaua aagccugagu

720

aggaaggcgg ccgcucgagc augcau

746

420

< 210 > SEQ ID NO 5
< 211 > LENGTH : 854
< 212 > TYPE : RNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 5

gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug guauccaagg

60

gggaggagga caacauggcg aucaucaagg aguucaugcg auucaaggug cacauggaag
guucggucaa cggacacgaa uuugaaaucg aaggagaggg ugaaggaagg cccuaugaag
ggacacagac cgcgaaacuc aaggucacga aagggggacc acuuccuuuc gccugggaca

120

240

uucuuucgcc ccaguuuaug uacgggucca aagcauaugu gaagcauccc gccgauauuc

300

cugacuaucu gaaacucagc uuucccgagg gauucaagug ggagcggguc augaacuuug

360

aggacggggg uguagucacc guaacccaag acucaagccu ccaagacggc gaguucaucu

420

acaaggucaa acugcggggg acuaacuuuc cgucggaugg gccggugaug cagaagaaaa

480

cgaugggaug ggaagcguca ucggagagga uguacccaga agauggugca uugaaggggg

540

agaucaagca gagacugaag uugaaagaug ggggacauua ugaugccgag gugaaaacga

600

cauacaaagc gaaaaagccg gugcagcuuc ccggagcgua uaaugugaau aucaaguugg

660

auauuacuuc acacaaugag gacuacacaa uugucgaaca guacgaacgc gcugagggua

720

gacacucgac gggaggcaug gacgaguugu acaaaugaua agcugccuuc ugcggggcuu

780

gccuucuggc caugcccuuc uucucucccu ugcaccugua ccucuugguc uuugaauaaa

840

gccugaguag gaag

854

180

US 10,898,574 B2

95

96
- continued

< 210 > SEQ ID NO 6
< 211 > LENGTH : 924
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 6

tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga

60

aaagaagagt aagaagaaat ataagagcca ccatggtatc caagggggag gaggacaaca

120

tggcgatcat caaggagttc atgcgattca aggtgcacat ggaaggttcg gtcaacggac

180

acgaatttga aatcgaagga gagggtgaag gaaggcccta tgaagggaca cagaccgcga
aactcaaggt cacgaaaggg ggaccacttc ctttcgcctg ggacattctt tcgccccagt

240

ttatgtacgg gtccaaagca tatgtgaagc atcccgccga tattcctgac tatctgaaac

360

tcagctttcc cgagggattc aagtgggagc gggtcatgaa ctttgaggac gggggtgtag
tcaccgtaac ccaagactca agcctccaag acggcgagtt catctacaag gtcaaactgc

420

gggggactaa ctttccgtcg gatgggccgg tgatgcagaa gaaaacgatg ggatgggaag

540

cgtcatcgga gaggatgtac ccagaagatg gtgcattgaa gggggagatc aagcagagac

600

tgaagttgaa agatggggga cattatgatg ccgaggtgaa aacgacatac aaagcgaaaa

660

agccggtgca gcttcccgga gcgtataatg tgaatatcaa gttggatatt acttcacaca
atgaggacta cacaattgtc gaacagtacg aacgcgctga gggtagacac tcgacgggag
gcatggacga gttgtacaaa tgataagctg ccttctgcgg ggcttgcctt ctggccatgo

720
780

ccttcttctc tcccttgcac ctgtacctct tggtctttga ataaagcctg agtaggaagg

900

cggccgctcg agcatgcatc taga

924

300

480

840

< 210 > SEQ ID NO 7
< 211 > LENGTH : 725
< 212 > TYPE : RNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 7

gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug ggagugcacg

60

agugucccgc gugguugugg uugcugcugu cgcucuugag ccucccacug ggacugccug
ugcugggggc accacccaga uugaucugcg acucacgggu acuugagagg uaccuucuug

120
180

aagccaaaga agccgaaaac aucacaaccg gaugcgccga gcacugcucc cucaaugaga

240

acauuacugu accggauaca aaggucaauu ucuaugcaug gaagagaaug gaaguaggac

300

agcaggccgu cgaagugugg caggggcucg cgcuuuuguc ggaggcggug uugcgggguc

360

aggcccuccu cgucaacuca ucacagccgu gggagccccu ccaacuucau gucgauaaag

420

cggugucggg gcuccgcagc uugacgacgu ugcuucgggc ucugggcgca caaaaggagg

480

cuauuucgcc gccugacgcg gccuccgcgg caccccuccg aacgaucacc gcggacacgu

540

uuaggaagcu uuuuagagug uacagcaauu uccuccgcgg aaagcugaaa uuguauacug

600

gugaagcgug uaggacaggg gaucgcugau aagcugccuu cugcggggcu ugccuucugg

660

ccaugcccuu cuucucuccc uugcaccugu accucuuggu cuuugaauaa agccugagua

720

ggaag

725

< 210 > SEQ ID NO 8
< 211 > LENGTH : 1536
< 212 > TYPE : RNA

< 213 > ORGANISM : Homo sapiens

US 10,898,574 B2
98

97
- continued
< 400 > SEQUENCE : 8
gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaau gcagcgcguc

60

aacaugauua uggccgaauc gccgggacuc aucacaaucu gccucuuggg uuaucucuug

120

ucggcagaau guaccguguu cuuggaucac gaaaacgcga acaaaauucu uaaucgcccg

180

aagcgguaua acuccgggaa acuugaggag uuugugcagg gcaaucuuga acgagagugc

240

auggaggaga aaugcuccuu ugaggaggcg agggaagugu uugaaaacacagagcgaaca

300

acggaguuuu ggaagcaaua cguag?uggg gaccagugug agucgaaucc gugccucaau

360

gggggaucau guaaagauga caucaauagc uaugaaugcu ggugcccguu uggguuugaa

420

gggaagaacu gugagcugga ugugacgugc aacaucaaaa acggacgcug ugagcaguuu

480

uguaagaacu cggcugacaa uaagguagua ugcucgugca cagagggaua ccggcuggcg

540

gagaaccaaa aaucgugcga gcccgcaguc ccguucccuu gugggagggu gagcguguca

600

cagacuagca aguugacgag agcggagacu guauuccccg acguggacua cgucaacagc
accgaagccg aaacaaucou cgauaacauc acgcagagca cucaguccuu caaugacuuu

660

acgagggucg uaggugguga ggacgcgaaa cccggucagu uccccuggca ggugguauug

780

aacggaaaag ucgaugccuu uuguggaggu uccauuguca acgagaagug gauugucaca

840

gcggcacacu gcguagaaac aggagugaaa aucacgguag uggcgggaga gcauaacauu

900

gaagagacag agcacacgga acaaaagcga aaugucauca gaaucauucc acaccauaac
uauaacgcgg caaucaauaa guacaaucac gacaucgcac uuuuggagcu ugacgaaccu

960
1020

uuggugcuua auucguacgu caccccuauu uguauugccg acaaagagua uacaaacauc

1080

uucuugaaau ucggcuccgg guacguaucg ggcuggggca gaguguucca uaaggguaga
uccgcacugg uguugcaaua ccucagggug ccccucgugg aucgagecac uugucugcgg

1140
1200

uccaccaaau ucacaaucua caacaauaug uucugugcgg gauuccauga aggugggaga

1260

gauagcugcc agggagacuc aggggguccc cacgugacgg aagucgaggg gacgucauuu

1320

cugacgggaa uuaucucaug gggagaggaa ugugcgauga aggggaaaua uggcaucuac

1380

acuaaagugu cacgguaugu caauuggauc aaggaaaaga cgaaacucac gugaucagcc
agcgcugccu ucugcggggc uugccuucug gccaugcccu ucuucucucc cuugcaccug

1440

uaccucuugg ucuuugaaua aagccugagu aggaag

1536

720

1500

< 210 > SEQ ID NO 9
< 211 > LENGTH : 794
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 9

caagcttttg gaccctcgta cagaagctaa tacgactcac tatagggaaa taagagagaa

60

aagaagagta agaagaaata taagagecac catgggagtg cacgagtgtc ccgcgtggtt

120

gtggttgctgctgtcgctct tgagcctccc actgggactg cctgtgctgg gggcaccacc

180

cagattgate tgcgactcac gggtacttga gaggtacctt cttgaagcca aagaagccga

240

aaacatcaca accggatgcgccgagcactg ctccctcaat gagaacatta ctgtaccgga
tacaaaggtc aatttctatg catggaagag aatggaagta ggacagcagg ccgtcgaagt

300

gtggcagggg ctcgcgcttt tgtcggaggc ggtgttgcgg ggtcaggccc tcctcgtcaa

420

ctcatcacag ccgtgggagc ccctccaact tcatgtcgat aaagcggtgt cggggctccg

480

360

US 10,898,574 B2

99

100
- continued

cagettgacg acgttgcttc gggctctggg cgcacaaaag gaggotattt cgccgcctga

540

cgcggcctcc gcggcacccc tccgaacgat caccgcggac acgtttagga agctttttag

600

agtgtacagc aatttcctcc gcggaaagct gaaattgtat actggtgaag cgtgtaggac
aggggatcgc tgataagctg ccttctgcgg ggcttgcctt ctggccatgc ccttcttctc

660

tcccttgcac ctgtacctct tggtctttga ataaagcctg agtaggaagg cggccgctcg

780

agcatgcatc taga

794

720

< 210 > SEQ ID NO 10
< 211 > LENGTH : 1869
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 10

agcttttgga ccctcgtaca gaagctaata cgactcacta tagggaaata agagagaaaa

60

gaagagtaag aagaaatata agagccacca tggaagatgc gaagaacatc aagaagggac

120

ctgccccgtt ttaccctttg gaggacggta cagcaggaga acagctccac aaggcgatga

180

aacgctacgc cctggtcccc ggaacgattg cgtttaccga tgcacatatt gaggtagaca

240

tcacatacgc agaatacttc gaaatgtcgg tgaggctggc ggaagcgatg aagagatatg

300

gtcttaacac taatcaccgc atcgtggtgt gttcggagaa ctcattgcag tttttcatgo

360

cggtccttgg agcacttttc atcggggtcg cagtcgcgcc agcgaacgac atctacaatg

420

agcgggaact cttgaatagc atgggaatct cccagccgac ggtcgtgttt gtctccaaaa

480

aggggctgca gaaaatcctc aacgtgcaga agaagctccc cattattcaa aagatcatca

540

ttatggatag caagacagat taccaagggt tccagtcgat gtataccttt gtgacatcgc

600

atttgccgcc agggtttaac gagtatgact tcgtccccga gtcatttgac agagataaaa

660

ccatcgcgct gattatgaat tcctcgggta gcaccggttt gccaaagggg gtggcgttgc

720

cccaccgcac tgottgtgtg cggttctcgc acgctaggga tcctatcttt ggtaatcaga

780

tcattcccga cacagcaatc ctgtccgtgg taccttttca tcacggtttt ggcatgttca

840

cgactctcgg ctatttgatt tgcggtttca gggtcgtact tatgtatcgg ttcgaggaag

900

aactgttttt
ttttctcatt
agattgcctc
tccaccttcc
tcacgcccga

960

gtgccaacgc
aatctgcatg
gocaagcgct
gcgatcctta
ttctttgaag

1080

ccaaggtcgt agacctcgac acgggaaaaa ccctcggagt gaaccagagg ggcgagctct

1260

gcgtgagagg gccgatgatc atgtcaggtt acgtgaataa ccctgaagcg acgaatgcgc

1320

tgatcgacaa ggatgggtgg ttgcattcgg gagacattgc ctattgggat gaggatgagc

1380

acttctttat cgtagatega cttaagagct tgatcaaata caaaggctat caggtagcgc

1440

ctgccgagct cgagtcaatc ctgctccagc accccaacat tttcgacgcc ggagtggccg

1500

ggttgcccga tgacgacgcg ggtgagctgc cagcggccgt ggtagtcctc gaacatggga

1560

aaacaatgac cgaaaaggag atcgtggact acgtagcatc acaagtgacg actgegaaga

1620

aggtcccgaa ggcttgact gggaagcttg

1680

acgctcgcaa aatccgggaa atcctgatta aggcaaagaa aggcgggaaa atcgctgtct

1740

gataagctgc cttctgcggg gottgccttc tggccatgcc cttcttctct cccttgcacc

1800

??

agggg

gagatccttg caagattaca
ctttgcgaaa tcgacactta
agggggagcgccgcttagca
cggaattcgg cagggatacg
gggtgacgat aagccgggag

tagtc tt

agatccagtc ggccctcctt
ttgataagta tgacctttcc
aggaagtcggggaggcagtg
ggctcacgga gacaacatcc
ccgtcggaaa agtggtcccc

1020

1140
1200

US 10,898,574 B2
102

101
- continued
tgtacctctt ggtctttgaa taaagcctga gtaggaaggc ggccgctcga gcatgcatct

1860

agagggccc

1869

< 210 > SEQ ID NO 11
< 211 > LENGTH : 930
< 212 > TYPE : DNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 11

tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga
aaagaagagt aagaagaaat ataagagcca ccatggtgag caagggcgag gagctgttca

120

ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg

180

tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca

240

ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc

300

agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tocgccatgo

360

ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc

420

gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg

480

acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca

540

acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc

600

acaacatcga ggacggcagc gtgcagctcgccgaccacta ccagcagaac acccccatcg

660

gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca

720

aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga

780

tcactctcgg catggacgag ctgtacaagt aagctgcctt ctgcggggct tgccttctgg

840

ccatgccctt cttctctccc ttgcacctgt acctcttggt ctttgaataa agcctgagta

900

ggaaggcggc cgctcgagca tgcatctaga

930

60

< 210 > SEQ ID NO 12
< 211 > LENGTH : 716
< 212 > TYPE : RNA

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 12

gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug aacuuucucu

60

ugucaugggu gcacuggagc cuugcgcugc ugcuguaucu ucaucacgcu aaguggagcc

120

aggccgcacc cauggcggag gguggcggac agaaucacca cgaaguaguc aaauucaugg

180

acguguacca gaggucguau ugccauc?ga uugaaacucu uguggauauc uuucaagaau

240

accccgauga aaucgaguac auuuucaaac cgucgugugu cccucucaug aggugcgggg

300

gaugcugcaa ugaugaaggg uuggagugug uccccacgga ggagucgaau aucacaaugc

360

aaaucaugcg caucaaacca caucaggguc agcauauugg agagaugucc uuucuccago

420

acaacaaaug ugaguguaga ccgaagaagg accgagcccg acaggaaaac ccaugcggac

480

cgugcuccga gcggcgcaaa cacuuguucg uacaagaccc ccagacaugc aagugcucau

540

guaagaauac cgauucgcgg uguaaggcga gacagcugga auugaacgag cgcacgugua

600

ggugcgacaa gccuagacgg ugagcugccu ucugcggggc uugccuucug gccaugcccu

660

ucuucucucc cuugcaccug uaccucuugg ucuuugaaua aagccugagu aggaag

716

< 210 > SEQ ID NO 13

US 10,898,574 B2
104

103
- continued
< 211 > LENGTH : 30
< 212 > TYPE : PRT

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 13

Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln
1

10

5

15

Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gin Glu Ala
20

25

30

< 210 > SEQ ID NO 14
< 211 > LENGTH : 25
< 212 > TYPE : PRT

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 14

Met Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu
5

1

15

10

Cys Cys Leu Val Pro Val Ser Leu Ala
20

25

< 210 > SEQ ID NO 15
< 211 > LENGTH : 46
< 212 > TYPE : PRT

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 15

Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Ser Leu Ile Thr
5

1

10

15

Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu
20

25

30

Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg
35

40

45

< 210 > SEQ ID NO 16
< 211 > LENGTH : 21

< 212 > TYPE : PRT

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 16

Met Lys Gly Ser Leu Leu Leu Leu Leu Val Ser Asn Leu Leu Leu Cys
1

5

10

15

Gln Ser Val Ala Pro
20

< 210 > SEQ ID NO 17
< 211 > LENGTH : 24
< 212 > TYPE : PRT

< 213 > ORGANISM : Homo sapiens
< 400 > SEQUENCE : 17

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
5

1

Tyr Ser Arg Gly Val Phe Arg Arg
20

10

15

US 10,898,574 B2
105

106

1. A method of producing a polypeptide of interest in a
cell in a subject in need thereof, comprising administering to

the modified uridine is modified on the major groove face of
the uridine.
7. The pharmaceutical composition of claim 2 , wherein

We claim :

the subject a pharmaceutical composition comprising a

6. The pharmaceutical composition of claim 2 , wherein

modified
messenger
) suchmmRNA
that thecomprises
mmRNA 5 the modified uridine is a pyridine-4 -one ribonucleoside,
is introduced
into theRNA
cell ,(mmRNA
wherein the

5 - aza -uridine , 2 -thio - 5 - aza - uridine, 2 - thio -uridine, 4 -thio

a translatable region encoding the polypeptide of interest
and comprises the modified nucleoside 1 -methyl-pseudou
ridine
, and wherein the pharmaceutical composition com
prises an effective amount of the mmRNA providing for 10
increased polypeptide production and substantially reduced
innate immune response in the cell , as compared to a
composition comprising a corresponding unmodified
mRNA .
2. A pharmaceutical composition comprising:
a plurality of lipid nanoparticles comprising a cationic 15
lipid , a sterol, and a PEG - lipid ,
wherein the lipid nanoparticles comprise an mRNA
encoding a polypeptide, wherein the mRNA comprises
one or more uridines, one or more cytidines , one or
more adenosines, and one or more guanosines and 20
wherein substantially all uridines are modified uridines .
3. The pharmaceutical composition of claim 2 , wherein
the plurality of lipid nanoparticles further comprise a phos
phatidyl choline .

pseudouridine, 2 -thio -pseudouridine, 5 -hydroxy - uridine,
3 -methyl -uridine, 5 -carboxymethyl -uridine, 1 -carboxym
ethyl-pseudouridine, 5 -propynyl-uridine, 1 -propynyl
pseudouridine, 5 - taurinomethyl -uridine, 1-taurinomethyl
pseudouridine , 5 - taurinomethyl - 2 - thio - uridine, 1-taurino -4
thio -pseudouridine, 1 -methyl-pseudouridine, 4 - thio - 1
methyl -pseudouridine,
2 - thio - 1 -methyl- pseudouridine,

5. The pharmaceutical composition of claim 2 , wherein
the plurality of lipid nanoparticles has a mean lipid to

the mRNA further comprises an operably - linked signal

1 -methyl -1deaza -pseudouridine, 2- thio -1 -methyl-1 -deaza
pseudouridine, dihydro - uridine, dihydro - pseudouridine,
2 - thio -dihydro -uridine,
2 - thio - dihydro -pseudouridine,
2 -methoxy -uridine, 2 -methoxy - 4 - thio -uridine, 4 -methoxy
pseudouridine, 4 -methoxy - 2 -thio -pseudouridine , or
pseudouridine.
8. The pharmaceutical composition of claim 2 , wherein
the modified uridine is pseudouridine or 1 -methyl-pseudou
ridine.
9. The pharmaceutical composition of claim 2 , wherein
4. The pharmaceutical composition of claim 2, wherein 25 the10.modified
uridine is 1 -methyl
-pseudouridine
The pharmaceutical
composition
of claim. 2 , wherein
the sterol is cholesterol.

polynucleotide ratio ( wt /wt) of between 10 to 1 and 20 to 1 .

sequence .