Jul 2020 MODERNAUS107037891

US010703789B2
( 12 ) United States Patent ( 10 ) Patent No.: US 10,703,789 B2

De Fougerolles et al. (45 ) Date of Patent: * Jul. 7 , 2020
(54 ) MODIFIED POLYNUCLEOTIDES FOR THE (2013.01 ); A61K 38/36 ( 2013.01 ); A61K
PRODUCTION OF SECRETED PROTEINS 38/363 ( 2013.01 ); A61K 38/44 ( 2013.01) ;
A61K 38/4833 (2013.01); A61K 38/4846
( 71 ) Applicant: Moderna TX , Inc., Cambridge, MA (2013.01 ); A61K 39/3955 ( 2013.01); A61K
(US) 47/10 (2013.01); A61K 47/54 (2017.08 ); A61K
( 72 ) Inventors: Antonin De Fougerolles, Waterloo 47/542 (2017.08 ); A61K 48/0033 ( 2013.01 );
(BE ); Justin Guild , Framingham , MA A61K 48/0066 (2013.01); A61K 48/0075
(US) (2013.01 ); CO7K 14/47 (2013.01 ); CO7K
14/475 (2013.01); CO7K 14/505 (2013.01 );
(73 ) Assignee : Moderna TX , Inc., Cambridge , MA CO7K 14/525 (2013.01); C07K 14/56
(US) (2013.01 ); CO7K 14/565 (2013.01 ); CO7K
14/745 (2013.01 ); C07K 14/75 ( 2013.01) ;
( * ) Notice: Subject to any disclaimer , the term of this CO7K 16/2887 (2013.01 ); CO7K 16/32
patent is extended or adjusted under 35 (2013.01); CO7K 19/00 (2013.01); C12N
U.S.C. 154(b ) by 0 days . 9/0069 (2013.01); C12N 9/644 (2013.01 );
This patent is subject to a terminal dis C12N 15/85 (2013.01 ); C12N 15/88
claimer . ( 2013.01 ); C12Y 113/12007 (2013.01 ); C12Y
304/21005 (2013.01 ); C12Y 304/21022
(21 ) Appl. No.: 16 /438,978 (2013.01 ); A61K 9/0019 (2013.01 ); A61K
48/00 (2013.01); C12N 2840/00 (2013.01)
(22) Filed : Jun . 12 , 2019 (58) Field of Classification Search
CPC CO7H 21/02 , C12N 15/67; C12N 15/11
(65) Prior Publication Data See application file for complete search history.
US 2020/0017565 A1 Jan. 16 , 2020
(56 ) References Cited
Related U.S. Application Data
U.S. PATENT DOCUMENTS
(63 ) Continuation of application No. 14 /987,328 , filed on
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(51) Int. Cl.
A61K 48/00 ( 2006.01 ) FOREIGN PATENT DOCUMENTS
A61K 38/17 (2006.01) CA 2028849 A1 9/1991
A61K 47/54 ( 2017.01 ) CA 2473135 A1 6/2003
A61K 9/127 (2006.01)
COZK 14/535 ( 2006.01) (Continued )
C12N 15/88 (2006.01) OTHER PUBLICATIONS
A61K 9/50 ( 2006.01)
COZK 14/47 ( 2006.01 ) Anderson et al., “ Incorporation ofpseudouridine intomRNA enhances
A61K 31/7088 (2006.01) translation by diminishing PKR activation ,” Nucleic Acids Res .
CO7K 19/00 (2006.01) 38( 17 ): 5884-92 ( 2010 ).
CI2N 15/85 ( 2006.01 ) (Continued )
A61K 38/18 ( 2006.01)
A61K 38/19 (2006.01)
A61K 38/48 (2006.01 ) Primary Examiner — Antonio Galisteo Gonzalez
A61K 9/14 ( 2006.01 ) (74 ) Attorney, Agent, or Firm -Clark & Elbing LLP
A61K 47/10 ( 2017.01 )
A61K 38/21 (2006.01 )
A61K 38/36 ( 2006.01) (57) ABSTRACT
A61K 38/44 ( 2006.01)
A61K 39/395 ( 2006.01) A pharmaceutical composition which has a plurality of lipid
(Continued ) nanoparticles that has a mean particle size ofbetween 80 nm
(52) U.S. CI. and 160 nm and contains a modified mRNA encoding a
CPC CO7K 14/535 (2013.01 ); A61K 9/1271 polypeptide. The lipid nanoparticles include a cationic lipid ,
(2013.01); AKIK 9/1272 ( 2013.01); A6IK a neutral lipid , a cholesterol, and a PEG lipid . The mRNA
9/1277 (2013.01); A61K 9/14 (2013.01); A61K contains a 5'-cap , 5'-UTR , N1-methyl-pseudouridine, a
9/5031 (2013.01) ; A61K 31/7088 (2013.01 ); 3 '-UTR , and a poly - A region with at least 100 nucleotides.
A61K 38/1767 ( 2013.01) ; A61K 38/1816
( 2013.01 ); A61K 38/1866 (2013.01); A61K 14 Claims, 14 Drawing Sheets
38/191 (2013.01); A61K 38/193 (2013.01);
A61K 38/212 ( 2013.01 ); A61K 38/215 Specification includes a Sequence Listing .
US 10,703,789 B2
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Related U.S. Application Data CO7K 14/56 (2006.01)

C07K 14/565 (2006.01)
continuation of application No. 14 /750,004 , filed on CO7K 14/745 (2006.01 )
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14 , 2012, provisional application No. 61/737,168 , A61K 31/7115 (2006.01)
filed on Dec. 14 , 2012, provisional application No. COZK 14/005 ( 2006.01 )
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US 10,703,789 B2
2
MODIFIED POLYNUCLEOTIDES FOR THE Plasma Membrane Proteins, U.S. Provisional Patent Appli
PRODUCTION OF SECRETED PROTEINS cation No.61/737,152, filed Dec. 14 , 2012 , entitled Modi
fied Polynucleotides for the Production of Plasma Mem
CROSS REFERENCE TO RELATED brane Proteins, U.S. Provisional Patent Application No.
APPLICATIONS 5 61/618,885 , filed Apr. 2 , 2012 , entitled Modified Polynucle
otides for the Production of Cytoplasmic and Cytoskeletal
This application is a continuation of U.S. application Ser. Proteins, U.S. Provisional Patent Application No. 61/681,
No. 14 / 987,328 , filed Jan. 4, 2016 , entitled Modified Poly 658 , filed Aug. 10 , 2012, entitled Modified Polynucleotides
nucleotides for the Production of Secreted Proteins , now for the Production of Cytoplasmic and Cytoskeletal Pro
U.S. Pat. No. 10,385,106 , which is a continuation of U.S. 10 teins , U.S. Provisional Patent Application No. 61/737,155 ,
application Ser . No. 14 /750,004 , filed Jun . 25 , 2015, entitled filed Dec. 14 , 2012 , entitled Modified Polynucleotides for
Modified Polynucleotides for the Production of Secreted the Production of Cytoplasmic and Cytoskeletal Proteins ,
Proteins, now U.S. Pat. No. 9,828,416 , which is a continu U.S. Provisional Patent Application No. 61/618,896 , filed
ation of U.S. application Ser. No. 13 /791,921, filed Mar. 9 , Apr. 2, 2012 , entitled Modified Polynucleotides for the
2013 , entitled Modified Polynucleotides for the Production 15 Production of IntracellularMembrane Bound Proteins, U.S.
of Secreted Proteins , now U.S. Pat. No. 9,192,651 , which Provisional Patent Application No. 61/668,157, filed Jul. 5 ,
claims priority to U.S. Provisional Patent Application No. 2012, entitled Modified Polynucleotides for the Production
61/681,742 , filed , Aug. 10 , 2012 , entitled Modified Poly of Intracellular Membrane Bound Proteins, U.S. Provisional
nucleotides for the Production of Oncology -Related Proteins Patent Application No. 61/681,661, filed Aug. 10 , 2012 ,
and Peptides, U.S. Provisional Patent Application No. 20 entitled Modified Polynucleotides for the Production of
61/737,224 , filed Dec. 14 , 2012 , entitled Terminally Opti Intracellular Membrane Bound Proteins , U.S. Provisional
mized Modified RNAs, International Application No PCT/ Patent Application No. 61/737,160 , filed Dec. 14, 2012 ,
US2012 / 069610 , filed Dec. 14 , 2012 , entitled Modified entitled Modified Polynucleotides for the Production of
Nucleoside, Nucleotide, and Nucleic Acid Compositions, Intracellular Membrane Bound Proteins, U.S. Provisional
U.S. Provisional Patent Application No. 61/618,862, filed 25 Patent Application No. 61 /618,911, filed Apr. 2, 2012 ,
Apr. 2 , 2012 , entitled Modified Polynucleotides for the entitled Modified Polynucleotides for the Production of
Production of Biologics, U.S. Provisional PatentApplication Nuclear Proteins , U.S. Provisional Patent Application No.
No. 61/681,645 , filed Aug. 10 , 2012 , entitled Modified 61/681,667 , filed Aug. 10 , 2012 , entitled Modified Poly
Polynucleotides for the Production of Biologics, U.S. Pro nucleotides for the Production of Nuclear Proteins , U.S.
visional Patent Application No. 61/ 737,130 , filed Dec. 14 , 30 Provisional Patent Application No. 61/737,168, filed Dec.
2012 , entitled Modified Polynucleotides for the Production 14 , 2012 , entitled Modified Polynucleotides for the Produc
of Biologics , U.S. Provisional Patent Application No. tion ofNuclear Proteins, U.S. Provisional PatentApplication
61/618,866 , filed Apr. 2 , entitled Modified Polynucle No. 61/618,922 , filed Apr. 2 , 2012 , entitled Modified Poly
otides for the Production of Antibodies, U.S. Provisional nucleotides for the Production of Proteins , U.S. Provisional
Patent Application No. 61/681,647 , filed Aug. 10 , 2012 , 35 Patent Application No. 61/681,675 , filed Aug. 10 , 2012 ,
entitled Modified Polynucleotides for the Production of entitled Modified Polynucleotides for the Production of
Antibodies, U.S. Provisional Patent Application No. 61/737, Proteins, U.S. Provisional Patent Application No. 61/737 ,
134 , filed Dec. 14 , 2012 , entitled Modified Polynucleotides 174, filed Dec. 14 , 2012 , entitled Modified Polynucleotides
for the Production of Antibodies, U.S. Provisional Patent for the Production of Proteins, U.S. Provisional Patent
Application No. 61/618,868 , filed Apr. 2, 2012 , entitled 40 Application No. 61/618,935, filed Apr. 2, 2012 , entitled
Modified Polynucleotides for the Production of Vaccines , Modified Polynucleotides for the Production of Proteins
U.S. Provisional Patent Application No. 61/681,648 , filed Associated with Human Disease , U.S. Provisional Patent
Aug. 10 , 2012 , entitled Modified Polynucleotides for the Application No. 61/681,687 , filed Aug. 10 , 2012, entitled
Production of Vaccines, U.S. Provisional Patent Application Modified Polynucleotides for the Production of Proteins
No. 61/ 737,135 , filed Dec. 14 , 2012 , entitled Modified 45 Associated with Human Disease , U.S. Provisional Patent
Polynucleotides for the Production of Vaccines, U.S. Pro Application No. 61 /737,184, filed Dec. 14 , 2012 , entitled
visional Patent Application No. 61/618,870 , filed Apr. 2 , Modified Polynucleotides for the Production of Proteins
2012 , entitled Modified Polynucleotides for the Production Associated with Human Disease , U.S. Provisional Patent
of Therapeutic Proteins and Peptides , U.S. Provisional Pat Application No. 61/618,945, filed Apr. 2, 2012, entitled
ent Application No. 61 /681,649, filed August 10,2012, 50 Modified Polynucleotides for the Production of Proteins
entitled Modified Polynucleotides for the Production of Associated with Human Disease, U.S. Provisional Patent
Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No. 61/681,696 , filed Aug. 10 , 2012 , entitled
Application No. 61 /737,139, filed Dec. 14 , 2012 ,Modified Modified Polynucleotides for the Production of Proteins
Polynucleotides for the Production of Therapeutic Proteins Associated with Human Disease , U.S. Provisional Patent
and Peptides, U.S. Provisional Patent Application No. 55 Application No. 61/737,191, filed Dec. 14 , 2012, entitled
61/618,873 , filed Apr. 2 , 2012 , entitled Modified Polynucle Modified Polynucleotides for the Production of Proteins
otides for the Production of Secreted Proteins , U.S. Provi Associated with Human Disease , U.S. Provisional Patent
sional Patent Application No. 61 /681,650 , filed Aug. 10 , Application No. 61/618,953, filed Apr. 2, 2012, entitled
2012 , entitled Modified Polynucleotides for the Production Modified Polynucleotides for the Production of Proteins
of Secreted Proteins, U.S. Provisional Patent Application 60 Associated with Human Disease, U.S. Provisional Patent
No. 61/737,147, filed Dec. 14 , 2012 , entitled Modified Application No. 61/681,704, filed Aug. 10 , 2012 , entitled
Polynucleotides for the Production of Secreted Proteins , Modified Polynucleotides for the Production of Proteins
U.S. Provisional Patent Application No. 61 /618,878 , filed Associated with Human Disease , U.S. Provisional Patent
Apr. 2, 2012, entitled Modified Polynucleotides for the Application No. 61/737,203 , filed Dec. 14 , 2012, entitled
Production of Plasma Membrane Proteins , U.S. Provisional 65 Modified Polynucleotides for the Production of Proteins
Patent Application No. 61/681,654 , filed Aug. 10 , 2012 , Associated with Human Disease , U.S. Provisional Patent
entitled Modified Polynucleotides for the Production of Application No. 61/618,961, filed Apr. 2 , 2012 , entitled
US 10,703,789 B2
3 4
Dosing Methods for Modified mRNA , U.S. Provisional facture and /or formulation of polynucleotides, primary con
Patent Application No. 61/648,286 , filed May 17 , 2012 , structs and modified mRNA molecules (mmRNA ).
entitled Dosing Methods for Modified mRNA, the contents
of each ofwhich are herein incorporated by reference in its BACKGROUND OF THE INVENTION
entirety . 5
There are multiple problems with prior methodologies of
REFERENCE TO SEQUENCE LISTING effecting protein expression . For example , introduced DNA
The instant application contains a Sequence Listing which can integrate into host cell genomic DNA at some frequency,
resulting in alterations and /or damage to the host cell
has beenincorporated
hereby submitted byelectronically
reference in initsASCII
entiretyformat and is 10 genomic DNA. Alternatively, the heterologous deoxyribo
. Said ASCII
copy, created Jun . 10 , 2019 , is named nucleic acid (DNA ) introduced into a cell can be inherited by
51165_008004_Sequence Listing_ST25. daughter cells ( whether or not the heterologous DNA has
integrated into the chromosome) or by offspring .
REFERENCE TO LENGTHY TABLE 15 In addition , assuming proper delivery and no damage or
integration into the host genome, there are multiple steps
The specification includes a lengthy Table 6. Lengthy which must occur before the encoded protein is made . Once
Table 6 has been submitted via EFS -Web in electronic inside the cell , DNA must be transported into the nucleus
format as follows: File name: M304TBL.txt, Date created : where it is transcribed into RNA . The RNA transcribed from
Mar. 9, 2013; File size: 205,684 Bytes and is incorporated 20 DNA must then enter the cytoplasm where it is translated
herein by reference in its entirety . Please refer to the end of into protein . Not only do themultiple processing steps from
the specification for access instructions . administered DNA to protein create lag times before the
LENGTHY TABLES
The patent contains a lengthy table section . A copy of the table is available in electronic form from the
USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10703789B2). An electronic copy
of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19 (b )(3 ).
This application is also related to International Publica generation of the functional protein , each step represents an
tion No. PCT/US2012/58519 , filed Oct. 3 , 2012 , entitled 35 opportunity for error and damage to the cell. Further, it is
Modified Nucleosides, Nucleotides , and Nucleic Acids, and
Uses Thereof and International Publication No. PCT/
known to be difficult to obtain DNA expression in cells as
US2012 /69610 , filed Dec. 14 , 2012 , entitled Modified DNA frequently enters a cell but is not expressed or not
Nucleoside, Nucleotide, and Nucleic Acid Compositions . expressed
a
at reasonable rates or concentrations. This can be
particular problem when DNA is introduced into primary
The instant application is also related to co -pending 40 cells or modified cell lines .
applications, each filed concurrently herewith on Mar. 9 ,
2013 and having International Publication No. WO /2013/ In the early 1990's Bloom and colleagues successfully
151666, entitled Modified Polynucleotides for the Produc rescued vasopressin -deficient rats by injecting in vitro
tion of Biologics and Proteins Associated with Human transcribed vasopressin mRNA into the hypothalamus (Sci
Disease ; International Publication No. WO /2013 / 151667 , 45 ence 255 : 996-998; 1992). However , the low levels of
entitled Modified Polynucleotides; International Publication translation and the immunogenicity of the molecules ham
No. WO /2013 / 151663, entitled Modified Polynucleotides pered the development of mRNA as a therapeutic and efforts
for the Production of Membrane Proteins ; International
Publication No. PCT/US2013/ 151669, entitled Modified have since focused on alternative applications that could
instead exploit these pitfalls , i.e. immunization with mRNAs
Polynucleotides for the Production of Cytoplasmic and
Cytoskeletal Proteins; International Publication No. PCT) 50 coding for cancer antigens.
US2013 / 151670 , entitled Modified Polynucleotides for the Others have investigated the use of mRNA to deliver a
Production of Nuclear Proteins; International Publication polypeptide of interest and shown that certain chemical
No. WO /2013 /151664 , entitled Modified Polynucleotides modifications ofmRNA molecules, particularly pseudouri
for the Production of Proteins ; International Publication No. dine and 5 -methyl-cytosine , have reduced immunostimula
WO /2013 /151665, entitled Modified Polynucleotides for the 55 tory effect.
Production of Proteins Associated with Human Disease ; These studies are disclosed in , for example, Ribostem
International Publication No. WO /2013 / 151671, entitled Limited in United Kingdom patentapplication serialnumber
Modified Polynucleotides for the Production of Cosmetic 0316089.2 filed on Jul. 9 , 2003 now abandoned , PCT
Proteins and Peptides; and International Publication No. application number PCT/GB2004 /002981 filed on Jul. 9 ,
WO /2013 /151672
Production , entitled-Related
of Oncology ModifiedProteins
Polynucleotides
and Peptidesfor, the
the 60 2004 published as W02005005622, U.S. patent application
contents of each of which are herein incorporated by refer national
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application serial number EP2007024312 filed Dec. 14 , 2011 published as US20110311472 all of which are herein
2007 now abandoned , PCT application number PCT/ 10 incorporated by reference in their entirety.
EP2008 /01059 filed on Dec. 12 , 2008 published as Notwithstanding these reports which are limited to a
WO2009077134 , European patent application national selection of chemical modifications including pseudouridine
phase entry serial number EP 2008861423 filed on Jun. 2, and 5 -methylcytosine , there remains a need in the art for
2010 published as EP2240572 , U.S. patent application Ser. therapeutic modalities to address the myriad of barriers
No. 12/735,060 filed Nov. 24, 2010 published as 15 surrounding the efficacious modulation of intracellular
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DE 10 2005 046 490 filed Sep. 28 , 2005 , PCT application peptides or fragments thereof.
PCT/EP2006 /0448 filed Sep. 28 , 2006 published as To this end, the inventors have shown that certain modi
WO2007036366 , national phase European patent fied mRNA sequences have the potential as therapeutics
EP1934345 published Mar. 21 , 2012 and national phase U.S. 20 with benefits beyond just evading, avoiding or diminishing
patent application Ser . No. 11 /992,638 filed Aug. 14 , 2009 the immune response . Such studies are detailed in published
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PCT/US2011/ 32679 filed Apr. 15 , 2011 published as 25 PCT/US2011/054617 filed Oct. 3, 2011 , the contents of
WO20110130624 ; Shire Human Genetic Therapeutics in which are incorporated herein by reference in their entirety .
U.S.patent application Ser. No. 12 /957,340 filed on Nov. 20 , The present invention addresses this need by providing
2010 published as US20110244026 ; Sequitur Inc. in PCT nucleic acid based compounds or polynucleotides which
application PCT/US1998 /019492 filed on Sep. 18 , 1998 encode a polypeptide of interest ( e.g., modified mRNA or
published as W01999014346 ; The Scripps Research Insti- 30 mmRNA ) and which have structural and /or chemical fea
tute in PCT application number PCT/US2010 /00567 filed on tures that avoid one or more of the problems in the art, for
Feb. 24 , 2010 published as WO2010098861, and U.S. patent example , features which are useful for optimizing formula
application Ser. No. 13 /203,229 filed Nov. 3, 2011 published tion and delivery of nucleic acid -based therapeutics while
as US20120053333 ; Ludwig -Maximillians University in retaining structural and functional integrity, overcoming the
PCT application number PCT/EP2010/004681 filed on Jul. 35 threshold of expression , improving expression rates, half life
30 , 2010 published as WO2011012316 ; Cellscript Inc. in and /or protein concentrations, optimizing protein localiza
U.S. Pat. No. 8,039,214 filed Jun . 30 , 2008 and granted Oct. tion , and avoiding deleterious bio -responses such as the
18 , 2011, U.S. patent application Ser. No. 12/ 962,498 filed immune response and/or degradation pathways.
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12 / 962,468 filed on Dec. 7, 2010 published as 40 SUMMARY OF THE INVENTION
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published as US20120009649 , and PCT applications PCTas/ Described herein are compositions, methods, processes,
US2010 /59305 filed Dec. 7 , 2010 published kits and devices for the design , preparation , manufacture
WO2011071931 and PCT/US2010 / 59317 filed on Dec. 7 , and/or formulation of modified mRNA (mmRNA ) mol
2010 published as WO2011071936 ; The Trustees of the 45 ecules.
University of Pennsylvania in PCT application number The details of various embodiments of the invention are
PCT/US2006 / 32372 filed on Aug. 21, 2006 published as set forth in the description below . Other features , objects ,
WO2007024708 , and U.S. patent application national phase and advantages of the invention will be apparent from the
entry Ser. No. 11 /990,646 filed on Mar. 27, 2009 published description and the drawings, and from the claims.
as US20090286852 ; Curevac GMBH in German patent 50
application serial numbers DE10 2001 027 283.9 filed Jun . BRIEF DESCRIPTION OF THE DRAWINGS
5 , 2001, DE 10 2001 062 480.8 filed Dec. 19 , 2001, and DE
20 2006 051 516 filed Oct. 31, 2006 all abandoned , Euro The foregoing and other objects , features and advantages
pean patent numbers EP1392341 granted Mar. 30 , 2005 and will be apparent from the following description of particular
EP1458410 granted Jan. 2 , 2008 , PCT application numbers 55 embodiments of the invention , as illustrated in the accom
PCT/EP2002/06180 filed Jun . 5 , 2002 published as panying drawings in which like reference characters refer to
WO2002098443 , PCT/EP2002 / 14577 filed on Dec. 19 , the same parts throughout the different views. The drawings
2002 published as WO2003051401, PCT/EP2007 /09469 are not necessarily to scale, emphasis instead being placed
filed on Dec. 31 , 2007 published as WO2008052770 , PCT/ upon illustrating the principles of various embodiments of
EP2008 /03033 filed on Apr. 16 , 2008 published as 60 the invention .
WO2009127230 , PCT/EP2006 /004784 filed on May 19 , FIG . 1 is a schematic of a primary construct of the present
2005 published as WO2006122828 , PCT/ EP2008 /00081 invention .
filed on Jan. 9 , 2007 published as WO2008083949 ,and U.S. FIG . 2 illustrates lipid structures in the prior art useful in
patent application Ser. No. 10 /729,830 filed on Dec. 5 , 2003 the present invention . Shown are the structures for 98N12-5
published as US20050032730 , Ser. No. 10/870,110 filed on 65 (TETA5 -LAP ), DLin -DMA, DLin -K -DMA (2,2 -Dilinoleyl
Jun . 18 , 2004 published as US20050059624, Ser. No. 4 - dimethylaminomethyl-[ 1,3 ]-dioxolane ), DLin -KC2
11/ 914,945 filed on Jul. 7 , 2008 published as DMA, DLin -MC3 -DMA and C12-200 .
US 10,703,789 B2
7 8
FIG . 3 is a representative plasmid useful in the IVT applications of mmRNA technology ; 61/533,537 filed on
reactions taught herein . The plasmid contains Insert 64818 ,Sep. 12 , 2011 teaching antimicrobial applications of
designed by the instant inventors. mmRNA technology ; 61 /533,554 filed on Sep. 12 , 2011
FIG . 4 is a gel profile ofmodified mRNA encapsulated in teaching viral applications ofmmRNA technology, 61/542,
PLGA microspheres . 5 533 filed on Oct. 3, 2011 teaching various chemical modi
FIG . 5 is a histogram of Factor IX protein production fications for use in mmRNA technology ;61/570,690 filed on
PLGA formulation Factor IX modified mRNA . Dec. 14 , 2011 teaching mobile devices for use in making or
FIGS. 6A -6C are histograms showing VEGF protein using mmRNA technology ; 61/570,708 filed on Dec. 14 ,
production in human keratinocyte cells after transfection of 2011 teaching the use of mmRNA in acute care situations ;
modified mRNA at a range of doses. FIG . 6A shows protein 10 61/ 576,651 filed on Dec. 16 , 2011 teaching terminal modi
production after transfection of modified mRNA comprising fication architecture for mmRNA ;61/576,705 filed on Dec.
natural nucleoside triphosphate (NTP ). FIG . 6B shows pro 16 , 2011 teaching delivery methods using lipidoids for
tein production after transfection of modified mRNA fully mmRNA ;61/ 578,271 filed on Dec. 21 , 2011 teachingmeth
modified with pseudouridine (Pseudo -U ) and 5 -methylcy ods to increase the viability of organs or tissues using
tosine (5 mC ). FIG . 6C shows protein production after 15 mmRNA ; 61/581,322 filed on Dec. 29 , 2011 teaching
transfection of modified mRNA fully modified with mmRNA encoding cell penetrating peptides; 61/581,352
N1-methyl-pseudouridine (N1-methyl-Pseudo - U ) and filed on Dec. 29, 2011 teaching the incorporation of cyto
5 -methylcytosine (5 m ). toxic nucleosides in mmRNA and 61/631,729 filed on Jan.
FIG . 7 is a histogram of VEGF protein production in 10 , 2012 teaching methods of using mmRNA for crossing
HEK293 cells . 20 the blood brain barrier; all ofwhich are herein incorporated
FIGS . 8A - 8B are histograms of VEGF expression and by reference in their entirety .
IFN -alpha induction after transfection of VEGF modified Provided herein , in part, are polynucleotides, primary
mRNA in peripheral blood mononuclear cells (PBMC ). FIG . constructs and / ormmRNA encoding polypeptides of interest
8A shows VEGF expression . FIG . 8B shows IFN -alpha which have been designed to improve one or more of the
induction . 25 stability and /or clearance in tissues, receptor uptake and /or
FIG . 9 is a histogram of VEGF protein production in kinetics, cellular access by the compositions, engagement
HeLa cells from VEGF modified mRNA . with translational machinery , mRNA half-life , translation
FIG . 10 is a histogram of VEGF protein production from efficiency, immune evasion , protein production capacity ,
lipoplexed VEGF modified mRNA in mice . secretion efficiency (when applicable ), accessibility to cir
FIG . 11 is a histogram of G -CSF protein production in 30 culation , protein half-life and /or modulation of a cell's
HeLa cells from G -CSF modified mRNA . status, function and /or activity .
FIG . 12 is a histogram of Factor IX protein production in
HeLa cell supernatant from Factor IX modified mRNA. I. Compositions of the Invention (mmRNA )
FIG . 13 shows Table 8 showing the chemical faces of
canonical nucleotides . 35 The present invention provides nucleic acid molecules ,
specifically polynucleotides, primary constructs and /or
DETAILED DESCRIPTION mmRNA which encode one or more polypeptides of interest.
The term “ nucleic acid ,” in its broadest sense, includes any
It is of great interest in the fields of therapeutics , diag compound and/or substance that comprise a polymer of
nostics, reagents and for biological assays to be able to 40 nucleotides . These polymers are often referred to as poly
deliver a nucleic acid , e.g., a ribonucleic acid (RNA ) inside nucleotides. Exemplary nucleic acids or polynucleotides of
a cell , whether in vitro , in vivo , in situ or ex vivo , such as the invention include, but are not limited to , ribonucleic
to cause intracellular translation of the nucleic acid and acids (RNAs), deoxyribonucleic acids (DNAs), threose
production of an encoded polypeptide of interest. Of par nucleic acids ( TNAs), glycol nucleic acids (GNAs), peptide
ticular importance is the delivery and function of a non- 45 nucleic acids (PNAs ), locked nucleic acids (LNAs, includ
integrative polynucleotide. ing LNA having a B -D -ribo configuration , a -LNA having an
Described herein are compositions ( including pharmaceu A -L -ribo configuration (a diastereomer of LNA ), 2'- amino
tical compositions ) and methods for the design , preparation , LNA having a 2'-amino functionalization , and 2'-amino -a
manufacture and/or formulation of polynucleotides encod LNA having a 2'-amino functionalization ) or hybrids
ing one or more polypeptides of interest . Also provided are 50 thereof.
systems, processes, devices and kits for the selection ,design In preferred embodiments, the nucleic acid molecule is a
and /or utilization of the polynucleotides encoding the poly messenger RNA (mRNA ). As used herein , the term “ mes
peptides of interest described herein . senger RNA ” (mRNA ) refers to any polynucleotide which
According to the present invention , these polynucleotides encodes a polypeptide of interest and which is capable of
are preferably modified as to avoid the deficiencies of other 55 being translated to produce the encoded polypeptide of
polypeptide - encoding molecules of the art. Hence these interest in vitro , in vivo, in situ or ex vivo .
polynucleotides are referred to as modified mRNA or Traditionally , the basic components of an mRNA mol
mmRNA . ecule include at least a coding region, a 5'UTR , a 3'UTR , a
The use of modified polynucleotides in the fields of 5 ' cap and a poly - A tail. Building on this wild type modular
antibodies, viruses, veterinary applications and a variety of 60 structure, the present invention expands the scope of func
in vivo settings has been explored by the inventors and these tionality of traditional mRNA molecules by providing poly
studies are disclosed in for example, co -pending and co nucleotides or primary RNA constructs which maintain a
owned U.S. provisional patent application Ser. Nos. 61/470 , modular organization , but which comprise one or more
451 filed Mar. 31, 2011 teaching in vivo applications of structural and / or chemical modifications or alterations
mmRNA ; 61/ 517,784 filed on Apr. 26 , 2011 teaching engi- 65 which impart useful properties to the polynucleotide includ
neered nucleic acids for the production of antibody poly ing, in some embodiments, the lack of a substantial induc
peptides ; 61/519,158 filed May 17 , 2011 teaching veterinary tion of the innate immune response of a cell into which the
US 10,703,789 B2
9 10
polynucleotide is introduced . As such , modified mRNA hexapeptide, a heptapeptide, an octapeptide, a nonapeptide,
molecules of the present invention are termed “mmRNA.” or a decapeptide . In another embodiment, the length may be
As used herein , a " structural” feature or modification is one sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30,
in which two or more linked nucleotides are inserted , 10-30 , 2-25 , 5-25, 10-25, or 10-20 amino acids. The length
deleted , duplicated , inverted or randomized in a polynucle- 5 may be sufficient to encode for a peptide of at least 11, 12 ,
otide, primary construct or mmRNA without significant 13 , 14 , 15, 17, 20 , 25 or 30 amino acids, or a peptide that is
chemical modification to the nucleotides themselves . no longer than 40 amino acids, e.g.no longer than 35 , 30 , 25 ,
Because chemical bonds will neces essarily be broken and 20 , 17 , 15 , 14 , 13, 12 , 11 or 10 amino acids. Examples of
reformed to effect a structuralmodification, structural modi dipeptides that the polynucleotide sequences can encode or
fications are of a chemical nature and hence are chemical 10 include , but are not limited to , carnosine and anserine.
modifications. However, structural modifications will result Generally, the length of the first region encoding the
in a different sequence of nucleotides. For example, the polypeptide of interest of the present invention is greater
polynucleotide “ ATCG ” may be chemically modified to than about 30 nucleotides in length (e.g., at least or greater
“ AT-5meC -G ” . The same polynucleotide may be structurally than about 35 , 40 , 45 , 50 , 55 , 60, 70 , 80 , 90 , 100 , 120 , 140 ,
modified from “ ATCG ” to “ ATCCCG ” . Here, the dinucle- 15 160 , 180 , 200 , 250 , 300 , 350 , 400 , 450 , 500 , 600 , 700 , 800 ,
otide “ CC ” has been inserted , resulting in a structural 900, 1,000 , 1,100 , 1,200, 1,300 , 1,400 , 1,500, 1,600 , 1,700 ,
modification to the polynucleotide. 1,800, 1,900 , 2,000 , 2,500, and 3,000 , 4,000, 5,000 , 6,000 ,
mmRNA Architecture 7,000 , 8,000, 9,000 , 10,000, 20,000 , 30,000, 40,000, 50,000 ,
The mmRNA of the present invention are distinguished 60,000 , 70,000 , 80,000 , 90,000 or up to and including
from wild type mRNA in their functional and/or structural 20 100,000 nucleotides).As used herein ,the “ first region ” may
design features which serve to , as evidenced herein , over be referred to as a “ coding region ” or “ region encoding” or
come existing problems of effective polypeptide production simply the “ first region .”
using nucleic acid -based therapeutics. In some embodiments, the polynucleotide, primary con
FIG . 1 shows a representative polynucleotide primary struct, or mmRNA includes from about 30 to about 100,000
construct 100 of the present invention . As used herein , the 25 nucleotides (e.g., from 30 to 50 , from 30 to 100 , from 30 to
term " primary construct” or “ primary mRNA construct” 250 , from 30 to 500 , from 30 to 1,000 , from 30 to 1,500 ,
refers to a polynucleotide transcript which encodes one or from 30 to 3,000 , from 30 to 5,000 , from 30 to 7,000 , from
more polypeptides of interest and which retains sufficient 30 to 10,000 , from 30 to 25,000, from 30 to 50,000 , from 30
structural and/or chemical features to allow the polypeptide to 70,000 , from 100 to 250 , from 100 to 500 , from 100 to
of interest encoded therein to be translated . Primary con- 30 1,000 , from 100 to 1,500 , from 100 to 3,000 , from 100 to
structs may be polynucleotides of the invention . When 5,000 , from 100 to 7,000, from 100 to 10,000 , from 100 to
structurally or chemically modified , the primary construct 25,000 , from 100 to 50,000 , from 100 to 70,000 , from 100
may be referred as an mmRNA . to 100,000 , from 500 to 1,000 , from 500 to 1,500 , from 500
Returning to FIG . 1, the primary construct 100 here to 2,000, from 500 to 3,000 , from 500 to 5,000 , from 500 to
contains a first region of linked nucleotides 102 that is 35 7,000 , from 500 to 10,000 , from 500 to 25,000 , from 500 to
flanked by a first flanking region 104 and a second flaking 50,000 , from 500 to 70,000 , from 500 to 100,000 , from
region 106. As used herein , the “ first region ” may be referred 1,000 to 1,500 , from 1,000 to 2,000, from 1,000 to 3,000 ,
to as a “ coding region ” or “ region encoding " or simply the from 1,000 to 5,000 , from 1,000 to 7,000 , from 1,000 to
" first region.” This first region may include , but is not 10,000 , from 1,000 to 25,000 , from 1,000 to 50,000, from
limited to , the encoded polypeptide of interest. The poly- 40 1,000 to 70,000 , from 1,000 to 100,000 , from 1,500 to 3,000 ,
peptide of interest may comprise at its 5 ' terminus one or from 1,500 to 5,000 , from 1,500 to 7,000 , from 1,500 to
more signal sequences encoded by a signal sequence region 10,000, from 1,500 to 25,000 , from 1,500 to 50,000 , from
103. The flanking region 104 may comprise a region of 1,500 to 70,000 , from 1,500 to 100,000 , from 2,000 to 3,000 ,
linked nucleotides comprising one or more complete or from 2,000 to 5,000 , from 2,000 to 7,000 , from 2,000 to
incomplete 5'UTRs sequences. The flanking region 104may 45 10,000 , from 2,000 to 25,000 , from 2,000 to 50,000 , from
also comprise a 5' terminal cap 108. The second flanking 2,000 to 70,000 , and from 2,000 to 100,000 ).
region 106 may comprise a region of linked nucleotides According to the present invention , the first and second
comprising one or more complete or incomplete 3' UTRs. flanking regions may range independently from 15-1,000
The flanking region 106 may also comprise a 3' tailing nucleotides in length (e.g., greater than 30 , 40 , 45 , 50 , 55 ,
sequence 110 . 50 60 , 70 , 80 , 90 , 100 , 120 , 140 , 160 , 180 , 200 , 250 , 300 , 350 ,
Bridging the 5 ' terminus of the first region 102 and the 400 , 450 , 500 , 600 , 700 , 800 , and 900 nucleotides or at least
first flanking region 104 is a first operational region 105 . 30 , 40 , 45 , 50 , 55 , 60, 70 , 80 , 90 , 100 , 120 , 140 , 160, 180 ,
Traditionally this operational region comprises a Start 200 , 250 , 300, 350, 400, 450 , 500 , 600 , 700, 800 , 900 , and
codon . The operational region may alternatively comprise 1,000 nucleotides ).
any translation initiation sequence or signal including a Start 55 According to the present invention , the tailing sequence
codon . may range from absent to 500 nucleotides in length ( e.g., at
Bridging the 3 ' terminus of the first region 102 and the least 60, 70 , 80 , 90 , 120 , 140, 160 , 180 , 200 , 250 , 300 , 350 ,
second flanking region 106 is a second operational region 400 , 450 , or 500 nucleotides). Where the tailing region is a
107. Traditionally this operational region comprises a Stop polyA tail , the length may be determined in units of or as a
codon . The operational region may alternatively comprise 60 function of polyA Binding Protein binding . In this embodi
any translation initiation sequence or signal including a Stop ment, the polyA tail is long enough to bind at least 4
codon . According to the present invention , multiple serial monomers of PolyA Binding Protein . PolyA Binding Protein
stop codons may also be used . monomers bind to stretches of approximately 38 nucleo
Generally , the shortest length of the first region of the tides . As such , it has been observed that polyA tails of about
primary construct of the present invention can be the length 65 80 nucleotides and 160 nucleotides are functional.
of a nucleic acid sequence that is sufficient to encode for a According to the present invention , the capping region
dipeptide, a tripeptide , a tetrapeptide, a pentapeptide , a may comprise a single cap or a series ofnucleotides forming
US 10,703,789 B2
11 12
the ??? . In this embodiment the capping region may be from er's protocol. After the addition of the 3'-modified nucleo
1 to 10 , e.g. 2-9 , 3-8 , 4-7 , 1-5 , 5-10 , or at least 2 , or 10 ortide , the two polynucleotide, primary construct or mmRNA
fewer nucleotides in length . In some embodiments, the cap species may be combined in an aqueous solution , in the
is absent. presence or absence of copper, to form a new covalent
According to the present invention , the first and second 5 linkage via a click chemistry mechanism as described in the
operational regions may range from 3 to 40 , e.g., 5-30, literature .
10-20 , 15 , or at least 4 , or 30 or fewer nucleotides in length In another example , more than two polynucleotides may
and may comprise , in addition to a Start and /or Stop codon , be linked together using a functionalized linker molecule.
one or more signal and /or restriction sequences. For example , a functionalized saccharide molecule may be
Cyclic mmRNA 10 chemically modified to contain multiple chemical reactive
According to the present invention , a primary constructor groups (SH— , NH2— , N3, etc .... ) to react with the cognate
mmRNA may be cyclized , or concatemerized , to generate a moiety on a 3'- functionalized mRNA molecule (i.e., a 3'-ma
translation competent molecule to assist interactions leimide ester, 3'-NHS -ester, alkynyl). The number of reac
between poly - A binding proteins and 5 '-end binding pro tive groups on the modified saccharide can be controlled in
teins . The mechanism of cyclization or concatemerization 15 a stoichiometric fashion to directly control the stoichiomet
may occur through at least 3 different routes : 1) chemical, 2 ) ric ratio of conjugated polynucleotide, primary construct or
enzymatic , and 3 ) ribozyme catalyzed . The newly formed mmRNA .
5-/3 '-linkage may be intramolecular or intermolecular. mmRNA Conjugates and Combinations
In the first route , the 5 '-end and the 3 '- end of the nucleic In order to further enhance protein production , primary
acid contain chemically reactive groups that, when close 20 constructs or mmRNA of the present invention can be
together , form a new covalent linkage between the 5 '-end designed to be conjugated to other polynucleotides, dyes ,
and the 3'-end of the molecule . The 5 '- end may contain an intercalating agents (e.g. acridines ), cross- linkers (e.g. pso
NHS -ester reactive group and the 3 '-end may contain a ralene, mitomycin C ), porphyrins ( TPPC4, texaphyrin , Sap
3'-amino -terminated nucleotide such that in an organic sol phyrin ), polycyclic aromatic hydrocarbons (e.g., phenazine ,
vent the 3'-amino -terminated nucleotide on the 3'-end of a 25 dihydrophenazine ), artificial endonucleases ( e.g. EDTA ),
synthetic mRNA molecule will undergo a nucleophilic alkylating agents , phosphate , amino, mercapto , PEG ( e.g. ,
attack on the 5 '-NHS -ester moiety forming a new 5:-/3' PEG -40K ),MPEG , [MPEG ]2, polyamino, alkyl, substituted
amide bond . alkyl, radiolabeled markers , enzymes, haptens (e.g. biotin ),
In the second route , T4 RNA ligase may be used to transport/absorption facilitators (e.g., aspirin , vitamin E ,
enzymatically link a 5'-phosphorylated nucleic acid mol- 30 folic acid ), synthetic ribonucleases, proteins, e.g., glycopro
ecule to the 3'-hydroxyl group of a nucleic acid forming a teins , or peptides, e.g. , molecules having a specific affinity
new phosphorodiester linkage. In an example reaction , 1 ug for a co - ligand, or antibodies e.g. , an antibody , that binds to
of a nucleic acid molecule is incubated at 37 ° C. for 1 hour a specified cell type such as a cancer cell , endothelial cell ,
with 1-10 units of T4 RNA ligase (New England Biolabs , or bone cell, hormones and hormone receptors, non- peptidic
Ipswich , Mass.) according to the manufacturer's protocol. 35 species, such as lipids, lectins, carbohydrates , vitamins,
The ligation reaction may occur in the presence of a split cofactors, or a drug .
oligonucleotide capable of base-pairing with both the 5'- and Conjugation may result in increased stability and /or half
3'- region in juxtaposition to assist the enzymatic ligation life and may be particularly useful in targeting the poly
reaction . nucleotides, primary constructs ormmRNA to specific sites
In the third route, either the 5'- or 3 '-end of the cDNA 40 in the cell, tissue or organism .
template encodes a ligase ribozyme sequence such that According to the present invention , the mmRNA or pri
during in vitro transcription , the resultant nucleic acid mol mary constructs may be administered with , or further encode
ecule can contain an active ribozyme sequence capable of one or more of RNAi agents , siRNAs, shRNAs, miRNAs,
ligating the 5 '-end of a nucleic acid molecule to the 3'-end miRNA binding sites , antisense RNAs, ribozymes, catalytic
of a nucleic acid molecule . The ligase ribozyme may be 45 DNA , RNA , RNAs that induce triple helix formation ,
derived from the Group 1 Intron , Group I Intron , Hepatitis aptamers or vectors , and the like .
Delta Virus, Hairpin ribozyme or may be selected by Bifunctional mmRNA
SELEX ( systematic evolution of ligands by exponential In one embodiment of the invention are bifunctional
enrichment). The ribozyme ligase reaction may take 1 to 24 polynucleotides (e.g., bifunctional primary constructs or
hours at temperatures between 0 and 37 ° C. 50 bifunctional mmRNA ). As the name implies, bifunctional
mmRNA Multimers polynucleotides are those having or capable of at least two
According to the present invention, multiple distinct functions. These molecules may also by convention be
polynucleotides, primary constructs or mmRNA may be referred to as multi- functional.
linked together through the 3 '-end using nucleotides which The multiple functionalities of bifunctional polynucle
are modified at the 3'-terminus. Chemical conjugation may 55 otides may be encoded by the RNA ( the function may not
be used to control the stoichiometry of delivery into cells. manifest until the encoded product is translated ) or may be
For example, the glyoxylate cycle enzymes, isocitrate lyase a property of the polynucleotide itself. It may be structural
and malate synthase ,may be supplied into HepG2 cells at a or chemical. Bifunctional modified polynucleotides may
1 : 1 ratio to alter cellular fatty acid metabolism . This ratio comprise a function that is covalently or electrostatically
may be controlled by chemically linking polynucleotides , 60 associated with the polynucleotides. Further , the two func
primary constructs or mmRNA using a 3'-azido terminated tions may be provided in the context of a complex of a
nucleotide on one polynucleotide, primary construct or mmRNA and another molecule .
mmRNA species and a C5-ethynyl or alkynyl-containing Bifunctional polynucleotides may encode peptides which
nucleotide on the opposite polynucleotide, primary construct are anti-proliferative. These peptides may be linear, cyclic,
or mmRNA species . The modified nucleotide is added 65 constrained or random coil. They may function as aptamers,
post-transcriptionally using terminal transferase (New Eng signaling molecules, ligands or mimics or mimetics thereof.
land Biolabs, Ipswich , Mass .) according to the manufactur Anti-proliferative peptides may , as translated , be from 3 to
US 10,703,789 B2
13 14
50 amino acids in length . They may be 5-40, 10-30 , or one or more amino acids which would mimic an activated
approximately 15 amino acids long. They may be single sequence . For example , glutamate may serve as a mimic for
chain , multichain or branched and may form complexes , phosphoro - threonine and /or phosphoro - serine . Alterna
aggregates or any multi -unit structure once translated . tively , variant mimics may result in deactivation or in an
Noncoding Polynucleotides and Primary Constructs 5 inactivated product containing the mimic , e.g., phenylala
As described herein , provided are polynucleotides and nine may act as an inactivating substitution for tyrosine ; or
primary constructs having sequences that are partially or alanine may act as an inactivating substitution for serine.
substantially not translatable , e.g. , having a noncoding “ Homology ” as it applies to amino acid sequences is
region . Such noncoding region may be the “ first region ” of defined as the percentage ofresidues in the candidate amino
the primary construct. Alternatively , the noncoding region 10 acid sequence that are identical with the residues in the
may be a region other than the first region. Such molecules amino acid sequence of a second sequence after aligning the
are generally not translated , but can exert an effecton protein sequences and introducing gaps, if necessary , to achieve the
production by one or more of binding to and sequestering maximum percent homology . Methods and computer pro
one or more translational machinery components such as a grams for the alignment are well known in the art. It is
ribosomal protein or a transfer RNA (TRNA ), thereby effec- 15 understood that homology depends on a calculation of
tively reducing protein expression in the cell or modulating percent identity but may differ in value due to gaps and
one or more pathways or cascades in a cell which in turn penalties introduced in the calculation .
alters protein levels . The polynucleotide or primary con By " homologs " as it applies to polypeptide sequences
structmay contain or encode one or more long noncoding means the corresponding sequence of other species having
RNA (lncRNA , or lincRNA ) or portion thereof, a small 20 substantial identity to a second sequence of a second species .
nucleolar RNA (sno -RNA ), micro RNA (miRNA ), small “ Analogs” is meant to include polypeptide variants which
interfering RNA (siRNA ) or Piwi-interacting RNA differ by one or more amino acid alterations, e.g., substitu
( piRNA ). tions , additions or deletions of amino acid residues that still
Polypeptides of Interest maintain one or more of the properties of the parent or
According to the present invention , the primary construct 25 starting polypeptide .
is designed to encode one or more polypeptides of interest The present invention contemplates several types of com
or fragments thereof. A polypeptide of interest may include, positions which are polypeptide based including variants
but is not limited to, whole polypeptides, a plurality of and derivatives . These include substitutional, insertional,
polypeptides or fragments of polypeptides, which indepen deletion and covalent variants and derivatives . The term
dently may be encoded by one or more nucleic acids, a 30 “ derivative” is used synonymously with the term “ variant”
plurality of nucleic acids, fragments of nucleic acids or but generally refers to a molecule that has been modified
variants of any of the aforementioned . As used herein , the and /or changed in any way relative to a reference molecule
term “ polypeptides of interest” refer to any polypeptide or starting molecule .
which is selected to be encoded in the primary construct of As such, mmRNA encoding polypeptides containing sub
the present invention . As used herein , " polypeptide” means 35 stitutions, insertions and /or additions, deletions and covalent
a polymer of amino acid residues (natural or unnatural) modifications with respect to reference sequences, in par
linked together most often by peptide bonds. The term , as ticular the polypeptide sequences disclosed herein , are
used herein , refers to proteins, polypeptides, and peptides of included within the scope of this invention . For example ,
any size , structure, or function . In some instances the sequence tags or amino acids, such as one or more lysines ,
polypeptide encoded is smaller than about 50 amino acids 40 can be added to the peptide sequences of the invention (e.g.,
and the polypeptide is then termed a peptide . If the poly at the N -terminal or C -terminal ends ). Sequence tags can be
peptide is a peptide, it will be at least about 2 , 3, 4 , or at least used for peptide purification or localization . Lysines can be
5 amino acid residues long . Thus , polypeptides include gene used to increase peptide solubility or to allow for bioti
products, naturally occurring polypeptides, synthetic poly nylation . Alternatively, amino acid residues located at the
peptides, homologs, orthologs, paralogs, fragments and 45 carboxy and amino terminal regions of the amino acid
other equivalents , variants, and analogs of the foregoing. A sequence of a peptide or protein may optionally be deleted
polypeptide may be a single molecule or may be a multi providing for truncated sequences . Certain amino acids (e.g.,
molecular complex such as a dimer, trimer or tetramer. They C -terminal or N -terminal residues ) may alternatively be
may also comprise single chain or multichain polypeptides deleted depending on the use of the sequence, as for
such as antibodies or insulin and may be associated or 50 example , expression of the sequence as part of a larger
linked . Most commonly disulfide linkages are found in sequence which is soluble , or linked to a solid support.
multichain polypeptides. The term polypeptide may also “ Substitutional variants” when referring to polypeptides
apply to amino acid polymers in which one or more amino are those that have at least one amino acid residue in a native
acid residues are an artificial chemical analogue of a corre or starting sequence removed and a different amino acid
sponding naturally occurring amino acid . 55 inserted in its place at the same position . The substitutions
The term “ polypeptide variant” refers to molecules which may be single , where only one amino acid in the molecule
differ in their amino acid sequence from a native or reference has been substituted , or they may be multiple, where two or
sequence. The amino acid sequence variants may possess more amino acids have been substituted in the same mol
substitutions, deletions, and /or insertions at certain positions ecule .
within the amino acid sequence, as compared to a native or 60 As used herein the term " conservative amino acid sub
reference sequence . Ordinarily, variants will possess at least stitution ” refers to the substitution of an amino acid that is
about 50 % identity (homology ) to a native or reference normally present in the sequence with a different amino acid
sequence, and preferably , they will be at least about 80 % , of similar size , charge , or polarity. Examples of conservative
more preferably at least about 90 % identical (homologous ) substitutions include the substitution of a non -polar (hydro
to a native or reference sequence . 65 phobic) residue such as isoleucine , valine and leucine for
In some embodiments “ variantmimics” are provided . As another non -polar residue. Likewise , examples of conserva
used herein , the term “ variantmimic ” is one which contains tive substitutions include the substitution of one polar (hy
US 10,703,789 B2
15 16
drophilic ) residue for another such as between arginine and As used herein when referring to polypeptides the term
lysine, between glutamine and asparagine , and between " fold ” refers to the resultant conformation of an amino acid
glycine and serine. Additionally , the substitution of a basic sequence upon energy minimization. A fold may occur at the
residue such as lysine , arginine or histidine for another, or secondary or tertiary level of the folding process. Examples
the substitution of one acidic residue such as aspartic acid or 5 of secondary level folds include beta sheets and alpha
glutamic acid for another acidic residue are additional
examples of conservative substitutions. Examples of non helices regions
. Examples of tertiary folds include domains and
formed due to aggregation or separation of energetic
conservative substitutions include the substitution of a non forces. Regions formed in this way include hydrophobic and
polar (hydrophobic ) amino acid residue such as isoleucine, hydrophilic pockets, and the like.
valine , leucine, alanine, methionine for a polar (hydrophilic ) 10 As used herein the term “ turn ” as it relates to protein
residue such as cysteine, glutamine, glutamic acid or lysine conformation means a bend which alters the direction of the
and / or a polar residue for a non -polar residue.
“ Insertional variants” when referring to polypeptides are backbone
two , three
of a peptide or polypeptide and may involve one,
or more amino acid residues .
those with one or more amino acids inserted immediately
adjacent to an amino acid at a particular position in a native 15 As used herein when referring to polypeptides the term
or starting sequence. “ Immediately adjacent” to an amino “ loop ” refers to a structural feature of a polypeptide which
acid means connected to either the alpha-carboxy or alpha may serve to reverse the direction of the backbone of a
amino functional group of the amino acid . peptide or polypeptide . Where the loop is found in a
“ Deletional variants " when referring to polypeptides are polypeptide and only alters the direction of the backbone, it
those with one ormore amino acids in the native or starting 20 may comprise four ormore amino acid residues.Oliva et al.
amino acid sequence removed . Ordinarily , deletional vari have identified at least 5 classes ofprotein loops ( J.Mol Biol
ants will have one or more amino acids deleted in a 266 (4 ): 814-830 ; 1997 ). Loops may be open or closed .
particular region of the molecule . Closed loops or “ cyclic ” loops may comprise 2 , 3 , 4 , 5 , 6 ,
" Covalent derivatives” when referring to polypeptides 7 , 8 , 9 , 10 or more amino acids between the bridging
include modifications of a native or starting protein with an 25 moieties . Such bridging moieties may comprise a cysteine
organic proteinaceous or non -proteinaceous derivatizing cysteine bridge (Cys - Cys) typical in polypeptides having
agent, and/or post-translational modifications. Covalent disulfide bridges or alternatively bridging moieties may be
modifications are traditionally introduced by reacting tar non -protein based such as the dibromozylyl agents used
geted amino acid residues of the protein with an organic herein .
derivatizing agent that is capable of reacting with selected 30 As used herein when referring to polypeptides the term
side - chains or terminal residues, or by harnessing mecha “ half - loop ” refers to a portion of an identified loop having at
nisms of post- translational modifications that function in least half the number of amino acid resides as the loop from
selected recombinant host cells. The resultant covalent which it is derived . It is understood that loops may not
derivatives are useful in programs directed at identifying always contain an even number of amino acid residues .
residues important for biological activity , for immunoas- 35 Therefore , in those cases where a loop contains or is
says, or for the preparation of anti -protein antibodies for identified to comprise an odd number of amino acids, a
immunoaffinity purification of the recombinant glycopro half-loop of the odd -numbered loop will comprise the whole
tein . Such modifications are within the ordinary skill in the number portion or next whole number portion of the loop
art and are performed without undue experimentation . (number of amino acids of the loop /2 +/- 0.5 amino acids).
Certain post-translational modifications are the result of 40 For example , a loop identified as a 7 amino acid loop could
the action of recombinant host cells on the expressed poly produce half- loops of 3 amino acids or 4 amino acids
peptide. Glutaminyl and asparaginyl residues are frequently (7 /2 = 3.5 +/- 0.5 being 3 or 4 ).
post -translationally deamidated to the corresponding gluta As used herein when referring to polypeptides the term
myl and aspartyl residues. Alternatively , these residues are " domain ” refers to a motif of a polypeptide having one or
deamidated under mildly acidic conditions. Either form of 45 more identifiable structural or functional characteristics or
these residues may be present in the polypeptides produced properties (e.g., binding capacity , serving as a site for
in accordance with the present invention . protein -protein interactions ).
Other post - translational modifications include hydroxy As used herein when referring to polypeptides the term
lation of proline and lysine , phosphorylation of hydroxyl “half-domain ” means a portion of an identified domain
groups of seryl or threonyl residues, methylation of the 50 having at least half the number of amino acid resides as the
alpha -amino groups of lysine , arginine , and histidine side domain from which it is derived . It is understood that
chains ( T. E. Creighton , Proteins: Structure and Molecular domains may not always contain an even number of amino
Properties , W.H. Freeman & Co., San Francisco , pp . 79-86 acid residues . Therefore, in those cases where a domain
( 1983) ). contains or is identified to comprise an odd number of amino
" Features” when referring to polypeptides are defined as 55 acids, a half -domain of the odd -numbered domain will
distinct amino acid sequence-based components of a mol comprise the whole number portion or next whole number
ecule . Features of the polypeptides encoded by the mmRNA portion of the domain (number of amino acids of the
of the present invention include surface manifestations, local domain / 2 +/- 0.5 amino acids). For example , a domain iden
conformational shape , folds, loops, half-loops , domains, tified as a 7 amino acid domain could produce half-domains
half- domains , sites , termini or any combination thereof. 60 of 3 amino acids or 4 amino acids (7 /2 = 3.5 +/- 0.5 being 3 or
As used herein when referring to polypeptides the term 4 ). It is also understood that subdomains may be identified
“ surface manifestation” refers to a polypeptide based com within domains or half -domains, these subdomains possess
ponent of a protein appearing on an outermost surface. ing less than all of the structural or functional properties
As used herein when referring to polypeptides the term identified in the domains or half domains from which they
" local conformational shape” means a polypeptide based 65 were derived . It is also understood that the amino acids that
structuralmanifestation of a protein which is located within comprise any of the domain types herein need not be
a definable space of the protein . contiguous along the backbone of the polypeptide ( i.e.,
US 10.703.789 B2
19 20
2217 , 2218 , 2219,2220,2221 , 2222,2223, 2224,2225 , 2820 , 2821 , 2822,2823 , 2824,2825, 2826 , 2827,2828,
2226,2227, 2228, 2229 , 2230, 2231, 2232, 2233 , 2234 , 2829 , 2830, 2831, 2832,2833, 2834,2835 , 2836,2837 ,
2235, 2236 , 2237,2238,2239 , 2240, 2241 , 2242 , 2243, 2838,2839 , 2840, 2841,2842 , 2843, 2844,2845, 2846 ,
2244,2245 , 2246 , 2247,2248 , 2249,2250,2251,2252 , 2847,2848, 2849,2850 , 2851 , 2852,2853 , 2854,2855 ,
2253, 2254,2255, 2256,2257, 2258, 2259 , 2260,2261,5 2856, 2857, 2858, 2859, 2860, 2861, 2862 , 2863, 2864,
2262,2263 , 2264,2265,2266, 2267,2268 , 2269,2270 , 2865, 2866,2867,2868 , 2869 , 2870, 2871 , 2872 , 2873 ,
2271 , 2272,2273 , 2274,2275, 2276 , 2277,2278 , 2279 , 2874,2875, 2876,2877,2878 , 2879 , 2880 , 2881 , 2882,
2280 , 2281, 2282, 2283 , 2284,2285, 2286, 2287,2288, 2883 , 2884,2885, 2886,2887,2888,2889 , 2890 , 2891 ,
2289,2290 , 2291 , 2292,2293 , 2294,2295 , 2296,2297 , 2892,2893, 2894,2895, 2896 , 2897, 2898 , 2899,2900 ,
2298 , 2299 , 2300,2301 , 2302 , 2303 , 2304,2305 , 2306 , 10 2901,2902,2903, 2904,2905, 2906, 2907,2908,2909 ,
2307,2308, 2309, 2310 , 2311, 2312, 2313 , 2314,2315, 2910, 2911 , 2912 , 2913, 2914,2915 , 2916,2917, 2918,
2316 , 2317,2318, 2319, 2320,2321, 2322, 2323 , 2324 , 2919, 2920, 2921, 2922,2923, 2924,2925,2926 , 2927,
2325 , 2326, 2327, 2328, 2329, 2330, 2331, 2332 , 2333 , 2928, 2929 , 2930, 2931,2932,2933, 2934,2935, 2936,
2334,2335 , 2336, 2337,2338 , 2339,2340 , 2341, 2342 , 2937,2938,2939,2940 , 2941 , 2942,2943, 2944,2945 ,
2343 , 2344,2345, 2346,2347,2348, 2349, 2350,2351, 15 2946,2947,2948 , 2949,2950, 2951 , 2952 , 2953, 2954,
2352 , 2353, 2354,2355 , 2356 , 2357,2358, 2359 , 2360 , 2955 , 2956,2957,2958 , 2959 , 2960,2961, 2962,2963 ,
2361, 2362,2363 , 2364,2365 , 2366,2367,2368 , 2369 , 2964,2965,2966,2967,2968,2969,2970,2971,2972 ,
2370,2371, 2372 , 2373 , 2374, 2375 , 2376, 2377,2378 , 2973, 2974,2975,2976,2977,2978 , 2979,2980,2981,
2379, 2380,2381, 2382,2383, 2384,2385 , 2386 , 2387, 2982,2983, 2984,2985,2986,2987,2988, 2989 , 2990 ,
2388 , 2389, 2390, 2391 , 2392 , 2393 , 2394, 2395 , 2396 , 20 2991,2992,2993 , 2994,2995,2996,2997,2998 , 2999,
2397,2398 , 2399, 2400, 2401 , 2402, 2403, 2404,2405 , 3000 , 3001, 3002, 3003 , 3004,3005, 3006, 3007, 3008 ,
2406,2407,2408 , 2409,2410 , 2411 , 2412, 2413, 2414 , 3009, 3010, 3011,3012, 3013 , 3014,3015, 3016,3017,
2415, 2416, 2417, 2418 , 2419,2420 , 2421 , 2422 , 2423 , 3018,3019, 3020,3021,3022 , 3023 , 3024,3025 , 3026 ,
2424,2425 , 2426 , 2427,2428 , 2429 , 2430, 2431 , 2432 , 3027,3028 , 3029,3030,3031,3032 , 3033, 3034,3035 ,
2433, 2434,2435, 2436,2437,2438, 2439, 2440, 2441, 25 3036,3037,3038, 3039,3040,3041, 3042 , 3043, 3044 ,
2442 , 2443 , 2444,2445, 2446, 2447 , 2448 , 2449, 2450 , 3045, 3046, 3047,3048 , 3049,3050, 3051,3052, 3053,
2451 , 2452,2453, 2454,2455, 2456 , 2457, 2458, 2459 , 3054,3055 , 3056, 3057,3058 , 3059,3060 , 3061,3062 ,
2460,2461 , 2462, 2463 , 2464,2465, 2466 , 2467,2468, 3063,3064,3065, 3066,3067,3068, 3069,3070,3071 ,
2469,2470 , 2471 , 2472, 2473 , 2474,2475 , 2476 , 2477, 3072, 3073 , 3074,3075,3076,3077,3078 , 3079,3080 ,
2478 , 2479 , 2480, 2481, 2482, 2483, 2484,2485, 2486, 30 3081,3082, 3083, 3084,3085,3086,3087,3088,3089,
2487,2488, 2489, 2490 , 2491, 2492, 2493, 2494,2495, 3090,3091, 3092,3093 , 3094,3095,3096,3097,3098 ,
2496 , 2497, 2498, 2499 , 2500,2501, 2502 , 2503, 2504 , 3099,3100, 3101, 3102, 3103 , 3104,3105, 3106, 3107 ,
2505, 250 2507 , 19,2510, 2511 , 2512, 2513, 3108,3109,3110,3111,3112,3113,3114,3115 , 3116,3117,
2514,2515, 2516, 2517, 2518 , 2519, 2520, 2521 , 2522 , 3118, 3119,3120,3121, 3122, 3123, 3124,3125, 3126 ,
2523 , 2524,2525, 2526 , 2527,2528, 2529 , 2530, 2531, 35 3127,3128 , 3129,3130 , 3131,3132, 3133,3134,3135 ,
2532,2533 , 2534,2535, 2536, 2537,2538, 2539,2540 , 3136,3137,3138, 3139,3140,3141,3142,3143, 3144 ,
2541, 2542 , 2543, 2544,2545, 2546 , 2547,2548 , 2549 , 3145, 3146,3147,3148,3149,3150, 3151,3152, 3153 ,
2550, 2551, 2552, 2553, 2554, 2555, 2556 , 2557, 2558, 3154,3155, 3156 , 3157,3158, 3159,3160,3161,3162 ,
2559, 2560 , 2561, 2562,2563 , 2564,2565, 2566, 2567 , 3163,3164,3165,3166,3167,3168, 3169,3170 , 3171 ,
2568 , 2569, 2570, 2571 , 2572 , 2573, 2574,2575 , 2576 , 40 3172, 3173 , 3174,3175,3176,3177,3178,3179,3180,
2577,2578 , 2579,2580, 2581, 2582,2583 , 2584,2585 , 3181,3182, 3183, 3184,3185, 3186,3187,3188,3189 ,
2586,2587,2588, 2589,2590, 2591, 2592 , 2593 , 2594 , 3190,3191,3192, 3193 , 3194,3195, 3196,3197,3198,
2595,2596 , 2597,2598 , 2599, 2600,2601 , 2602, 2603, 3199 , 3200, 3201, 3202, 3203 , 3204,3205, 3206, 3207 ,
2604,2605 , 2606, 2607,2608 , 2609 , 2610, 2611 , 2612 , 3208, 3209,3210, 3211, 3212, 3213, 3214,3215, 3216,
2613, 2614,2615, 2616,2617, 2618, 2619, 2620, 2621, 45 3217, 3218, 3219,3220 , 3221, 3222 , 3223 , 3224,3225 ,
2622,2623 , 2624, 2625 , 2626, 2627,2628 , 2629, 2630, 3226,3227,3228,3229,3230 , 3231, 3232 , 3233 , 3234,
2631 , 2632,2633, 2634,2635, 2636, 2637,2638, 2639, 3235 , 3236,3237,3238 , 3239,3240 , 3241,3242 , 3243 ,
2640 , 2641, 2642,2643 , 2644,2645, 2646, 2647,2648 , 3244,3245, 3246,3247,3248,3249,3250, 3251 , 3252 ,
2649,2650, 2651 , 2652, 2653 , 2654,2655, 2656 , 2657, 3253, 3254,3255 , 3256,3257 , 3258 , 3259,3260 , 3261 ,
2658 , 2659, 2660, 2661,2662,2663 , 2664,2665,2666 , 50 3262 , 3263 , 3264,3265,3266,3267,3268 , 3269,3270 ,
2667,2668, 2669, 2670 , 2671, 2672, 2673 , 2674,2675, 3271,3272, 3273 , 3274,3275,3276,3277,3278,3279 ,
2676, 2677,2678 , 2679, 2680 , 2681, 2682,2683 , 2684, 3280,3281, 3282, 3283, 3284,3285 , 3286,3287,3288 ,
2685,2686,2687, 2688, 2689,2690 , 2691 , 2692 , 2693 , 3289,3290 , 3291, 3292 , 3293 , 3294,3295,3296,3297 ,
2694,2695,2696, 2697,2698 , 2699, 2700 , 2701 , 2702, 3298,3299 , 3300, 3301 , 3302 , 3303, 3304,3305, 3306,
2703 , 2704,2705, 2706,2707,2708, 2709, 2710 , 2711 , 55 3307, 3308 , 3309 , 3310, 3311,3312 , 3313,3314,3315,
2712,2713 , 2714,2715 , 2716, 2717, 2718 , 2719,2720 , 3316, 3317, 3318, 3319,3320, 3321, 3322 , 3323 , 3324,
2721,2722,2723, 2724,2725, 2726 , 2727,2728, 2729 , 3325,3326, 3327, 3328, 3329,3330, 3331,3332,3333,
2730, 2731 , 2732, 2733 , 2734,2735, 2736,2737,2738, 3334,3335,3336,3337,3338, 3339,3340,3341,3342,
2739 , 2740, 2741, 2742 , 2743 , 2744,2745, 2746, 2747 , 3343, 3344,3345, 3346,3347,3348, 3349,3350,3351 ,
2748,2749,2750, 2751,2752,2753 , 2754,2755, 2756 , 60 3352,3353, 3354,3355,3356,3357 , 3358,3359,3360 ,
2757,2758 , 2759,2760 , 2761 , 2762, 2763 , 2764,2765 , 3361,3362 , 3363, 3364,3365 , 3366,3367,3368 , 3369 ,
2766, 2767,2768 , 2769,2770,2771 , 2772 , 2773, 2774, 3370 , 3371,3372 , 3373 , 3374,3375,3376,3377,3378 ,
2775 , 2776 , 2777,2778 , 2779, 2780, 2781, 2782,2783 , 3379,3380, 3381, 3382 , 3383,3384,3385, 3386,3387 ,
2784,2785, 2786,2787,2788,2789,2790 , 2791,2792 , 3388,3389, 3390, 3391, 3392,3393 , 3394,3395,3396 ,
2793 , 2794,2795, 2796,2797,2798, 2799 , 2800,2801, 65 3397,3398 , 3399,3400 , 3401,3402 , 3403,3404,3405 ,
2802 , 2803, 2804,2805 , 2806,2807 , 2808 , 2809 , 2810 , 3406,3407, 3408 , 3409,3410,3411,3412, 3413 , 3414,
2811, 2812, 2813, 2814,2815 , 2816, 2817,2818, 2819, 3415, 3416,3417, 3418, 3419,3420,3421,3422, 3423,
US 10,703,789 B2
21 22
3424 , 3425 , 3426 , 3427 , 3428, 3429, 3430 , 3431 , 3432, monoclonal antibodies, cytokines, growth factors, enzymes,
3433 , 3434, 3435 , 3436 , 3437 , 3438 , 3439, 3440 , 3441, thrombolytics , and immunomodulators, among others.
3442 , 3443, 3444, 3445 , 3446 , 3447, 3448 , 3449, 3450 , According to the present invention , one or more biologics
3451, 3452 , 3453 , 3454, 3455 , 3456 , 3457, 3458 , 3459, currently being marketed or in developmentmay be encoded
3460 , 3461, 3462 , 3463 , 3464 , 3465 , 3466 , 3467 , 3468 , 5 by the polynucleotides , primary constructs or mmRNA of
the present invention . While not wishing to be bound by
3469, 3470 , 3471, 3472, 3473 , 3474, 3475 , 3476 , 3477, theory
3478, 3479 , 3480 , 3481, 3482 , 3483, 3484, 3485 , 3486 , , it is believed that incorporation of the encoding
3487 , 3488 , 3489, 3490 , 3491, 3492 , 3493 , 3494 , 3495 , polynucleotides of a known biologic into the primary con
3496 , 3497 . structs or mmRNA of the invention will result in improved
The term “ identity ” as known in the art, refers to a 10 purity
therapeutic efficacy due at least in part to the specificity ,
and /or selectivity of the construct designs .
relationship between the sequences of two or more peptides ,
as determined by comparing the sequences. In the art, Antibodies
The primary constructs ormmRNA disclosed herein , may
identity also means the degree of sequence relatedness encode one or more antibodies or fragments thereof. The
between peptides, as determined by the number of matches 15 term “ antibody” includes monoclonal antibodies ( including
between strings oftwo or more amino acid residues. Identity full length antibodies which have an immunoglobulin Fc
measures the percent of identical matches between the region ), antibody compositions with polyepitopic specific
smaller of two or more sequences with gap alignments ( if ity, multispecific antibodies (e.g., bispecific antibodies , dia
any ) addressed by a particular mathematicalmodel or com bodies, and single -chain molecules), as well as antibody
puter program ( i.e., “ algorithms” ). Identity of related pep- 20 fragments. The term “ immunoglobulin” (Ig ) is used inter
tides can be readily calculated by known methods. Such changeably with “ antibody ” herein . As used herein , the term
methods include , but are not limited to , those described in “ monoclonal antibody” refers to an antibody obtained from
ComputationalMolecular Biology , Lesk , A.M., ed ., Oxford a population of substantially homogeneous antibodies, i.e.,
University Press ,New York , 1988 ; Biocomputing: Informat the individual antibodies comprising the population are
ics and Genome Projects, Smith , D. W., ed .,Academic Press, 25 identical except for possible naturally occurring mutations
New York , 1993 ; Computer Analysis of Sequence Data, Part and / or post- translation modifications ( e.g., isomerizations,
1 , Griffin , A. M., and Griffin , H. G., eds., Humana Press , amidations) that may be present in minor amounts. Mono
New Jersey, 1994 ; Sequence Analysis in Molecular Biology , clonal antibodies are highly specific , being directed against
von Heinje , G., Academic Press, 1987 ; Sequence Analysis a single antigenic site .
Primer , Gribskov, M. and Devereux , J., eds ., M. Stockton 30 The monoclonal antibodies herein specifically include
Press, New York , 1991 ; and Carillo et al., SIAM J. Applied “ chimeric” antibodies ( immunoglobulins) in which a portion
Math . 48 , 1073 ( 1988). of the heavy and /or light chain is identical with or homolo
In some embodir its , the polypeptide variant may have gous to corresponding sequences in antibodies derived from
the same or a similar activity as the reference polypeptide . a particular species or belonging to a particular antibody
Alternatively , the variantmay have an altered activity ( e.g., 35 class or subclass, while the remainder of the chain (s) is (are )
increased or decreased ) relative to a reference polypeptide. identical with or homologous to corresponding sequences in
Generally, variants of a particular polynucleotide or poly antibodies derived from another species or belonging to
peptide of the invention will have at least about 40 % , 45 % , another antibody class or subclass, as well as fragments of
50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 91 % , such antibodies , so long as they exhibit the desired biologi
92 % , 93 % , 94 % , 95 % , 96 % , 97 % , 98 % , 99 % but less than 40 cal activity. Chimeric antibodies of interest herein include,
100 % sequence identity to that particular reference poly but are not limited to , “ primatized ” antibodies comprising
nucleotide or polypeptide as determined by sequence align variable domain antigen -binding sequences derived from a
ment programs and parameters described herein and known non -human primate ( e.g., Old World Monkey, Ape etc.) and
to those skilled in the art . Such tools for alignment include human constant region sequences.
those of the BLAST suite (Stephen F. Altschul, Thomas L. 45 An “ antibody fragment” comprises a portion of an intact
Madden , Alejandro A. Schäffer, Jinghui Zhang, Zheng antibody, preferably the antigen binding and /or the variable
Zhang , Webb Miller, and David J. Lipman (1997 ), “ Gapped region of the intact antibody. Examples of antibody frag
BLAST and PSI-BLAST: a new generation of protein data ments include Fab, Fab', F (ab'), and Fv fragments; diabod
base search programs”, Nucleic Acids Res. 25 :3389-3402.) ies; linear antibodies ; nanobodies ; single-chain antibody
Other tools are described herein , specifically in the defini- 50 molecules and multispecific antibodies formed from anti
tion of “ Identity.” body fragments.
Default parameters in the BLAST algorithm include , for Any of the five classes of immunoglobulins, IgA , IgD ,
example, an expect threshold of 10 , Word size of 28 , IgE , IgG and IgM ,may be encoded by the mmRNA of the
Match /Mismatch Scores 1, -2 , Gap costs Linear. Any filter invention , including the heavy chains designated alpha ,
can be applied as well as a selection for species specific 55 delta , epsilon , gamma and mu, respectively. Also included
repeats, e.g., Homo sapiens. are polynucleotide sequences encoding the subclasses,
Biologics gamma and mu. Hence any of the subclasses of antibodies
The polynucleotides, primary constructs or mmRNA dis may be encoded in part or in whole and include the follow
closed herein ,may encode one or more biologics. As used ing subclasses : IgG1, IgG2, IgG3 , IgG4, IgAl and IgA2.
herein , a “ biologic ” is a polypeptide-based molecule pro- 60 According to the present invention , one ormore antibod
duced by the methods provided herein and which may be ies or fragments currently being marketed or in development
used to treat , cure, mitigate , prevent, or diagnose a serious may be encoded by the polynucleotides, primary constructs
or life-threatening disease or medical condition . Biologics , or mmRNA of the present invention . While not wishing to
according to the present invention include, but are not be bound by theory, it is believed that incorporation into the
limited to , allergenic extracts (e.g. for allergy shots and 65 primary constructs of the invention will result in improved
tests ), blood components , gene therapy products , human therapeutic efficacy due at least in part to the specificity,
tissue or cellular products used in transplantation , vaccines, purity and selectivity of the mmRNA designs.
US 10,703,789 B2
23 24
Antibodies encoded in the polynucleotides , primary con invention may contain one or more detectable labels. The
structs or mmRNA of the invention may be utilized to treat polypeptides may be partially labeled or completely labeled
conditions or diseases in many therapeutic areas such as , but throughout. The polynucleotide , primary construct or
not limited to , blood , cardiovascular, CNS, poisoning ( in mmRNA may encode the detectable label completely , par
cluding antivenoms), dermatology, endocrinology, gastroin- 5 tially or not at all. The cell-penetrating peptide may also
testinal, medical imaging, musculoskeletal, oncology, include a signal sequence . As used herein , a “ signal
immunology , respiratory, sensory and anti - infective. sequence” refers to a sequence of amino acid residues bound
In one embodiment, primary constructs or mmRNA dis at the amino terminus of a nascent protein during protein
closed herein may encode monoclonal antibodies and/or translation . The signal sequence may be used to signal the
variants thereof. Variants of antibodiesmay also include, but 10 secretion of the cell -penetrating polypeptide.
are not limited to , substitutional variants , conservative In one embodiment, the polynucleotides , primary con
amino acid substitution , insertional variants, deletional vari structs or mmRNA may also encode a fusion protein . The
ants and /or covalent derivatives. In one embodiment, the
primary construct and /or mmRNA disclosed herein may fusion protein may be created by operably linking a charged
encode an immunoglobulin Fc region. In another embodi- 15 linked protein” torefers
a therapeutic protein . As used herein , “ operably
to the therapeutic protein and the charged
ment, the primary constructs and /or mmRNA may encode a protein being connected in such a way to permit the expres
variant immunoglobulin Fc region . As a non -limiting
example , the primary constructs and /or mmRNA may sion of the complex when introduced into the cell. As used
encode an antibody having a variant immunoglobulin Fc herein , “ charged protein ” refers to a protein that carries a
region as described in U.S. Pat. No. 8,217,147 herein 20 erably positive, therapeutic
, negative orprotein
overallmayneutral electrical charge. Pref
be covalently linked to the
incorporated by reference in its entirety.
Vaccines charged protein in the formation of the fusion protein . The
The primary constructs ormmRNA disclosed herein ,may ratio of surface charge to total or surface amino acids may
encode one or more vaccines . As used herein , a “ vaccine” is be approximately 0.1, 0.2 , 0.3 , 0.4 , 0.5, 0.5 , 0.7 , 0.8 or 0.9 .
a biological preparation that improves immunity to a par- 25 The cell-penetrating polypeptide encoded by the poly
ticular disease or infectious agent. According to the present nucleotides, primary constructs or mmRNA may form a
invention , one ormore vaccines currently being marketed or complex after being translated . The complex may comprise
in development may be encoded by the polynucleotides, a charged protein linked , e.g. covalently linked , to the
primary constructs or mmRNA of the present invention . cell-penetrating polypeptide. “ Therapeutic protein ” refers to
While not wishing to be bound by theory, it is believed that 30 a protein that, when administered to a cell has a therapeutic ,
incorporation into the primary constructs or mmRNA of the diagnostic, and/ or prophylactic effect and/or elicits a desired
invention will result in improved therapeutic efficacy due at biological and /or pharmacological effect.
least in part to the specificity , purity and selectivity of the In one embodiment, the cell-penetrating polypeptide may
construct designs . comprise a first domain and a second domain . The first
Vaccines encoded in the polynucleotides, primary con- 35 domain may comprise a supercharged polypeptide. The
structs or mmRNA of the invention may be utilized to treat second domain may comprise a protein -binding partner. As
conditions or diseases in many therapeutic areas such as, but used herein , “ protein -binding partner " includes, but is not
not limited to , cardiovascular, CNS, dermatology, endocri limited to , antibodies and functional fragments thereof,
nology, oncology , immunology , respiratory, and anti -infec scaffold proteins, or peptides. The cell -penetrating polypep
tive. 40 tide may further comprise an intracellular binding partner
Therapeutic Proteins or Peptides for the protein -binding partner. The cell-penetrating poly
The primary constructs or mmRNA disclosed herein , may peptide may be capable of being secreted from a cell where
encode one or more validated or “ in testing ” therapeutic the polynucleotide, primary construct or mmRNA may be
proteins or peptides. introduced . The cell -penetrating polypeptide may also be
According to the present invention , one or more thera- 45 capable of penetrating the first cell.
peutic proteins or peptides currently being marketed or in In a further embodiment, the cell- penetrating polypeptide
development may be encoded by the polynucleotides, pri is capable of penetrating a second cell. The second cell may
mary constructs or mmRNA of the present invention . While be from the same area as the first cell , or it may be from a
not wishing to be bound by theory , it is believed that different area . The area may include , but is not limited to ,
incorporation into the primary constructs or mmRNA of the 50 tissues and organs. The second cell may also be proximal or
invention will result in improved therapeutic efficacy due at distal to the first cell.
least in part to the specificity, purity and selectivity of the In one embodiment, the polynucleotides, primary con
construct designs . structs or mmRNA may encode a cell-penetrating polypep
Therapeutic proteins and peptides encoded in the poly tide which may comprise a protein -binding partner. The
nucleotides, primary constructs or mmRNA of the invention 55 protein binding partner may include, but is not limited to , an
may be utilized to treat conditions or diseases in many antibody , a supercharged antibody or a functional fragment.
therapeutic areas such as, but not limited to , blood , cardio The polynucleotides, primary constructs ormmRNA may be
vascular, CNS , poisoning (including antivenoms) , derma introduced into the cell where a cell-penetrating polypeptide
tology , endocrinology , genetic , genitourinary, gastrointesti comprising the protein -binding partner is introduced .
nal, musculoskeletal, oncology , and immunology, 60 Secreted Proteins
respiratory, sensory and anti - infective . Human and other eukaryotic cells are subdivided by
Cell -Penetrating Polypeptides membranes into many functionally distinct compartments.
The primary constructs or mmRNA disclosed herein , may Each membrane -bounded compartment, or organelle, con
encode one or more cell-penetrating polypeptides. As used tains different proteins essential for the function of the
herein , " cell -penetrating polypeptide " or CPP refers to a 65 organelle . The cell uses " sorting signals ,” which are amino
polypeptide which may facilitate the cellular uptake of acid motifs located within the protein , to target proteins to
molecules . A cell -penetrating polypeptide of the present particular cellular organelles .
US 10,703,789 B2
25 26
One type of sorting signal, called a signal sequence, a partner , scaffold protein , and other polypeptides taught
signal peptide , or a leader sequence, directs a class of herein or known in the art. In a preferred embodiment, the
proteins to an organelle called the endoplasmic reticulum polynucleotides are primary constructs of the present inven
(ER ). tion , including mmRNA which may be suitable for direct
Proteins targeted to the ER by a signal sequence can be 5 introduction into a target cell or culture which in turn may
released into the extracellular space as a secreted protein . synthesize the encoded polypeptides.
Similarly, proteins residing on the cell membrane can also be In certain embodiments, multiple variants of a protein ,
secreted into the extracellular space by proteolytic cleavage each with different amino acid modification (s), may be
of a “ linker” holding the protein to the membrane. While not produced and tested to determine the best variant in termsof
wishing to be bound by theory, the molecules of the present 10 pharmacokinetics , stability , biocompatibility , and /or bio
invention may be used to exploit the cellular trafficking logical activity, or a biophysical property such as expression
described above. As such , in some embodiments of the level. Such a library may contain 10 , 102, 103, 104, 105 , 106,
invention , polynucleotides, primary constructs or mmRNA 10%, 108, 109, or over 10º possible variants ( including, but
are provided to express a secreted protein . The secreted not limited to , substitutions, deletions of one or more
proteins may be selected from those described herein or 15 residues, and insertion of one or more residues).
those in US Patent Publication , 20100255574 , the contents Anti-Microbial and Anti- Viral Polypeptides
of which are incorporated herein by reference in their The polynucleotides, primary constructs and mmRNA of
entirety. the present invention may be designed to encode on or more
In one embodiment, these may be used in the manufacture antimicrobial peptides (AMP) or antiviral peptides (AVP ).
of large quantities of valuable human gene products. 20 AMPs and AVPs have been isolated and described from a
Plasma Membrane Proteins wide range of animals such as, but not limited to , microor
In some embodiments of the invention , polynucleotides, ganisms, invertebrates , plants , amphibians, birds, fish , and
primary constructs or mmRNA are provided to express a mammals (Wang et al., Nucleic Acids Res. 2009 ; 37 (Data
protein of the plasma membrane . base issue):D933-7) . For example, anti-microbial polypep
Cytoplasmic or Cytoskeletal Proteins 25 tides are described in Antimicrobial Peptide Database
In some embodiments of the invention , polynucleotides, (http://aps.unmc.edu/AP/main.php; Wang et al., Nucleic
primary constructs or mmRNA are provided to express a Acids Res. 2009 ; 37 (Database issue ):D933-7), CAMP:
cytoplasmic or cytoskeletal protein . Collection of Anti-Microbial Peptides (http ://www.bicnir
Intracellular Membrane Bound Proteins rh.res.in/antimicrobial/ ); Thomas et al., Nucleic Acids Res .
In some embodiments of the invention , polynucleotides , 30 2010 ; 38 (Database issue ):D774-80 ), U.S. Pat . Nos. 5,221 ,
primary constructs or mmRNA are provided to express an 732,5,447,914 , 5,519,115,5,607,914,5,714,577,5,734,015 ,
intracellular membrane bound protein . 5,798,336 , 5,821,224 , 5,849,490 , 5,856,127 , 5,905,187 ,
Nuclear Proteins 5,994,308 , 5,998,374 , 6,107,460, 6,191,254 , 6,211,148 ,
In some embodiments of the invention , polynucleotides , 6,300,489, 6,329,504 , 6,399,370, 6,476,189, 6,478,825 ,
primary constructs or mmRNA are provided to express a 35 6,492,328 , 6,514,701, 6,573,361, 6,573,361, 6,576,755 ,
nuclear protein . 6,605,698 , 6,624,140 , 6,638,531 , 6,642,203 , 6,653,280 ,
Proteins Associated with Human Disease 6,696,238 , 6,727,066 , 6,730,659, 6,743,598 , 6,743,769,
In some embodiments of the invention , polynucleotides, 6,747,007, 6,790,833 , 6,794,490 , 6,818,407, 6,835,536 ,
primary constructs or mmRNA are provided to express a 6,835,713 , 6,838,435 , 6,872,705 , 6,875,907, 6,884,776 ,
protein associated with human disease . 40 6,887,847 , 6,906,035 , 6,911,524, 6,936,432, 7,001,924 ,
Miscellaneous Proteins 7,071,293 , 7,078,380 , 7,091,185 , 7,094,759 , 7,166,769 ,
In some embodiments of the invention , polynucleotides, 7,244,710 , 7,314,858 , and 7,582,301 , the contents of which
primary constructs or mmRNA are provided to express a are incorporated by reference in their entirety .
protein with a presently unknown therapeutic function . The anti-microbial polypeptides described herein may
Targeting Moieties 45 block cell fusion and /or viral entry by one or more envel
In some embodiments of the invention , polynucleotides, oped viruses (e.g., HIV , HCV ). For example, the anti
primary constructs or mmRNA are provided to express a microbial polypeptide can comprise or consist of a synthetic
targeting moiety. These include a protein -binding partner or peptide corresponding to a region , e.g. , a consecutive
a receptor on the surface of the cell,which functions to target sequence of at least about 5 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , 45 ,
the cell to a specific tissue space or to interact with a specific 50 50, 55 , or 60 amino acids of the transmembrane subunit of
moiety, either in vivo or in vitro . Suitable protein -binding a viral envelope protein , e.g., HIV - 1 gp120 or gp41. The
partners include, but are not limited to , antibodies and amino acid and nucleotide sequences of HIV -1 gp120 or
functional fragments thereof, scaffold proteins , or peptides . gp41 are described in , e.g., Kuiken et al., ( 2008 ). “ HIV
Additionally, polynucleotide , primary construct or mmRNA Sequence Compendium ,” Los Alamos National Laboratory .
can be employed to direct the synthesis and extracellular 55 In some embodiments, the anti -microbial polypeptide
localization of lipids, carbohydrates , or other biological may have at least about 75 % , 80 % , 85 % , 90 % , 95 % , 100 %
moieties or biomolecules. sequence homology to the corresponding viral protein
Polypeptide Libraries sequence . In some embodiments , the anti-microbial poly
In one embodiment, the polynucleotides , primary con peptide may have at least about 75 % , 80 % , 85 % , 90 % , 95 % ,
structs or mmRNA may be used to produce polypeptide 60 or 100 % sequence homology to the corresponding viral
libraries . These libraries may arise from the production of a protein sequence .
population of polynucleotides , primary constructs or In other embodiments , the anti-microbial polypeptide
mmRNA , each containing various structural or chemical may comprise or consist of a synthetic peptide correspond
modification designs. In this embodiment, a population of ing to a region , e.g. , a consecutive sequence of at least about
polynucleotides, primary constructs or mmRNA may com- 65 5 , 10 , 15 , 20 , 25, 30 , 35, 40 , 45 , 50 , 55 , or 60 amino acids
prise a plurality of encoded polypeptides , including but not of the binding domain of a capsid binding protein . In some
limited to , an antibody or antibody fragment, protein binding embodiments , the anti -microbial polypeptide may have at
US 10,703,789 B2
27 28
least about 75 % , 80 % , 85 % , 90 % , 95 % , or 100 % sequence Gram -positive bacterium . In some embodiments , the anti
homology to the corresponding sequence of the capsid microbial polypeptide may be cytostatic to a virus, fungus,
binding protein . protozoan , parasite , prion , or a combination thereof. In some
The anti -microbial polypeptides described herein may embodiments , the anti-microbial polypeptide may be cyto
block protease dimerization and inhibit cleavage of viral 5 toxic to a virus, fungus, protozoan , parasite, prion , or a
proproteins (e.g. , HIV Gag -pol processing ) into functional combination thereof. In certain embodiments, the anti-mi
proteins thereby preventing release of one or more envel crobial polypeptide may be cytostatic and cytotoxic to a
oped viruses (e.g., HIV , HCV ). In some embodiments , the virus, fungus, protozoan , parasite, prion , or a combination
anti-microbial polypeptide may have at least about 75 % , thereof. In some embodiments , the anti-microbial polypep
80corresponding
% , 85 % , 90 %viral
, 95protein
% , 100sequence
% sequence . homology to the 10 tide may be cytotoxic to a tumor or cancer cell (e.g.,a human
In other embodiments , the anti -microbial polypeptide can tumor and /or cancer cell). In some embodiments, the anti
comprise or consist of a synthetic peptide corresponding to microbial cell ( e.g. ,
polypeptide may be cytostatic to a tumor or cancer
a human tumor and /or cancer cell). In certain
a region , e.g., a consecutive sequence of at least about 5, 10 , embodiments , the anti-microbial polypeptide may be cyto
15 , 20 , 25 , 30 , 35 , 40 , 45 , 50 , 55 , or 60 amino acids of the 15
binding domain of a protease binding protein . In some toxic and cytostatic to a tumor or cancer cell ( e.g., a human
embodiments, the anti -microbial polypeptide may have at tumor or cancer cell ). In some embodiments, the anti
least about 75 % , 80 % , 85 % , 90 % , 95 % , 100 % sequence microbial polypeptide (e.g., an anti -bacterial polypeptide )
homology to the corresponding sequence of the protease may be a secreted polypeptide .
binding protein . 20 In some embodiments , the anti-microbial polypeptide
The anti -microbial polypeptides described herein can comprises or consists of a defensin . Exemplary defensins
include an in vitro -evolved polypeptide directed against a include , but are not limited to , a -defensins (e.g., neutrophil
viral pathogen . defensin 1, defensin alpha 1, neutrophil defensin 3 , neutro
Anti-Microbial Polypeptides phil defensin 4 , defensin 5 , defensin 6 ), B -defensins (e.g.,
Anti-microbial polypeptides (AMPs) are small peptides 25 beta -defensin 1, beta -defensin 2, beta -defensin 103 , beta
of variable length , sequence and structure with broad spec defensin 107, beta - defensin 110 , beta - defensin 136 ), and
trum activity against a wide range of microorganisms 0 - defensins . In other embodiments, the anti -microbial poly
including , but not limited to , bacteria , viruses, fungi, pro peptide comprises or consists of a cathelicidin ( e.g.,
tozoa, parasites, prions, and tumor/cancer cells . (See , e.g., hCAP18 ) .
Zaiou , J Mol Med , 2007 ; 85 :317 ; herein incorporated by 30 Anti- Viral Polypeptides
reference in its entirety ). It has been shown that AMPs have Anti- viral polypeptides (AVPs ) are small peptides of
broad -spectrum of rapid onset of killing activities, with variable length , sequence and structure with broad spectrum
potentially low levels of induced resistance and concomitant activity against a wide range of viruses . See, e.g. , Zaiou , J
broad anti -inflammatory effects. MolMed , 2007 ; 85 :317 . It has been shown that AVPs have
In some embodiments , the anti -microbial polypeptide 35 a broad -spectrum of rapid onset of killing activities, with
(e.g., an anti -bacterial polypeptide ) may be under 10 kDa, potentially low levels of induced resistance and concomitant
e.g., under 8 kDa, 6 kDa, 4 kDa, 2 kDa, or 1 kDa. In some broad anti-inflammatory effects. In some embodiments , the
embodiments, the anti -microbial polypeptide (e.g., an anti anti- viral polypeptide is under 10 kDa, e.g. , under 8 kDa, 6
bacterial polypeptide ) consists of from about 6 to about 100 kDa, 4 kDa, 2 kDa, or 1 kDa. In some embodiments, the
amino acids, e.g., from about 6 to about 75 amino acids , 40 anti-viral polypeptide comprises or consists of from about 6
about 6 to about 50 amino acids , about 6 to about 25 amino to about 100 amino acids , e.g., from about 6 to about 75
acids, about 25 to about 100 amino acids, about 50 to about amino acids, about 6 to about 50 amino acids, about 6 to
100 amino acids, or about 75 to about 100 amino acids. In about 25 amino acids, about 25 to about 100 amino acids ,
certain embodiments , the anti-microbial polypeptide (e.g., about 50 to about 100 amino acids, or about 75 to about 100
an anti-bacterial polypeptide) may consist of from about 15 45 amino acids. In certain embodiments , the anti -viral poly
to about 45 amino acids. In some embodiments , the anti peptide comprises or consists of from about 15 to about 45
microbial polypeptide (e.g., an anti -bacterial polypeptide ) is amino acids . In some embodiments , the anti-viral polypep
substantially cationic . tide is substantially cationic . In some embodiments, the
In some embodiments , the anti-microbial polypeptide anti -viral polypeptide is substantially amphipathic . In cer
(e.g., an anti -bacterial polypeptide ) may be substantially 50 tain embodiments , the anti-viral polypeptide is substantially
amphipathic . In certain embodiments, the anti -microbial cationic and amphipathic . In some embodiments , the anti
polypeptide (e.g., an anti -bacterial polypeptide) may be viral polypeptide is cytostatic to a virus. In some embodi
substantially cationic and amphipathic. In some embodi ments , the anti-viral polypeptide is cytotoxic to a virus. In
ments , the anti-microbial polypeptide (e.g., an anti -bacterial some embodiments , the anti-viral polypeptide is cytostatic
polypeptide) may be cytostatic to a Gram -positive bacte- 55 and cytotoxic to a virus. In some embodiments , the anti -viral
rium . In some embodiments, the anti-microbial polypeptide polypeptide is cytostatic to a bacterium , fungus, protozoan ,
(e.g., an anti-bacterial polypeptide) may be cytotoxic to a parasite , prion , or a combination thereof. In some embodi
Gram -positive bacterium . In some embodiments , the anti ments , the anti- viral polypeptide is cytotoxic to a bacterium ,
microbial polypeptide ( e.g., an anti -bacterial polypeptide ) fungus, protozoan , parasite , prion or a combination thereof.
may be cytostatic and cytotoxic to a Gram -positive bacte- 60 In certain embodiments , the anti -viral polypeptide is cyto
rium . In some embodiments , the anti-microbial polypeptide static and cytotoxic to a bacterium , fungus, protozoan ,
(e.g., an anti-bacterial polypeptide) may be cytostatic to a parasite, prion , or a combination thereof. In some embodi
Gram -negative bacterium . In some embodiments , the anti ments, the anti -viral polypeptide is cytotoxic to a tumor or
microbial polypeptide (e.g. , an anti-bacterial polypeptide ) cancer cell (e.g., a human cancer cell ). In some embodi
may be cytotoxic to a Gram -negative bacterium . In some 65 ments, the anti-viral polypeptide is cytostatic to a tumor or
embodiments, the anti-microbial polypeptide (e.g., an anti cancer cell (e.g., a human cancer cell ). In certain embodi
bacterial polypeptide ) may be cytostatic and cytotoxic to a ments, the anti- viral polypeptide is cytotoxic and cytostatic
US 10,703,789 B2
29 30
to a tumor or cancer cell ( e.g., a human cancer cell ). In some pyrimidines, allopurinol, azathioprine, capecitabine , cyto
embodiments, the anti -viral polypeptide is a secreted poly sine arabinoside, fluorouracil , mercaptopurine, 6 -thiogua
peptide. nine, acyclovir , ara -adenosine, ribavirin , 7 -deaza -adenosine ,
Cytotoxic Nucleosides 7 -deaza - guanosine, 6 -aza -uracil , 6 -aza -cytidine, thymidine
In one embodiment, the polynucleotides, primary con- 5 ribonucleotide, 5 -bromodeoxyuridine, 2 -chloro -purine, and
structs or mmRNA of the present invention may incorporate inosine, or combinations thereof.
one or more cytotoxic nucleosides. For example , cytotoxic Flanking Regions: Untranslated Regions (UTRs)
nucleosides may be incorporated into polynucleotides , pri Untranslated regions (UTRs) of a gene are transcribed but
mary constructs or mmRNA such as bifunctional modified not translated . The 5'UTR starts at the transcription start site
RNAs or mRNAs. Cytotoxic nucleoside anticancer agents 10 and continues to the start codon but does not include the start
include, but are not limited to , adenosine arabinoside, cyt codon ; whereas, the 3'UTR starts immediately following the
arabine, cytosine arabinoside , 5 - fluorouracil , fludarabine , stop codon and continues until the transcriptional termina
floxuridine, FTORAFUR® (a combination of tegafur and tion signal . There is growing body of evidence about the
uracil ), tegafur ((RS) -5 - fluoro - 1-(tetrahydrofuran - 2 -yl)py regulatory roles played by the UTRs in terms of stability of
rimidine - 2,4 ( 1H ,3H ) -dione), and 6 -mercaptopurine. 15 the nucleic acid molecule and translation . The regulatory
A number of cytotoxic nucleoside analogues are in clini features of a UTR can be incorporated into the polynucle
cal use , or have been the subject of clinical trials, as otides, primary constructs and /or mmRNA of the present
anticancer agents . Examples of such analogues include , but invention to enhance the stability of the molecule . The
are not limited to , cytarabine , gemcitabine , troxacitabine, specific features can also be incorporated to ensure con
decitabine , tezacitabine, 2'-deoxy - 2'-methylidenecytidine 20 trolled down- regulation of the transcript in case they are
( DMDC ) , cladribine , clofarabine, 5 - azacytidine , 4'- thio -ara misdirected to undesired organs sites.
cytidine , cyclopentenylcytosine and 1-(2 -C -cyano - 2-deoxy 5 ' UTR and Translation Initiation
beta- D - arabino -pentofuranosyl) -cytosine. Another example Natural 5'UTRs bear features which play roles in for
of such a compound is fludarabine phosphate. These com translation initiation . They harbor signatures like Kozak
pounds may be administered systemically and may have side 25 sequences which are commonly known to be involved in the
effects which are typical of cytotoxic agents such as, but not process by which the ribosome initiates translation ofmany
limited to , little or no specificity for tumor cells over genes. Kozak sequences have the consensus CCR (A / G )
proliferating normal cells . CCAUGG , where R is a purine (adenine or guanine ) three
A number of prodrugs of cytotoxic nucleoside analogues bases upstream of the start codon ( AUG ), which is followed
are also reported in the art . Examples include, but are not 30 by another ‘G ’. 5'UTR also have been known to form
limited to , N4- behenoyl-1- beta -D - arabinofuranosylcyto secondary structures which are involved in elongation factor
sine, N4 -octadecyl- 1-beta -D -arabinofuranosylcytosine , binding
N4-palmitoyl- 1-(2 - C -cyano - 2 -deoxy -beta - D - arabino-pento By engineering the features typically found in abundantly
furanosyl) cytosine, and P -4055 ( cytarabine 5'-elaidic acid expressed genes of specific target organs, one can enhance
ester). In general, these prodrugs may be converted into the 35 the stability and protein production of the polynucleotides,
active drugs mainly in the liver and systemic circulation and primary constructs or mmRNA of the invention . For
display little or no selective release of active drug in the example , introduction of 5 ' UTR of liver-expressed mRNA ,
tumor tissue. For example, capecitabine, a prodrug of 5 -de such as albumin , serum amyloid A , Apolipoprotein A / B /E ,
oxy -5 - fluorocytidine ( and eventually of 5 -fluorouracil), is transferrin , alpha fetoprotein , erythropoietin , or Factor VIII,
metabolized both in the liver and in the tumor tissue . A series 40 could be used to enhance expression of a nucleic acid
of capecitabine analogues containing " an easily hydroly molecule , such as a mmRNA , in hepatic cell lines or liver.
sable radical under physiological conditions” has been Likewise , use of 5 'UTR from other tissue-specific mRNA to
claimed by Fujiu et al. (U.S. Pat. No. 4,966,891) and is improve expression in that tissue is possible for muscle
herein incorporated by reference . The series described by (MyoD , Myosin , Myoglobin , Myogenin , Herculin ), for
Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy- 45 endothelial cells ( Tie -1 , CD36 ), for myeloid cells ( C / EBP,
5 - fluorocytidine and the implication that these compounds AML1, G -CSF , GM -CSF , CD11b , MSR , Fr- 1, i-NOS ), for
will be activated by hydrolysis under normal physiological leukocytes (CD45 , CD18 ), for adipose tissue (CD36 ,
conditions to provide 5'-deoxy - 5- fluorocytidine . GLUT4, ACRP30 , adiponectin ) and for lung epithelial cells
A series of cytarabine N4- carbamates has been by (SP - A / B /C / D )
reported by Fadl et al (Pharmazie . 1995 , 50 , 382-7 , herein 50 Other non -UTR sequencesmay be incorporated into the 5'
incorporated by reference ) in which compounds were ( or 3' UTR ) UTRs. For example, introns or portions of
designed to convert into cytarabine in the liver and plasma. introns sequences may be incorporated into the flanking
WO 2004/041203, herein incorporated by reference , dis regions of the polynucleotides , primary constructs or
closes prodrugs of gemcitabine ,where some of the prodrugs mmRNA of the invention . Incorporation of intronic
are N4-carbamates . These compounds were designed to 55 sequences may increase protein production as well as
overcome the gastrointestinal toxicity of gemcitabine and mRNA levels .
were intended to provide gemcitabine by hydrolytic release 3 ' UTR and the AU Rich Elements
in the liver and plasma after absorption of the intact prodrug 3 ' UTRs are known to have stretches of Adenosines and
from the gastrointestinal tract. Nomura et al (Bioorg Med . Uridines embedded in them . These AU rich signatures are
Chem . 2003, 11 , 2453-61, herein incorporated by reference ) 60 particularly prevalent in genes with high rates of turnover.
have described acetal derivatives of 1-(3 -C -ethynyl-ß - D Based on their sequence features and functional properties ,
ribo -pentofaranosyl) cytosine which , on bioreduction , pro the AU rich elements (ARES ) can be separated into three
duced an intermediate that required further hydrolysis under classes (Chen et al, 1995): Class I AREs contain several
acidic
pound .
conditions to produce a cytotoxic nucleoside com dispersed copies of an AUUUA motif within U -rich regions.
65 C -Myc and MyoD contain class I AREs . Class II ARES
Cytotoxic nucleotides which may be chemotherapeutic possess two or more overlapping UUAUUUA(U / A )(U / A )
also include, but are not limited to , pyrazolo [3,4 -D ] nonamers. Molecules containing this type of AREs include
US 10,703,789 B2
31 32
GM -CSF and TNF - a . Class III ARES are less well defined . microRNA , microRNA target regions, and their expression
These U rich regions do not contain an AUUUAmotif. c -Jun patterns and role in biology have been reported (Bonauer et
and Myogenin are two well-studied examples of this class. al., Curr Drug Targets 2010 11:943-949 ; Anand and Cheresh
Most proteins binding to the AREs are known to destabilize Curr Opin Hematol 2011 18 : 171-176 ; Contreras and Rao
the messenger, whereas members of the ELAV family , most 5 Leukemia 2012 26 :404-413 (2011 Dec. 20. doi: 10.1038 /
notably HuR , have been documented to increase the stability leu.2011.356 ); Bartel Cell 2009 136 :215-233 ; Landgraf et
of mRNA . Hur binds to AREs of all the three classes . al, Cell , 2007 129: 1401-1414 ; each of which is herein
Engineering the HuR specific binding sites into the 3' UTR incorporated by reference in its entirety ).
of nucleic acid molecules will lead to HUR binding and thus , For example , if the nucleic acid molecule is an mRNA and
stabilization of the message in vivo . 10 is not intended to be delivered to the liver but ends up there ,
Introduction , removal or modification of 3 'UTR AU rich then miR - 122, a microRNA abundant in liver, can inhibit the
elements (ARES) can be used to modulate the stability of expression of the gene of interest if one or multiple target
polynucleotides, primary constructs or mmRNA of the sites of miR - 122 are engineered into the 3' UTR of the
invention . When engineering specific polynucleotides, pri polynucleotides, primary constructs or mmRNA . Introduc
mary constructs or mmRNA , one or more copies of an ARE 15 tion of one or multiple binding sites for different microRNA
can be introduced to make polynucleotides , primary con can be engineered to further decrease the longevity, stability,
structs or mmRNA of the invention less stable and thereby and protein translation of a polynucleotides , primary con
curtail translation and decrease production of the resultant structs or mmRNA .
protein . Likewise , AREs can be identified and removed or As used herein , the term “ microRNA site ” refers to a
mutated to increase the intracellular stability and thus 20 microRNA target site or a microRNA recognition site , or any
increase translation and production of the resultant protein . nucleotide sequence to which a microRNA binds or associ
Transfection experiments can be conducted in relevant cell ates. It should be understood that “ binding ” may follow
lines, using polynucleotides, primary constructs or mmRNA traditional Watson - Crick hybridization rules or may reflect
of the invention and protein production can be assayed at any stable association of the microRNA with the target
various time points post - transfection . For example , cells can 25 sequence at or adjacent to the microRNA site .
be transfected with different ARE -engineering molecules Conversely, for the purposes of the polynucleotides, pri
and by using an ELISA kit to the relevant protein and mary constructs or mmRNA of the present invention , micro
assaying protein produced at 6 hour, 12 hour, 24 hour, 48 RNA binding sites can be engineered out of ( i.e. removed
hour, and 7 days post-transfection . from ) sequences in which they naturally occur in order to
Incorporating microRNA Binding Sites 30 increase protein expression in specific tissues. For example ,
microRNAs (or miRNA ) are 19-25 nucleotide long non miR - 122 binding sites may be removed to improve protein
coding RNAs that bind to the 3'UTR of nucleic acid mol expression in the liver. Regulation of expression in multiple
ecules and down-regulate gene expression either by reduc tissues can be accomplished through introduction or removal
ing nucleic acid molecule stability or by inhibiting or one or several microRNA binding sites.
translation . The polynucleotides , primary constructs or 35 Examples of tissues where microRNA are known to
mmRNA of the invention may comprise one ormore micro regulate mRNA, and thereby protein expression , include ,
RNA target sequences,microRNA sequences, ormicroRNA but are not limited to , liver (miR - 122 ), muscle (miR - 133 ,
seeds. Such sequences may correspond to any known micro miR -206 , miR - 208 ), endothelial cells (miR -17-92 , miR
RNA such as those taught in US Publication US2005 / 126 ), myeloid cells (miR - 142-3p , miR - 142-5p , miR - 16 ,
0261218 and US Publication US2005 /0059005 , the contents 40 miR - 21 , miR - 223 , miR -24 , miR -27 ), adipose tissue ( let -7 ,
of which are incorporated herein by reference in their miR -30c), heart (miR - 1d , miR - 149), kidney (miR - 192,miR
entirety. 194 , miR -204 ), and lung epithelial cells (let-7 , miR - 133 ,
A microRNA sequence comprises a " seed ” region , i.e., a miR - 126 ). MicroRNA can also regulate complex biological
sequence in the region of positions 2-8 of the mature processes such as angiogenesis (miR -132 ) ( Anand and
microRNA , which sequence has perfect Watson -Crick 45 Cheresh Curr Opin Hematol 2011 18 : 171-176 ; herein incor
complementarity to the miRNA target sequence . A micro porated by reference in its entirety ). In the polynucleotides,
RNA seed may comprise positions 2-8 or 2-7 of the mature primary constructs or mmRNA of the present invention,
microRNA . In some embodiments, a microRNA seed may binding sites for microRNAs that are involved in such
comprise 7 nucleotides ( e.g., nucleotides 2-8 of the mature processes may be removed or introduced , in order to tailor
microRNA ), wherein the seed -complementary site in the 50 the expression of the polynucleotides, primary constructs or
corresponding miRNA target is flanked by an adenine (A ) mmRNA expression to biologically relevant cell types or to
opposed to microRNA position 1. In some embodiments, a the context of relevant biological processes. A listing of
microRNA seed may comprise 6 nucleotides ( e.g., nucleo MicroRNA , miR sequences and miR binding sites is listed
tides 2-7 of the mature microRNA ), wherein the seed in Table 9 of U.S. Provisional Application No.61/753,661
complementary site in the corresponding miRNA target is 55 filed Jan. 17, 2013 , in Table 9 of U.S. Provisional Applica
flanked by an adenine (A ) opposed to microRNA position 1. tion No. 61/754,159 filed Jan. 18 , 2013 , and in Table 7 of
See for example , Grimson A , Farh K K , Johnston W K , U.S. Provisional Application No. 61/758,921 filed Jan. 31,
Garrett - Engele P, Lim L P , Bartel D P ; Mol Cell. 2007 Jul. 2013 , each of which are herein incorporated by reference in
6 ; 27 ( 1 ): 91-105; each of which is herein incorporated by their entireties .
reference in their entirety . The bases of the microRNA seed 60 Examples of use of microRNA to drive tissue or disease
have complete complementarity with the target sequence . specific gene expression are listed (Getner and Naldini,
By engineering microRNA target sequences into the 3'UTR Tissue Antigens. 2012 , 80 :393-403 ; herein incorporated by
of polynucleotides, primary constructs or mmRNA of the reference in its entirety ). In addition ,microRNA seed sites
invention one can target the molecule for degradation or can be incorporated into mRNA to decrease expression in
reduced translation , provided the microRNA in question is 65 certain cells which results in a biological improvement. An
available. This process will reduce the hazard of off target example of this is incorporation of miR - 142 sites into a
effects upon nucleic acid molecule delivery . Identification of UGT1A1-expressing lentiviral vector. The presence of miR
US 10,703,789 B2
33 34
142 seed sites reduced expression in hematopoietic cells , in the 5'-ppp -5' cap . Additionalmodified guanosine nucleo
and as a consequence reduced expression in antigen -presen tides may be used such as a -methyl- phosphonate and
tating cells , leading to the absence of an immune response seleno -phosphate nucleotides.
against the virally expressed UGT1A1 (Schmitt et al., Gas Additional modifications include , but are not limited to ,
troenterology 2010 ; 139: 999-1007; Gonzalez - Asequinolaza 5 2'-O -methylation of the ribose sugars of 5'-terminal and/or
et al. Gastroenterology 2010 , 139:726-729; both herein 5 - anteterminal nucleotides of the mRNA (as mentioned
incorporated by reference in its entirety ). Incorporation of above ) on the 2'-hydroxyl group of the sugar ring . Multiple
miR -142 sites into modified mRNA could not only reduce distinct 5 '-cap structures can be used to generate the 5 '-cap
expression of the encoded protein in hematopoietic cells ,but of a nucleic acid molecule, such as an mRNA molecule .
could also reduce or abolish immune responses to the 10 Cap analogs , which herein are also referred to as synthetic
mRNA - encoded protein . Incorporation of miR -142 seed cap analogs, chemical caps, chemical cap analogs, or struc
sites (one ormultiple ) into mRNA would be important in the tural or functional cap analogs , differ from natural (i.e.
case of treatment of patients with complete protein deficien endogenous, wild -type or physiological) 5'-caps in their
cies (UGT1A1 type I, LDLR - deficient patients, CRIM chemical structure , while retaining cap function . Cap ana
negative Pompe patients, etc.). 15 logs may be chemically i.e. non -enzymatically ) or enzy
Lastly , through an understanding of the expression pat matically synthesized and /or linked to a nucleic acid mol
terns of microRNA in different cell types, polynucleotides, ecule .
primary constructs or mmRNA can be engineered formore For example , the Anti -Reverse Cap Analog ( ARCA ) cap
targeted expression in specific cell types or only under contains two guanines linked by a 5'- 5'- triphosphate group ,
specific biological conditions. Through introduction of tis- 20 wherein one guanine contains an N7methyl group as well as
sue -specific microRNA binding sites , polynucleotides, pri a 3'-O -methyl group ( i.e., N7,3'-O -dimethyl-guanosine - 5'
mary constructs ormmRNA could be designed that would triphosphate -5 -guanosine (m’G - 3'mppp -G ; which may
be optimal for protein expression in a tissue or in the context equivaliently be designated 3 ' O -Me-m7G (5 )ppp ( 5 )G ). The
of a biological condition. 3-0 atom of the other, unmodified , guanine becomes linked
Transfection experiments can be conducted in relevant 25 to the 5'-terminal nucleotide of the capped nucleic acid
cell lines, using engineered polynucleotides, primary con molecule (e.g. an mRNA or mmRNA ). The N7- and 3'-0
structs or mmRNA and protein production can be assayed at methlyated guanine provides the terminal moiety of the
various time points post-transfection . For example , cells can capped nucleic acid molecule (e.g. mRNA or mmRNA ).
be transfected with different microRNA binding site- engi Another exemplary cap is mCAP, which is similar to
neering polynucleotides, primary constructs or mmRNA and 30 ARCA but has a 2'-O -methyl group on guanosine ( i.e.,
by using an ELISA kit to the relevant protein and assaying N7,2-0 - dimethyl- guanosine-5 '-triphosphate -5 '- guanosine,
protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 m °Gm -ppp-G ).
hour and 7 days post- transfection . In vivo experiments can While cap analogs allow for the concomitant capping of
also be conducted using microRNA -binding site -engineered a nucleic acid molecule in an in vitro transcription reaction ,
molecules to examine changes in tissue -specific expression 35 up to 20 % of transcripts can remain uncapped . This , as well
of formulated polynucleotides, primary constructs or as the structural differences of a cap analog from an endog
mmRNA . enous 5 '-cap structures of nucleic acids produced by the
5 ' Capping endogenous, cellular transcription machinery, may lead to
The 5 ' cap structure of an mRNA is involved in nuclear reduced translational competency and reduced cellular sta
export, increasing mRNA stability and binds the mRNA Cap 40 bility.
Binding Protein (CBP ), which is responsible for mRNA Polynucleotides, primary constructs and mmRNA of the
stability in the cell and translation competency through theinvention may also be capped post-transcriptionally , using
association of CBP with poly ( A ) binding protein to form the
enzymes, in order to generate more authentic 5 '-cap struc
mature cyclic mRNA species. The cap further assists the tures. As used herein , the phrase “ more authentic ” refers to
removal of 5 ' proximal introns removal during mRNA 45 a feature that closely mirrors or mimics, either structurally
splicing. or functionally, an endogenous or wild type feature . That is ,
Endogenous mRNA molecules may be 5'- end capped a “more authentic” feature is better representative of an
generating a 5'-ppp -5'-triphosphate linkage between a ter endogenous, wild -type, natural or physiological cellular
minal guanosine cap residue and the 5 '-terminal transcribed function and/or structure as compared to synthetic features
sense nucleotide of the mRNA molecule. This 5'- guanylate 50 or analogs, etc., of the prior art, or which outperforms the
cap may then be methylated to generate an N7-methyl corresponding endogenous ,wild -type, natural or physiologi
guanylate residue. The ribose sugars of the terminal and/or cal feature in one or more respects. Non -limiting examples
anteterminal transcribed nucleotides of the 5 ' end of the of more authentic 5'cap structures of the present invention
mRNA may optionally also be 2 - O -methylated . 5'-decap are those which , among other things, have enhanced binding
ping through hydrolysis and cleavage of the guanylate cap 55 of cap binding proteins, increased half life, reduced suscep
structure may target a nucleic acid molecule , such as an tibility to 5' endonucleases and /or reduced 5'decapping, as
mRNA molecule , for degradation . compared to synthetic 5'cap structures known in the art (or
Modifications to the polynucleotides, primary constructs, to a wild -type, natural or physiological 5'cap structure ). For
and mmRNA of the present invention may generate a example , recombinant Vaccinia Virus Capping Enzyme and
non -hydrolyzable cap structure preventing decapping and 60 recombinant 2'-O -methyltransferase enzyme can create a
thus increasing mRNA half - life. Because cap structure canonical 5'-5'-triphosphate linkage between the 5'-terminal
hydrolysis requires cleavage of 5'-ppp -5 ' phosphorodiester nucleotide of an mRNA and a guanine cap nucleotide
linkages, modified nucleotides may be used during the wherein the cap guanine contains an N7methylation and the
capping reaction . For example , a Vaccinia Capping Enzyme 5'- terminal nucleotide of the mRNA contains a 2 -O -methyl.
from New England Biolabs (Ipswich , Mass.) may be used 65 Such a structure is termed the Capl structure . This cap
with a - thio - guanosine nucleotides according to the manu results in a higher translational-competency and cellular
facturer's instructions to create a phosphorothioate linkage stability and a reduced activation of cellular pro -inflamma
US 10,703,789 B2
35 36
tory cytokines, as compared , e.g., to other 5'cap analog Generally , the length of a poly - A tail of the present
structures known in the art. Cap structures include, but are invention is greater than 30 nucleotides in length . In another
not limited to, 7mG (5')ppp (5')N ,pN2p (cap 0 ), 7mG (5')ppp embodiment, the poly - A tail is greater than 35 nucleotides in
(5')NlmpNp ( cap 1), and 7mG (5')-ppp (5')NlmpN2mp ( cap length ( e.g., at least or greater than about 35 , 40 , 45 , 50 , 55 ,
2 ). 5 60 , 70 , 80 , 90 , 100, 120 , 140 , 160, 180 , 200 , 250 , 300 , 350 ,
Because the polynucleotides, primary constructs or 400, 450 , 500, 600 , 700 , 800 , 900, 1,000 , 1,100, 1,200 ,
mmRNA may be capped post- transcriptionally , and because 2,5001,300 , 1,400 , 1,500 , 1,600 , 1,700 , 1,800 , 1,900 , 2,000 ,
, and 3,000 nucleotides ). In some embodiments, the
this process is more efficient, nearly 100 % of the polynucle polynucleotide
otides, primary constructs or mmRNA may be capped . This , primary construct, or mmRNA includes
is in contrast to ~ 80 % when a cap analog is linked to an 10 50from, fromabout
30
30 to about 3,000 nucleotides ( e.g., from 30 to
to 100 , from 30 to 250 , from 30 to 500, from 30
mRNA in the course of an in vitro transcription reaction .
According to the present invention , 5 ' terminal caps may tofrom75030, from 30 to 1,000 , from 30 to 1,500, from 30 to 2,000 ,
include endogenous caps or cap analogs. According to the to 500, from 50 to, from
to 2,500 50 to 100 , from 50 to 250 , from 50
750 , from 50 to 1,000 , from 50 to 1,500 ,
present invention , a 5'terminalcap may comprise a guanine 15 from 50 to 2,000 , from 50 to 2,500, from 50 to 3,000 , from
analog . Useful guanine analogs include , but are not limited 100 to 500, from 100 to 750 , from 100 to 1,000 , from 100
to , inosine, N1-methyl-guanosine , 2'fluoro - guanosine, to 1,500, from 100 to 2,000 , from 100 to 2,500 , from 100 to
7 -deaza- guanosine, 8 -oxo -guanosine, 2 -amino - guanosine, 3,000 , from 500 to 750 , from 500 to 1,000 , from 500 to
LNA -guanosine, and 2 -azido -guanosine. 1,500 , from 500 to 2,000 , from 500 to 2,500 , from 500 to
Viral Sequences 20 3,000 , from 1,000 to 1,500 , from 1,000 to 2,000 , from 1,000
Additional viral sequences such as,but not limited to , the to 2,500 , from 1,000 to 3,000 , from 1,500 to 2,000 , from
translation enhancer sequence of the barley yellow dwarf 1,500 to 2,500 , from 1,500 to 3,000, from 2,000 to 3,000 ,
virus (BYDV -PAV ) , the Jaagsiekte sheep retrovirus ( JSRV ) from 2,000 to 2,500 , and from 2,500 to 3,000 ).
and/or the Enzootic nasal tumor virus (See e.g., International In one embodiment, the poly- A tail is designed relative to
Pub . No.WO2012129648 ; herein incorporated by reference 25 the length of the overall polynucleotides, primary constructs
in its entirety ) can be engineered and inserted in the 3'UTR or mmRNA . This design may be based on the length of the
of the polynucleotides, primary constructs or mmRNA of the coding region , the length of a particular feature or region
invention and can stimulate the translation of the construct ( such as the first or flanking regions), or based on the length
in vitro and in vivo . Transfection experiments can be con of the ultimate product expressed from the polynucleotides,
ducted in relevantcell lines at and protein production can be 30 primary constructs or mmRNA .
assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7 In this context the poly - A tail may be 10 , 20 , 30 , 40 , 50 ,
post-transfection . 60, 70 , 80 , 90 , or 100 % greater in length than the poly
IRES Sequences nucled es, primary constructs or mmRNA or feature
Further, provided are polynucleotides, primary constructs thereof. The poly - A tail may also be designed as a fraction
or mmRNA which may contain an internal ribosome entry 35 of polynucleotides, primary constructs or mmRNA to which
site (IRES). First identified as a feature Picorna virus RNA , it belongs. In this context, the poly - A tail may be 10 , 20 , 30 ,
IRES plays an important role in initiating protein synthesis 40, 50 , 60, 70 , 80 , or 90 % or more of the total length of the
in absence of the 5' cap structure. An IRES may act as the construct or the total length of the construct minus the
sole ribosome binding site, or may serve as one of multiple poly - A tail. Further, engineered binding sites and conjuga
ribosome binding sites of an mRNA . Polynucleotides, pri- 40 tion of polynucleotides, primary constructs or mmRNA for
mary constructs or mmRNA containing more than one Poly - A binding protein may enhance expression .
functional ribosome binding site may encode several pep Additionally, multiple distinct polynucleotides, primary
tides or polypeptides that are translated independently by the constructs or mmRNA may be linked together to the PABP
ribosomes (“ multicistronic nucleic acid molecules” ). When ( Poly - A binding protein ) through the 3'-end using modified
polynucleotides, primary constructs or mmRNA are pro- 45 nucleotides at the 3'-terminus of the poly - A tail. Transfection
vided with an IRES , further optionally provided is a second experiments can be conducted in relevant cell lines at and
translatable region. Examples of IRES sequences that can be protein production can be assayed by ELISA at 12 hr, 24 hr,
used according to the invention include without limitation , 48 hr, 72 hr and day 7 post- transfection .
those from picornaviruses (e.g. FMDV ), pest viruses In one embodiment, the polynucleotide primary con
(CFFV ), polio viruses (PV ), encephalomyocarditis viruses 50 structs of the present invention are designed to include a
(ECMV), foot-and-mouth disease viruses (FMDV ), hepatitis polyA -G Quartet. The G - quartet is a cyclic hydrogen bonded
C viruses (HCV ), classical swine fever viruses (CSFV ), array of four guanine nucleotides that can be formed by
murine leukemia virus (MLV ), simian immune deficiency G -rich sequences in both DNA and RNA. In this embodi
viruses (SIV ) or cricket paralysis viruses (CrPV ). the G -quartet is incorporated at the end of the poly - A
Poly - A Tails 55
tail. The resultantmmRNA construct is assayed for stability ,
During RNA processing , a long chain of adenine nucleo protein production and other parameters including half-life
tides (poly - A tail) may be added to a polynucleotide such as at various time points. It has been discovered that the
an mRNA molecules in order to increase stability. Immedi polyA -G quartet results in protein production equivalent to
ately after transcription , the 3' end of the transcriptmay be at least 75 % of that seen using a poly - A tail of 120
cleaved to free a 3' hydroxyl. Then poly - A polymerase adds 60nucleotides alone .
a chain of adenine nucleotides to the RNA . The process, Quantification
called polyadenylation , adds a poly - A tail that can be In one embodiment, the polynucleotides , primary con
between , for example , approximately 100 and 250 residues structs or mmRNA of the present invention may be quanti
long. fied in exosomes derived from one or more bodily fluid . As
It has been discovered that unique poly - A tail lengths 65 used herein “ bodily fluids” include peripheral blood , serum ,
provide certain advantages to the polynucleotides, primary plasma, ascites, urine , cerebrospinal fluid (CSF ), sputum ,
constructs or mmRNA of the present invention . saliva, bone marrow , synovial fluid , aqueous humor, amni
US 10,703,789 B2
37 38
otic fluid , cerumen , breast milk , broncheoalveolar lavage undergo purification and clean -up processes. The steps of
fluid , semen , prostatic fluid , cowper's fluid or pre- ejacula which are provided in more detail below .
tory fluid , sweat, fecal matter, hair, tears, cyst fluid , pleural Gene Construction
and peritoneal fluid , pericardial fluid , lymph , chyme, chyle, The step of gene construction may include, but is not
bile , interstitial fluid, menses, pus, sebum , vomit, vaginal 5 limited to gene synthesis , vector amplification, plasmid
secretions, mucosal secretion , stool water, pancreatic juice , purification , plasmid linearization and clean -up , and cDNA
lavage fluids from sinus cavities, bronchopulmonary aspi template synthesis and clean -up .
rates , blastocyl cavity fluid , and umbilical cord blood . Alter Gene Synthesis
natively, exosomes may be retrieved from an organ selected Once a polypeptide of interest , or target, is selected for
from the group consisting of lung, heart, pancreas, stomach , 10 production , a primary construct is designed . Within the
intestine, bladder, kidney, ovary, testis , skin , colon , breast, primary construct, a first region of linked nucleosides encod
prostate , brain , esophagus, liver, and placenta . ing the polypeptide of interestmay be constructed using an
In the quantification method , a sample of notmore than 2 open reading frame (ORF ) of a selected nucleic acid (DNA
mL is obtained from the subject and the exosomes isolated or RNA ) transcript. The ORF may comprise the wild type
by size exclusion chromatography, density gradient centrifu- 15 ORF, an isoform , variant or a fragment thereof. As used
gation , differential centrifugation , nanomembrane ultrafil herein , an " open reading frame” or “ ORF” is meant to refer
tration , immunoabsorbent capture, affinity purification , to a nucleic acid sequence (DNA or RNA ) which is capable
microfluidic separation , or combinations thereof. In the ofencoding a polypeptide of interest .ORFs often begin with
analysis, the level or concentration of polynucleotide, the start codon , ATG and end with a nonsense or termination
primary construct or mmRNA may be an expression level, 20 codon or signal.
presence , absence, truncation or alteration of the adminis Further , the nucleotide sequence of the first region may be
tered construct. It is advantageous to correlate the level with codon optimized . Codon optimization methods are known in
one or more clinical phenotypes or with an assay for a the art and may be useful in efforts to achieve one or more
human disease biomarker . The assay may be performed of several goals. These goals include to match codon fre
using construct specific probes, cytometry , qRT-PCR, real- 25 quencies in target and host organisms to ensure proper
time PCR , PCR , flow cytometry, electrophoresis,mass spec folding , bias GC content to increase mRNA stability or
trometry , or combinations thereofwhile the exosomes may reduce secondary structures, minimize tandem repeat
be isolated using immunohistochemical methods such as codons or base runs that may impair gene construction or
enzyme linked immunosorbent assay ( ELISA ) methods. expression , customize transcriptional and translational con
Exosomes may also be isolated by size exclusion chroma- 30 trol regions, insert or remove protein trafficking sequences ,
tography , density gradient centrifugation , differential cen remove /add post translation modification sites in encoded
trifugation , nanomembrane ultrafiltration , immunoabsorbent protein ( e.g. glycosylation sites ), add , remove or shuffle
capture, affinity purification , microfluidic separation , or protein domains , insert or delete restriction sites, modify
combinations thereof. ribosome binding sites and mRNA degradation sites , to
These methods afford the investigator the ability to moni- 35 adjusttranslational rates to allow the various domains of the
tor, in real time, the level of polynucleotides, primary protein to fold properly, or to reduce or eliminate problem
constructs or mmRNA remaining or delivered. This is pos secondary structures within the mRNA . Codon optimization
sible because the polynucleotides, primary constructs or tools , algorithms and services are known in the art, non
mmRNA of the present invention differ from the endog limiting examples include services from GeneArt (Life
enous forms due to the structural or chemicalmodifications. 40 Technologies), DNA2.0 (Menlo Park Calif .) and /or propri
II . Design and Synthesis of mmRNA etary methods. In one embodiment, the ORF sequence is
optimized using optimization algorithms. Codon options for
each amino acid are given in Table 1.
Polynucleotides, primary constructs or mmRNA for use in
accordance with the invention may be prepared according to 45 TABLE 1
any available technique including , but not limited to chemi
cal synthesis , enzymatic synthesis, which is generally Codon Options
termed in vitro transcription ( IVT) or enzymatic or chemical Single
cleavage of a longer precursor, etc.Methods ofsynthesizing Letter
RNAs are known in the art (see , e.g., Gait , M. J. (ed .) 50 Amino Acid Code Codon Options
Oligonucleotide synthesis : a practical approach , Oxford Isoleucine I ATT , ATC , ATA
[Oxfordshire ], Washington , D.C .: IRL Press, 1984 ; and
Herdewijn , P. (ed .) Oligonucleotide synthesis: methods and Leucine L CTT , CTC , CTA , CTG ,
applications, Methods in Molecular Biology, v. 288 (Clifton , TTA , TTG
N.J.) Totowa, N.J.: Humana Press, 2005 ; both of which are 55 Valine V GTT , GTC , GTA , GTG
incorporated herein by reference ).
The process of design and synthesis of the primary Phenylalanine F TTT , TTC
constructs of the invention generally includes the steps of
gene construction , mRNA production (either with or without Methionine M ATG
modifications) and purification . In the enzymatic synthesis 60
method , a target polynucleotide sequence encoding the Cysteine ? TGT , TGC
polypeptide of interest is first selected for incorporation into Alanine A GCT, GCC , GCA , GCG
a vector which will be amplified to produce a cDNA
template . Optionally, the target polynucleotide sequence Glycine G GGT , GGC , GGA , GGG
and /or any flanking sequences may be codon optimized . The 65 Proline P CCT , CCC , CCA , CCG
cDNA template is then used to produce mRNA through in
vitro transcription (IVT). After production , the mRNA may
US 10,703,789 B2
39 40
TABLE 1 - continued including none , may be codon optimized and any may
independently contain one or more different structural or
Codon Options chemical modifications, before and /or after codon optimi
Single zation . Combinations of features may be included in the first
Letter 5 and second flanking regions and may be contained within
Amino Acid Code Codon Options other features . For example , the ORF may be flanked by a
5 ' UTR which may contain a strong Kozak translational
Threonine T ACT, ACC , ACA , ACG initiation signal and /or a 3 ' UTR which may include an
Serine S TCT , TCC , TCA , TCG , oligo (dT ) sequence for templated addition of a poly - A tail.
AGT, AGC 10 5'UTR may comprise a first polynucleotide fragment and a
second polynucleotide fragment from the same and /or dif
Tyrosine Y TAT, TAC ferent genes such as the 5'UTRs described in US Patent
Tryptophan Application Publication No. 20100293625, herein incorpo
W TGG
rated by reference in its entirety .
Glutamine CAA , CAG Tables 2 and 3 provide a listing of exemplary UTRs which
15
may be utilized in the primary construct of the present
Asparagine N AAT , AAC invention as flanking regions. Shown in Table 2 is a listing
Histidine H CAT, CAC
of a 5'-untranslated region of the invention . Variants of 5 '
UTRsmay be utilized wherein one or more nucleotides are
Glutamic acid E GAA , GAG added or removed to the termini, including A , T , C or G.
20
Aspartic acid D GAT, GAC TABLE 2
Lysine K AAA , AAG 5 ' - Untranslated Regions
Arginine R CGT, CGC, CGA , CGG , SEQ
AGA , AGG 25 5 ' UTR Name / ID
Identifier Description Sequence NO .
Selenocysteine Sec UGA in mRNA in
presence of Selenocystein 5UTR - 001 Upstream GGGAAATAAGAGAGAAAAGAA 1
insertion element ( SECIS ) UTR GAGTAAGAAGAAATATAAGAG
CCACC
Stop codons Stop TAA , TAG , TGA 30
SUTR - 002 Upstream GGGAGATCAGAGAGAAAAGAA 2
UTR GAGTAAGAAGAAATATAAGAG
Features , which may be considered beneficial in some CCACC
embodiments of the present invention , may be encoded by 5UTR - 003 Upstream GGAATAAAAGTCTCAACACAA 3
the primary construct and may flank the ORF as a first or 35 UTR CATATACAAAACAAACGAATC
second flanking region . The flanking regions may be incor TCAAGCAATCAAGCATTCTAC
porated into the primary construct before and /or after opti TTCTATTGCAGCAATTTAAAT
CATTTCTTTTAAAGCAAAAGC
mization of the ORF. It is not required that a primary AATTTTCTGAAAATTTTCACC
construct contain both a 5 ' and 3' flanking region . Examples ATTTACGAACGATAGCAAC
of such features include, but are not limited to , untranslated
regions (UTRs ), Kozak sequences, an oligo ( dT ) sequence , 40 SUTR - 004 Upstream
UTR
GGGAGACAAGCUUGGCAUUCC
GGUACUGUUGGUAAAGCCACC
4
and detectable tags and may include multiple cloning sites

which may have Xbal recognition .
In some embodiments, a 5 'UTR and /or a 3' UTR may be Shown in Table 3 is a representative listing of 3'-untrans
provided as flanking regions .Multiple 5 ' or 3 ' UTRsmay be 45 lated regions of the invention . Variants of 3 'UTRs may be
included in the flanking regions and may be the same or of utilized wherein one or more nucleotides are added or
different sequences. Any portion of the flanking regions, removed to the termini, including A , T, C or G.
TABLE 3
3 ' - Untranslated Regions
3! SEQ
UTR Name / ID
3UTR Creatine GCGCCTGCCCACCTGCCACCGACTGCTGGAAC 5
001 Kinase CCAGCCAGTGGGAGGGCCTGGCCCACCAGAG
TCCTGCTCCCTCACTCCTCGCCCCGCCCCCTGT
CCCAGAGTCCCACCTGGGGGCTCTCTCCACCC
TTCTCAGAGTTCCAGTTTCAACCAGAGTTCCA
ACCAATGGGCTCCATCCTCTGGATTCTGGCCA
ATGAAATATCTCCCTGGCAGGGTCCTCTTCTT
TTCCCAGAGCTCCACCCCAACCAGGAGCTCTA
GTTAATGGAGAGCTCCCAGCACACTCGGAGC
TTGTGCTTTGTCTCCACGCAAAGCGATAAATA
AAAGCATTGGTGGCCTTTGGTCTTTGAATAAA
GCCTGAGTAGGAAGTCTAGA
3UTR Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTGGG 6
002 CCCCGGGTTCAAGAGAGAGCGGGGTCTGATC
US 10,703,789 B2
41 42
TABLE 3 - continued
3 ' - Untranslated Reqions
31 SEO
UTR Name / ID
TCGTGTAGCCATATAGAGTTTGCTTCTGAGTG
TCTGCTTTGTTTAGTAGAGGTGGGCAGGAGGA
GCTGAGGGGCTGGGGCTGGGGTGTTGAAGTT
GGCTTTGCATGCCCAGCGATGCGCCTCCCTGT
GGGATGTCATCACCCTGGGAACCGGGAGTGG
CCCTTGGCTCACTGTGTTCTGCATGGTTTGGA
TCTGAATTAATTGTCCTTTCTTCTAAATCCCAA
CCGAACTTCTTCCAACCTCCAAACTGGCTGTA
ACCCCAAATCCAAGCCATTAACTACACCTGAC
AGTAGCAATTGTCTGATTAATCACTGGCCCCT
TGAAGACAGCAGAATGTCCCTTTGCAATGAG
GAGGAGATCTGGGCTGGGCGGGCCAGCTGGG
GAAGCATTTGACTATCTGGAACTTGTGTGTGC
CTCCTCAGGTATGGCAGTGACTCACCTGGTTT
TAATAAAACAACCTGCAACATCTCATGGTCTT
TGAATAAAGCCTGAGTAGGAAGTCTAGA
3UTR a - actin ACACACTCCACCTCCAGCACGCGACTTCTCAG 7
003 GACGACGAATCTTCTCAATGGGGGGGCGGCT
GAGCTCCAGCCACCCCGCAGTCACTTTCTTTG
TAACAACTTCCGTTGCTGCCATCGTAAACTGA
CACAGTGTTTATAACGTGTACATACATTAACT
TATTACCTCATTTTGTTATTTTTCGAAACAAAG
CCCTGTGGAAGAAAATGGAAAACTTGAAGAA
GCATTAAAGTCATTCTGTTAAGCTGCGTAAAT
GGTCTTTGAATAAAGCCTGAGTAGGAAGTCTA
GA
3 UTR Albumin CATCACATTTAAAAGCATCTCAGCCTACCATG 8

004 AGAATAAGAGAAAGAAAATGAAGATCAAAA
GCTTATTCATCTGTTTTTCTTTTTCGTTGGTGT
AAAGCCAACACCCTGTCTAAAAAACATAAAT
TTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTT
CAATTAATAAAAAATGGAAAGAATCTAATAG
AGTGGTACAGCACTGTTATTTTTCAAAGATGT
GTTGCTATCCTGAAAATTCTGTAGGTTCTGTG
GAAGTTCCAGTGTTCTCTCTTATTCCACTTCGG
TAGAGGATTTCTAGTTTCTTGTGGGCTAATTA
AATAAATCATTAATACTCTTCTAATGGTCTTT
GAATAAAGCCTGAGTAGGAAGTCTAGA
3UTR a - globin GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCAT 9
005 GCCCTTCTTCTCTCCCTTGCACCTGTACCTCTT
GGTCTTTGAATAAAGCCTGAGTAGGAAGGCG
GCCGCTCGAGCATGCATCTAGA
3 UTR G - CSF GCCAAGCCCTCCCCATCCCATGTATTTATCTC 10

006 TATTTAATATTTATGTCTATTTAAGCCTCATAT
TTAAAGACAGGGAAGAGCAGAACGGAGCCCC
AGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTT
TCATTCTCCTGCCTGTAGCAGTGAGAAAAAGC
TCCTGTCCTCCCATCCCCTGGACTGGGAGGTA
GATAGGTAAATACCAAGTATTTATTACTATGA
CTGCTCCCCAGCCCTGGCTCTGCAATGGGCAC
TGGGATGAGCCGCTGTGAGCCCCTGGTCCTGA
GGGTCCCCACCTGGGACCCTTGAGAGTATCAG
GTCTCCCACGTGGGAGACAAGAAATCCCTGTT
TAATATTTAAACAGCAGTGTTCCCCATCTGGG
TCCTTGCACCCCTCACTCTGGCCTCAGCCGAC
TGCACAGCGGCCCCTGCATCCCCTTGGCTGTG
AGGCCCCTGGACAAGCAGAGGTGGCCAGAGC
TGGGAGGCATGGCCCTGGGGTCCCACGAATTT
GCTGGGGAATCTCGTTTTTCTTCTTAAGACTTT
TGGGACATGGTTTGACTCCCGAACATCACCGA
CGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGA
CACCTGCCCTGCCCCCACGAGGGTCAGGACTG
TGACTCTTTTTAGGGCCAGGCAGGTGCCTGGA
CATTTGCCTTGCTGGACGGGGACTGGGGATGT
GGGAGGGAGCAGACAGGAGGAATCATGTCAG
GCCTGTGTGTGAAAGGAAGCTCCACTGTCACC
CTCCACCTCTTCACCCCCCACTCACCAGTGTC
CCCTCCACTGTCACATTGTAACTGAACTTCAG
GATAATAAAGTGTTTGCCTCCATGGTCTTTGA
US 10,703,789 B2
43 44
TABLE 3 - continued
31 SEO
UTR Name / ID
ATAAAGCCTGAGTAGGAAGGCGGCCGCTCGA
GCATGCATCTAGA
3 UTR Colla2 ; ACTCAATCTAAATTAAAAAAGAAAGAAATTT 11
007 collagen , GAAAAAACTTTCTCTTTGCCATTTCTTCTTCTT
type I , CTTTTTTAACTGAAAGCTGAATCCTTCCATTTC
alpha 2 TTCTGCACATCTACTTGCTTAAATTGTGGGCA
AAAGAGAAAAAGAAGGATTGATCAGAGCATT
GTGCAATACAGTTTCATTAACTCCTTCCCCCG
CTCCCCCAAAAATTTGAATTTTTTTTTCAACAC
TCTTACACCTGTTATGGAAAATGTCAACCTTT
GTAAGAAAACCAAAATAAAAATTGAAAAATA
AAAACCATAAACATTTGCACCACTTGTGGCTT
TTGAATATCTTCCACAGAGGGAAGTTTAAAAC
CCAAACTTCCAAAGGTTTAAACTACCTCAAAA
CACTTTCCCATGAGTGTGATCCACATTGTTAG
GTGCTGACCTAGACAGAGATGAACTGAGGTC
CTTGTTTTGTTTTGTTCATAATACAAAGGTGCT
AATTAATAGTATTTCAGATACTTGAAGAATGT
TGATGGTGCTAGAAGAATTTGAGAAGAAATA
CTCCTGTATTGAGTTGTATCGTGTGGTGTATTT
TTTAAAAAATTTGATTTAGCATTCATATTTTCC
ATCTTATTCCCAATTAAAAGTATGCAGATTAT
TTGCCCAAATCTTCTTCAGATTCAGCATTTGTT
CTTTGCCAGTCTCATTTTCATCTTCTTCCATGG
TTCCACAGAAGCTTTGTTTCTTGGGCAAGCAG
AAAAATTAAATTGTACCTATTTTGTATATGTG
AGATGTTTAAATAAATTGTGAAAAAAATGAA
ATAAAGCATGTTTGGTTTTCCAAAAGAACATA
T
3UTR Col6a2 ; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTC 12

008 collagen , GTGAGCCCACCCCGTCCATGGTGCTAAGCGG
type VI , GCCCGGGTCCCACACGGCCAGCACCGCTGCTC
alpha 2 ACTCGGACGACGCCCTGGGCCTGCACCTCTCC
AGCTCCTCCCACGGGGTCCCCGTAGCCCCGGC
CCCCGCCCAGCCCCAGGTCTCCCCAGGCCCTC
CGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGC
CATCCCAAGGCTCCTGACCTACCTGGCCCCTG
AGCTCTGGAGCAAGCCCTGACCCAATAAAGG
CTTTGAACCCAT
3 UTR RPN1 ; GGGGCTAGAGCCCTCTCCGCACAGCGTGGAG 13

009 ribophorin I ACGGGGCAAGGAGGGGGGTTATTAGGATTGG
TGGTTTTGTTTTGCTTTGTTTAAAGCCGTGGGA
AAATGGCACAACTTTACCTCTGTGGGAGATGC
AACACTGAGAGCCAAGGGGTGGGAGTTGGGA
TAATTTTTATATAAAAGAAGTTTTTCCACTTTG
AATTGCTAAAAGTGGCATTTTTCCTATGTGCA
GTCACTCCTCTCATTTCTAAAATAGGGACGTG
GCCAGGCACGGTGGCTCATGCCTGTAATCCCA
GCACTTTGGGAGGCCGAGGCAGGCGGCTCAC
GAGGTCAGGAGATCGAGACTATCCTGGCTAA
CACGGTAAAACCCTGTCTCTACTAAAAGTACA
AAAAATTAGCTGGGCGTGGTGGTGGGCACCT
GTAGTCCCAGCTACTCGGGAGGCTGAGGCAG
GAGAAAGGCATGAATCCAAGAGGCAGAGCTT
GCAGTGAGCTGAGATCACGCCATTGCACTCCA
GCCTGGGCAACAGTGTTAAGACTCTGTCTCAA
ATATAAATAAATAAATAAATAAATAAATAAA
TAAATAAAAATAAAGCGAGATGTTGCCCTCA
AA
3 UTR LRP1 ; low GGCCCTGCCCCGTCGGACTGCCCCCAGAAAG 14

010 density CCTCCTGCCCCCTGCCAGTGAAGTCCTTCAGT
lipoprotein GAGCCCCTCCCCAGCCAGCCCTTCCCTGGCCC
receptor CGCCGGATGTATAAATGTAAAAATGAAGGAA
related TTACATTTTATATGTGAGCGAGCAAGCCGGCA
protein 1 AGCGAGCACAGTATTATTTCTCCATCCCCTCC
TGCCTGCTCCTTGGCACCCCCATGCTGCCT
CAGGGAGACAGGCAGGGAGGGCTTGGGGCTG
CACCTCCTACCCTCCCACCAGAACGCACCCCA
CTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCC
CTGTATAAGACACTTTGCCAAGGCTCTCCCCT
US 10,703,789 B2
45 46
TABLE 3 - continued
31 SEO
UTR Name / ID
CTCGCCCCATCCCTGCTTGCCCGCTCCCACAG
CTTCCTGAGGGCTAATTCTGGGAAGGGAGAG
TTCTTTGCTGCCCCTGTCTGGAAGACGTGGCT
CTGGGTGAGGTAGGCGGGAAAGGATGGAGTG
TTTTAGTTCTTGGGGGAGGCCACCCCAAACCC
CAGCCCCAACTCCAGGGGCACCTATGAGATG
GCCATGCTCAACCCCCCTCCCAGACAGGCCCT
CCCTGTCTCCAGGGCCCCCACCGAGGTTCCCA
GGGCTGGAGACTTCCTCTGGTAAACATTCCTC
CAGCCTCCCCTCCCCTGGGGACGCCAAGGAG
GTGGGCCACACCCAGGAAGGGAAAGCGGGCA
GCCCCGTTTTGGGGACGTGAACGTTTTAATAA
TTTTTGCTGAATTCCTTTACAACTAAATAACA
CAGATATTGTTATAAATAAAATTGT
3 UTR Nnt1 ; ATATTAAGGATCAAGCTGTTAGCTAATAATGC 15

011 cardiotrophin CACCTCTGCAGTTTTGGGAACAGGCAAATAA
like cytokine AGTATCAGTATACATGGTGATGTACATCTGTA
factor 1 GCAAAGCTCTTGGAGAAAATGAAGACTGAAG
AAAGCAAAGCAAAAACTGTATAGAGAGATTT
TTCAAAAGCAGTAATCCCTCAATTTTAAAAAA
GGATTGAAAATTCTAAATGTCTTTCTGTGCAT
ATTTTTTGTGTTAGGAATCAAAAGTATTTTAT
AAAAGGAGAAAGAACAGCCTCATTTTAGATG
TAGTCCTGTTGGATTTTTTATGCCTCCTCAGTA
ACCAGAAATGTTTTAAAAAACTAAGTGTTTAG
GATTTCAAGACAACATTATACATGGCTCTGAA
ATATCTGACACAATGTAAACATTGCAGGCACC
TGCATTTTATGTTTTTTTTTTCAACAAATGTGA
CTAATTTGAAACTTTTATGAACTTCTGAGCTG
TCCCCTTGCAATTCAACCGCAGTTTGAATTAA
TCATATCAAATCAGTTTTAATTTTTTAAATTGT
ACTTCAGAGTCTATATTTCAAGGGCACATTTT
CTCACTACTATTTTAATACATTAAAGGACTAA
ATAATCTTTCAGAGATGCTGGAAACAAATCAT
TTGCTTTATATGTTTCATTAGAATACCAATGA
AACATACAACTTGAAAATTAGTAATAGTATTT
TTGAAGATCCCATTTCTAATTGGAGATCTCTT
TAATTTCGATCAACTTATAATGTGTAGTACTA
TATTAAGTGCACTTGAGTGGAATTCAACATTT
GACTAATAAAATGAGTTCATCATGTTGGCAAG
TGATGTGGCAATTATCTCTGGTGACAAAAGAG
TAAAATCAAATATTTCTGCCTGTTACAAATAT
CAAGGAAGACCTGCTACTATGAAATAGATGA
CATTAATCTGTCTTCACTGTTTATAATACGGA
TGGATTTTTTTTCAAATCAGTGTGTGTTTTGAG
GTCTTATGTAATTGATGACATTTGAGAGAAAT
GGTGGCTTTTTTTAGCTACCTCTTTGTTCATTT
AAGCACCAGTAAAGATCATGTCTTTTTATAGA
AGTGTAGATTTTCTTTGTGACTTTGCTATCGTG
CCTAAAGCTCTAAATATAGGTGAATGTGTGAT
GAATACTCAGATTATTTGTCTCTCTATATAATT
AGTTTGGTACTAAGTTTCTCAAAAAATTATTA
ACACATGAAAGACAATCTCTAAACCAGAAAA
AGAAGTAGTACAAATTTTGTTACTGTAATGCT
CGCGTTTAGTGAGTTTAAAACACACAGTATCT
TTTGGTTTTATAATCATTTCTATTTTGCTGTG
CCTGAGATTAAGATCTGTGTATGTGTGTGTGT
GTGTGTGTGCGTTTGTGTGTTAAAGCAGAAAA
GACTTTTTTAAAAGTTTTAAGTGATAAATGCA
ATTTGTTAATTGATCTTAGATCACTAGTAAAC
TCAGGGCTGAATTATACCATGTATATTCTATT
AGAAGAAAGTAAACACCATCTTTATTCCTGCC
CTTTTTCTTCTCTCAAAGTAGTTGTAGTTATAT
CTAGAAAGAAGCAATTTTGATTTCTTGAAAAG
GTAGTTCCTGCACTCAGTTTAAACTAAAAATA
ATCATACTTGGATTTTATTTATTTTTGTCATAG
TAAAAATTTTAATTTATATATATTTTTATTTAG
TATTATCTTATTCTTTGCTATTTGCCAATCCTT
TGTCATCH GTGTTAAATGAATTGAAAAT
CATGCCCTGTTCATTTTATTTTACTTTATTGGT
TAGGATATTTAAAGGATTTTTGTATATATAAT
TTCTTAAATTAATATTCCAAAAGGTTAGTGGA
CTTAGATTATAAATTATGGCAAAAATCTAAAA
US 10,703,789 B2
47 48
TABLE 3 - continued
31 SEO
UTR Name / ID
ACAACAAAAATGATTTTTATACATTCTATTTC
ATTATTCCTCTTTTTCCAATAAGTCATACAATT
GGTAGATATGACTTATTTTATTTTTGTATTATT
CACTATATCTTTATGATATTTAAGTATAAATA
ATTAAAAAAATTTATTGTACCTTATAGTCTGT
CACCAAAAAAAAAAAATTATCTGTAGGTAGT
GAAATGCTAATGTTGATTTGTCTTTAAGGGCT
TGTTAACTATCCTTTATTTTCTCATTTGTCTTA
AATTAGGAGTTTGTGTTTAAATTACTCATCTA
AGCAAAAAATGTATATAAATCCCATTACTGG
GTATATACCCAAAGGATTATAAATCATGCTGC
TATAAAGACACATGCACACGTATGTTTATTGC
AGCACTATTCACAATAGCAAAGACTTGGAAC
CAACCCAAATGTCCATCAATGATAGACTTGAT
TAAGAAAATGTGCACATATACACCATGGAAT
ACTATGCAGCCATAAAAAAGGATGAGTTCAT
GTCCTTTGTAGGGACATGGATAAAGCTGGAA
ACCATCATTCTGAGCAAACTATTGCAAGGACA
GAAAACCAAACACTGCATGTTCTCACTCATAG
GTGGGAATTGAACAATGAGAACACTTGGACA
CAAGGTGGGGAACACCACACACCAGGGCCTG
TCATGGGGTGGGGGGAGTGGGGAGGGATAGC
ATTAGGAGATATACCTAATGTAAATGATGAGT
TAATGGGTGCAGCACACCAACATGGCACATG
TATACATATGTAGCAAACCTGCACGTTGTGCA
CATGTACCCTAGAACTTAAAGTATAATTAAAA
AAAAAAAGAAAACAGAAGCTATTTATAAAGA
AGTTATTTGCTGAAATAAATGTGATCTTTCCC
ATTAAAAAAATAAAGAAATTTTGGGGTAAAA
AAACACAATATATTGTATTCTTGAAAAATTCT
AAGAGAGTGGATGTGAAGTGTTCTCACCACA
AAAGTGATAACTAATTGAGGTAATGCACATA
TTAATTAGAAAGATTTTGTCATTCCACAATGT
ATATATACTTAAAAATATGTTATACACAATAA
ATACATACATTAAAAAATAAGTAAATGTA
3UTR Col6a1 ; CCCACCCTGCACGCCGGCACCAAACCCTGTCC 16
012 collagen , TCCCACCCCTCCCCACTCATCACTAAACAGAG
type VI, TAAAATGTGATGCGAATTTTCCCGACCAACCT
alpha 1 GATTCGCTAGATTTTTTTTAAGGAAAAGCTTG
GAAAGCCAGGACACAACGCTGCTGCCTGCTTT
GTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTT
GGCATCACCTGCGCAGGGCCCTCTGGGGCTCA
GCCCTGAGCTAGTGTCACCTGCACAGGGCCCT
CTGAGGCTCAGCCCTGAGCTGGCGTCACCTGT
GCAGGGCCCTCTGGGGCTCAGCCCTGAGCTG
GCCTCACCTGGGTTCCCCACCCCGGGCTCTCC
TGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCC
TGCGCAGCTCCTTCCCTAGGCACCTCTGTGCT
GCATCCCACCAGCCTGAGCAAGACGCCCTCTC
GGGGCCTGTGCCGCACTAGCCTCCCTCTCCTC
TGTCCCCATAGCTGGTTTTTCCCACCAATCCTC
ACCTAACAGTTACTTTACAATTAAACTCAAAG
CAAGCTCTTCTCCTCAGCTTGGGGCAGCCATT
GGCCTCTGTCTCGTTTTGGGAAACCAAGGTCA
GGAGGCCGTTGCAGACATAAATCTCGGCGAC
TCGGCCCCGTCTCCTGAGGGTCCTGCTGGTGA
CCGGCCTGGACCTTGGCCCTACAGCCCTGGAG
GCCGCTGCTGACCAGCACTGACCCCGACCTCA
GAGAGTACTCGCAGGGGCGCTGGCTGCACTC
AAGACCCTCGAGATTAACGGTGCTAACCCCGT
CTGCTCCTCCCTCCCGCAGAGACTGGGGCCTG
GACTGGACATGAGAGCCCCTTGGTGCCACAG
AGGGCTGTGTCTTACTAGAAACAACGCAAAC
CTCTCCTTCCTCAGAATAGTGATGTGTTCGAC
GTTTTATCAAAGGCCCCCTTTCTATGTTCATGT
TAGTTTTGCTCCTTCTGTGTTTTTTTCTGAACC
ATATCCATGTTGCTGACTTTTCCAAATAAAGG
TTTTCACTCCTCTC
3UTR Calr ; AGAGGCCTGCCTCCAGGGCTGGA?TGAGGCC 17
013 calreticulin TGAGCGCTCCTGCCGCAGAGCTGGCCGCGCC
AAATAATGTCTCTGTGAGACTCGAGAACTTTC
ATTTTTTTCCAGGCTGGTTCGGATTTGGGGTG
US 10,703,789 B2
49 50
TABLE 3 - continued
31 SEO
UTR Name / ID
GATTTTGGTTTTGTTCCCCTCCTCCACTCTCCC
CCACCCCCTCCCCGCCCTTTTTTTTTTTTTTTTT
TAAACTGGTATTTTATCTTTGATTCTCCTTCAG
CCCTCACCCCTGGTTCTCATCTTTCTTGATCAA
CATCTTTTCTTGCCTCTGTCCCCTTCTCTCATC
TCTTAGCTCCCCTCCAACCTGGGGGGCAGTGG
TGTGGAGAAGCCACAGGCCTGAGATTTCATCT
GCTCTCCTTCCTGGAGCCCAGAGGAGGGCAG
CAGAAGGGGGTGGTGTCTCCAACCCCCCAGC
ACTGAGGAAGAACGGGGCTCTTCTCATTTCAC
CCCTCCCTTTCTCCCCTGCCCCCAGGACTGGG
CCACTTCTGGGTGGGGCAGTGGGTCCCAGATT
GGCTCACACTGAGAATGTAAGAACTACAAAC
AAAATTTCTATTAAATTAAATTTTGTGTCTCC
3UTR Collal ; CTCCCTCCATCCCAACCTGGCTCCCTCCCACC 18

014 collagen , CAACCAACTTTCCCCCCAACCCGGAAACAGA
type I , CAAGCAACCCAAACTGAACCCCCTCAAAAGC
alpha 1 CAAAAAATGGGAGACAATTTCACATGGACTT
TGGAAAATATTTTTTTCCTTTGCATTCATCTCT
CAAACTTAGTTTTTATCTTTGACCAACCGAAC
ATGACCAAAAACCAAAAGTGCATTCAACCTT
ACCAAAAAAAAAAAAAAAAAAAGAATAAAT
AAATAACTTTTTAAAAAAGGAAGCTTGGTCCA
CTTGCTTGAAGACCCATGCGGGGGTAAGTCCC
TTTCTGCCCGTTGGGCTTATGAAACCCCAATG
CTGCCCTTTCTGCTCCTTTCTCCACACCCCCCT
TGGGGCCTCCCCTCCACTCCTTCCCAAATCTG
TCTCCCCAGAAGACACAGGAAACAATGTATT
GTCTGCCCAGCAATCAAAGGCAATGCTCAAA
CACCCAAGTGGCCCCCACCCTCAGCCCGCTCC
TGCCCGCCCAGCACCCCCAGGCCCTGGGGGA
CCTGGGGTTCTCAGACTGCCAAAGAAGCCTTG
CCATCTGGCGCTCCCATGGCTCTTGCAACATC
TCCCCTTCGTTTTTGAGGGGGTCATGCCGGGG
GAGCCACCAGCCCCTCACTGGGTTCGGAGGA
GAGTCAGGAAGGGCCACGACAAAGCAGAAAC
ATCGGATTTGGGGAACGCGTGTCAATCCCTTG
TGCCGCAGGGCTGGGCGGGAGAGACTGTTCT
GTTCCTTGTGTAACTGTGTTGCTGAAAGACTA
CCTCGTTCTTGTCTTGATGTGTCACCGGGGCA
ACTGCCTGGGGGCGGGGATGGGGGCAGGGTG
GAAGCGGCTCCCCATTTTATACCAAAGGTGCT
ACATCTATGTGATGGGTGGGGTGGGGAGGGA
ATCACTGGTGCTATAGAAATTGAGATGCCCCC
CCAGGCCAGCAAATGTTCCTTTTTGTTCAAAG
TCTATTTTTATTCCTTGATATTTTTCTTTTTTTT
TTTTTTTTTTTGTGGATGGGGACTTGTGAATTT
TTCTAAAGGTGCTATTTAACATGGGAGGAGA
GCGTGTGCGGCTCCAGCCCAGCCCGCTGCTCA
CTTTCCACCCTCTCTCCACCTGCCTCTGGCTTC
TCAGGCCTCTGCTCTCCGACCTCTCTCCTCTGA
AACCCTCCTCCACAGCTGCAGCCCATCCTCCC
GGCTCCCTCCTAGTCTGTCCTGCGTCCTCTGTC
CCCGGGTTTCAGAGACAACTTCCCAAAGCAC
AAAGCAGTTTTTCCCCCTAGGGGTGGGAGGA
AGCAAAAGACTCTGTACCTATTTTGTATGTGT
ATAATAATTTGAGATGTTTTTAATTATTTTGAT
TGCTGGAATAAAGCATGTGGAAATGACCCAA
ACATAATCCGCAGTGGCCTCCTAATTTCCTTC
TTTGGAGTTGGGGGAGGGGTAGACATGGGGA
AGGGGCTTTGGGGTGATGGGCTTGCCTTCCAT
TCCTGCCCTTTCCCTCCCCACTATTCTCTTCTA
GATCCCTCCATAACCCCACTCCCCTTTCTCTCA
CCCTTCTTATACCGCAAACCTTTCTACTTCCTC
TTTCATTTTCTATTCTTGCAATTTCCTTGCACC
TTTTCCAAATCCTCTTCTCCCCTGCAATACCAT
ACAGGCAATCCACGTGCACAACACACACACA
CACTCTTCACATCTGGGGTTGTCCAAACCTCA
TACCCACTCCCC CCCATCCACTCTCO
ACCCCCTGGATGCCCTGCACTTGGTGGCGGTG
GGATGCTCATGGATACTGGGAGGGTGAGGGG
AGTGGAACCCGTGAGGAGGACCTGGGGGCCT
CTCCTTGAACTGACATGAAGGGTCATCTGGCC
US 10,703,789 B2
51 52
TABLE 3 - continued
31 SEO
UTR Name / ID
TCTGCTCCCTTCTCACCCACGCTGACCTCCTGC
CGAAGGAGCAACGCAACAGGAGAGGGGTCTG
CTGAGCCTGGCGAGGGTCTGGGAGGGACCAG
GAGGAAGGCGTGCTCCCTGCTCGCTGTCCTGG
CCCTGGGGGAGTGAGGGAGACAGACACCTGG
GAGAGCTGTGGGGAAGGCACTCGCACCGTGC
TCTTGGGAAGGAAGGAGACCTGGCCCTGCTC
ACCACGGACTGGGTGCCTCGACCTCCTGAATC
CCCAGAACACAACCCCCCTGGGCTGGGGTGG
TCTGGGGAACCATCGTGCCCCCGCCTCCCGCC
TACTCCTTTTTAAGCTT
3 UTR Plodl ; TTGGCCAGGCCTGACCCTCTTGGACCTTTCTT 19
015 procollagen CTTTGCCGACAACCACTGCCCAGCAGCCTCTG
lysine , 2 GGACCTCGGGGTCCCAGGGAACCCAGTCCAG
oxoglutarate 5 CCTCCTGGCTGTTGACTTCCCATTGCTCTTGGA
dioxygenase 1 GCCACCAATCAAAGAGATTCAAAGAGATTCC
TGCAGGCCAGAGGCGGAACACACCTTTATGG
CTGGGGCTCTCCGTGGTGTTCTGGACCCAGCC
CCTGGAGACACCATTCACTTTTACTGCTTTGT
AGTGACTCGTGCTCTCCAACCTGTCTTCCTGA
AAAACCAAGGCCCCCTTCCCCCACCTCTTCCA
TGGGGTGAGACTTGAGCAGAACAGGGGCTTC
CCCAAGTTGCCCAGAAAGACTGTCTGGGTGA
GAAGCCATGGCCAGAGCTTCTCCCAGGCACA
GGTGTTGCACCAGGGACTTCTGCTTCAAGTTT
TGGGGTAAAGACACCTGGATCAGACTCCAAG
GGCTGCCCTGAGTCTGGGACTTCTGCCTCCAT
GGCTGGTCATGAGAGCAAACCGTAGTCCCCT
GGAGACAGCGACTCCAGAGAACCTCTTGGGA
GACAGAAGAGGCATCTGTGCACAGCTCGATC
TTCTACTTGCCTGTGGGGAGGGGAGTGACAG
GTCCACACACCACACTGGGTCACCCTGTCCTG
GATGCCTCTGAAGAGAGGGACAGACCGTCAG
AAACTGGAGAGTTTCTATTAAAGGTCATTTAA
????
3UTR Nucbl; TCCTCCGGGACCCCAGCCCTCAGGATTCCTGA 20

016 nucleobindin 1 TGCTCCAAGGCGACTGATGGGCGCTGGATGA
AGTGGCACAGTCAGCTTCCCTGGGGGCTGGTG
TCATGTTGGGCTCCTGGGGCGGGGGCACGGC
CTGGCATTTCACGCATTGCTGCCACCCCAGGT
CCACCTGTCTCCACTTTCACAGCCTCCAAGTC
TGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTT
GCCTTCTCTCGTGTCCAGTGAGGTGCTCAGTG
ATCGGCTTAACTTAGAGAAGCCCGCCCCCTCC
CCTTCTCCGTCTGTCCCAAGAGGGTCTGCTCT
GAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGC
TGCCTGGGTTGTGGCCGCCCTAGCATCCTGTA
TGCCCACAGCTACTGGAATCCCCGCTGCTGCT
CCGGGCCAAGCTTCTGGTTGATTAATGAGGGC
ATGGGGTGGTCCCTCAAGACCTTCCCCTACCT
TTTGTGGAACCAGTGATGCCTCAAAGACAGTG
TCCCCTCCACAGCTGGGTGCCAGGGGCAGGG
GATCCTCAGTATAGCCGGTGAACCCTGATACC
AGGAGCCTGGGCCTCCCTGAACCCCTGGCTTC
CAGCCATCTCATCGCCAGCCTCCTCCTGGACC
TCTTGGCCCCCAGCCCCTTCCCCACACAGCCC
CAGAAGGGTCCCAGAGCTGACCCCACTCCAG
GACCTAGGCCCAGCCCCTCAGCCTCATCTGGA
GCCCCTGAAGACCAGTCCCACCCACCTTTCTG
GCCTCATCTGACACTGCTCCGCATCCTGCTGT
GTGTCCTGTTCCATGTTCCGGTTCCATCCAAA
TACACTTTCTGGAACAAA
3 UTR a - globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC 21

017 TTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCT
GCACCCGTACCCCCGTGGTCTTTGAATAAAGT
CTGAGTGGGCGGC
65
It should be understood that those listed in the previous be incorporated into the respective first or second flanking
tables are examples and that any UTR from any gene may region of the primary construct . Furthermore ,multiple wild
US 10,703,789 B2
53 54
type UTRs of any known gene may be utilized . It is also Vector Amplification
within the scope of the present invention to provide artificial The vector containing the primary construct is then ampli
UTRs which are not variants of wild type genes. These fied and the plasmid isolated and purified using methods
UTRs or portions thereof may be placed in the same known in the art such as, but not limited to , a maxi prep
orientation as in the transcript from which they were 5 using the Invitrogen PURELINKTM HiPure Maxiprep Kit
selected or may be altered in orientation or location . Hence ( Carlsbad , Calif.).
a 5' or 3 ' UTR may be inverted , shortened , lengthened ,made
chimeric with one or more other 5 ' UTRs or 3 ' UTRs . As Plasmid Linearization
used herein , the term “ altered ” as it relates to a UTR The plasmid may then be linearized using methods known
sequence, means that the UTR has been changed in some 10 in the art such as, but not limited to, the use of restriction
way in relation to a reference sequence. For example, a 3' or enzymes and buffers . The linearization reaction may be
5 ' UTR may be altered relative to a wild type or native UTR purified using methods including , for example Invitrogen's
by the change in orientation or location as taught above or PURELINKTM PCR Micro Kit (Carlsbad , Calif.), and HPLC
may be altered by the inclusion of additional nucleotides , based purification methods such as , but not limited to , strong
deletion of nucleotides, swapping or transposition ofnucleo- 15 anion exchange HPLC ,weak anion exchange HPLC , reverse
tides. Any of these changes producing an “ altered ” UTR phase HPLC (RP -HPLC), and hydrophobic interaction
(whether 3' or 5') comprise a variant UTR . HPLC (HIC -HPLC ) and Invitrogen's standard
In one embodiment, a double , triple or quadruple UTR PURELINKTM PCR Kit ( Carlsbad , Calif .). The purification
such as a 5' or 3' UTR may be used . As used herein , a method may be modified depending on the size of the
“ double” UTR is one in which two copies of the sameUTR 20 linearization reaction which was conducted . The linearized
are encoded either in series or substantially in series. For plasmid is then used to generate cDNA for in vitro tran
example , a double beta -globin 3' UTR may be used as scription (IVT ) reactions .
described in US Patent publication 20100129877, the con
tents of which are incorporated herein by reference in its cDNA Template Synthesis
entirety. 25 A cDNA template may be synthesized by having a lin
It is also within the scope of the present invention to have earized plasmid undergo polymerase chain reaction (PCR ).
patterned UTRs. As used herein “ patterned UTRs” are those Table 4 is a listing of primers and probes that may be
UTRs which reflect a repeating or alternating pattern , such usefully in the PCR reactions of the present invention . It
as ABABAB or AABBAABBAABB or ABCABCABC or should be understood that the listing is not exhaustive and
variants thereof repeated once, twice, ormore than 3 times . 30 that primer-probe design for any amplification is within the
In these patterns , each letter, A , B , or C represent a different skill of those in the art. Probes may also contain chemically
UTR at the nucleotide level. modified bases to increase base- pairing fidelity to the target
In one embodiment, flanking regions are selected from a molecule and base -pairing strength. Such modificationsmay
family of transcripts whose proteins share a common func include 5 -methyl-Cytidine, 2,6 -di-amino-purine , 2 - fluoro ,
tion, structure , feature of property . For example, polypep- 35 phosphoro -thioate, or locked nucleic acids.
tides of interestmay belong to a family of proteins which are
expressed in a particular cell, tissue or at some time during TABLE 4
development. The UTRs from any of these genes may be
swapped for any other UTR of the same or different family Primers and Probes
of proteins to create a new chimeric primary transcript. As 40 Primer / Hybridi SEQ
used herein , a “ family of proteins” is used in the broadest Probe zation ID
sense to refer to a group of two or more polypeptides of Identifier Sequence ( 5 ' - 3 ' ) target NO .
interest which share at least one function , structure , feature , UFP TTGGACCCTCGTACAG cDNA 22
localization , origin , or expression pattern . AAGCTAATACG Template
After optimization (if desired ), the primary construct 45
components are reconstituted and transformed into a vector URP Tx160CTTCCTACTCAG
GCTTTATTCAAAGACC
cDNA
Template
23
such as, but not limited to , plasmids, viruses , cosmids, and A
artificial chromosomes. For example , the optimized con
structmay be reconstituted and transformed into chemically GBA1 CCTTGACCTTCTGGAA Acid 24
competent E. coli , yeast, neurospora ,maize, drosophila , etc. 50 CTTC glucocere
where high copy plasmid -like or chromosome structures brosidase
occur by methods described herein . GBA2 CCAAGCACTGAAACGG Acid 25
The untranslated region may also include translation ATAT glucocere
enhancer elements ( TEE ). As a non -limiting example , the brosidase
TEE may include those described in US Application No. 55 LUC1 GATGAAAAGTGCTCCA Luciferase 26
20090226470 , herein incorporated by reference in its AGGA
entirety, and those known in the art .
Stop Codons LUC2 AACCGTGATGAAAAGG Luciferase 27
In one embodiment, the primary constructs of the present TACC
invention may include at least two stop codons before the 3' 60 LUC3 TCATGCAGATTGGAAA Luciferase 28
untranslated region (UTR ). The stop codon may be selected GGTC
from TGA , TAA and TAG . In one embodiment, the primary
constructs of the present invention include the stop codon GCSF1 CTTCTTGGACTGTCCA
GAGG
G - CSF 29
TGA and one additional stop codon . In a further embodi

ment the addition stop codon may be TAA . In another 65 GCSF2 GCAGTCCCTGATACAA G - CSF 30
embodiment, the primary constructs of the present invention GAAC
include three stop codons.
US 10,703,789 B2
55 56
TABLE 4 - continued amino acid substitution, insertional variants , deletional vari
ants and /or covalent derivatives.
Primers and Probes In one embodiment, the primary construct may be
Primer / Hybridi SEQ designed to be recognized by the wild type or variant RNA
Probe zation ID 5 polymerases. In doing so , the primary construct may be
Identifier Sequence ( 5 ' - 3 ' ) target NO . modified to contain sites or regions of sequence changes
from the wild type or parent primary construct.
GCSF3 GATTGAAGGTGGCTCG G - CSF 31 In one embodiment, the primary construct may be
???? designed to include at least one substitution and/ or insertion
* UFP is universal forward primer ; URP is universal
10 upstream of an RNA polymerase binding or recognition site,
primer .
reverse
downstream of the RNA polymerase binding or recognition
site, upstream of the TATA box sequence, downstream of the
In one embodiment, the cDNA may be submitted for TATA box sequence of the primary construct but upstream of
sequencing analysis before undergoing transcription. the coding region of the primary construct, within the
mRNA Production 15 5'UTR , before the 5'UTR and /or after the 5'UTR .
The process of mRNA or mmRNA production may In one embodiment, the 5'UTR of the primary construct
include, but is not limited to , in vitro transcription , cDNA may be replaced by the insertion of at least one region and /or
template removal and RNA clean -up , and mRNA capping string of nucleotides of the same base. The region and/or
and / or tailing reactions . string of nucleotides may include, but is not limited to , at
In Vitro Transcription 20 least 3 , at least 4 , at least 5 , at least 6 , at least 7 or at least
The cDNA produced in the previous step may be tran 8 nucleotides and the nucleotides may be natural and /or
scribed using an in vitro transcription (IVT) system . The unnatural. As a non -limiting example , the group of nucleo
system typically comprises a transcription buffer, nucleotide tides may include 5-8 adenine , cytosine , thymine , a string of
triphosphates (NTPs), an RNase inhibitor and a polymerase . any of the other nucleotides disclosed herein and /or combi
The NTPs may be manufactured in house , may be selected 25 nations thereof.
from a supplier, or may be synthesized as described herein . In one embodiment, the 5'UTR of the primary construct
The NTPsmay be selected from ,but are not limited to , those may be replaced by the insertion of at least two regions
described herein including natural and unnatural (modified ) and /or strings of nucleotides of two different bases such as,
NTPs. The polymerase may be selected from , but is not but not limited to , adenine , cytosine , thymine , any of the
limited to , T7 RNA polymerase , T3 RNA polymerase and 30 other nucleotides disclosed herein and /or combinations
mutant polymerases such as ,but not limited to , polymerases thereof. For example , the 5'UTR may be replaced by insert
able to incorporate modified nucleic acids. ing 5-8 adenine bases followed by the insertion of 5-8
RNA Polymerases cytosine bases . In another example , the 5'UTR may be
Any number of RNA polymerases or variants may be used replaced by inserting 5-8 cytosine bases followed by the
in the design of the primary constructs of the present 35 insertion of 5-8 adenine bases.
invention . In one embodiment, the primary constructmay include at
RNA polymerases may bemodified by inserting or delet least one substitution and/or insertion downstream of the
ing amino acids of the RNA polymerase sequence . As a transcription start site which may be recognized by an RNA
non - limiting example , the RNA polymerase may be modi polymerase. As a non -limiting example, at least one substi
fied to exhibit an increased ability to incorporate a 2 '-modi- 40 tution and /or insertion may occur downstream the transcrip
fied nucleotide triphosphate compared to an unmodified tion start site by substituting at least one nucleic acid in the
RNA polymerase (see International Publication region just downstream of the transcription start site (such
WO2008078180 and U.S. Pat. No. 8,101,385 ; herein incor as, but not limited to , +1 to +6 ). Changes to region of
porated by reference in their entireties ). nucleotides just downstream of the transcription start site
Variants may be obtained by evolving an RNA poly- 45 may affect initiation rates, increase apparent nucleotide
merase , optimizing the RNA polymerase amino acid and /or triphosphate (NTP ) reaction constant values , and increase
nucleic acid sequence and /or by using other methods known the dissociation of short transcripts from the transcription
in the art. As a non - limiting example, T7 RNA polymerase complex curing initial transcription (Brieba et al, Biochem
variants may be evolved using the continuous directed istry (2002 ) 41: 5144-5149; herein incorporated by reference
evolution system set out by Esvelt et al. (Nature (2011 ) 50 in its entirety ). The modification , substitution and / or inser
472 (7344):499-503 ; herein incorporated by reference in its tion of at least one nucleic acid may cause a silentmutation
entirety ) where clones of 17 RNA polymerase may encode of the nucleic acid sequence or may cause a mutation in the
at least one mutation such as , but not limited to , lysine at amino acid sequence .
position 93 substituted for threonine (K93T), 14M , AZT , In one embodiment, the primary construct may include
E63V , V64D , A65E , D66Y , 176N , C125R , S128R , A136T, 55 the substitution of at least 1 , at least 2 , at least 3 , at least 4 ,
N165S , G175R , H176L , Y178H , F182L , L196F, G198V, at least 5 , at least 6 , at least 7 , at least 8 , at least 9 , at least
D208Y, E222K , S228A , Q239R , T243N , G259D , M2671, 10 , at least 11 , at least 12 or at least 13 guanine bases
G280C , H300R , D351A , A354S , E356D , L360P, A383V, downstream of the transcription start site .
Y385C , D388Y, S397R , M401T , N410S , K450R , P451T, In one embodiment, the primary construct may include
G452V , E484A , H523L , H524N , G542V , E565K , K577E , 60 the substitution of at least 1, at least 2, at least 3 , at least 4 ,
K577M , N601S , S684Y, L6991, K713E , N748D , Q754R , at least 5 or at least 6 guanine bases in the region just
E775K , A827V , D851N or L864F. As another non - limiting downstream of the transcription start site. As a non -limiting
example, T7 RNA polymerase variants may encode at least example , if the nucleotides in the region are GGGAGA the
mutation as described in U.S. Pub . Nos. 20100120024 and guanine bases may be substituted by at least 1, at least 2, at
20070117112 ; herein incorporated by reference in their 65 least 3 or at least 4 adenine nucleotides . In another non
entireties. Variants of RNA polymerase may also include , limiting example , if the nucleotides in the region are
but are not limited to , substitutional variants , conservative GGGAGA the guanine bases may be substituted by at least
US 10,703,789 B2
57 58
1 , at least 2 , at least 3 or at least 4 cytosine bases . In another assurance and quality control.mRNA or mmRNA clean -up
non - limiting example , if the nucleotides in the region are may be performed bymethods known in the arts such as, but
GGGAGA the guanine bases may be substituted by at least not limited to , AGENCOURT® beads (Beckman Coulter
1, at least 2 , at least 3 or at least 4 thymine, and /or any of 5
Genomics, Danvers , Mass .), poly - T beads, LNATM oligo - T
the nucleotides described herein . capture probes (EXIQON® Inc , Vedbaek , Denmark ) or
In one embodiment, the primary construct may include at HPLC based purification methods such as, but not limited to ,
least one substitution and/ or insertion upstream of the start strong anion exchange HPLC , weak anion exchange HPLC ,
codon .For the purpose of clarity, one of skill in the art would reverse phase HPLC (RP -HPLC ), and hydrophobic interac
appreciate that the start codon is the first codon of the protein 10 tion HPLC (HIC -HPLC ). The term “ purified ” when used in
coding region whereas the transcription start site is the site relation to a polynucleotide such as a “ purified mRNA or
where transcription begins. The primary construct may mmRNA ” refers to one that is separated from at least one
include, but is not limited to , at least 1 , at least 2 , at least 3 , contaminant. As used herein , a " contaminant” is any sub
at least 4 , at least 5 , at least 6 , at least 7 or at least 8 15 stance which makes another unfit, impure or inferior. Thus ,
substitutions and /or insertions of nucleotide bases. The a purified polynucleotide ( e.g., DNA and RNA ) is present in
nucleotide bases may be inserted or substituted at 1 , at least a form or setting different from that in which it is found in
1, at least 2 , at least 3 , at least 4 or at least 5 locations nature , or a form or setting different from that which existed
upstream of the start codon . The nucleotides inserted and / or 20
prior to subjecting it to a treatment or purification method.
substituted may be the same base (e.g., all A or all C or all A quality assurance and /or quality control check may be
T or all G ), two different bases ( e.g., A and C , A and T, or conducted using methods such as, but not limited to , gel
C and T ), three different bases ( e.g., A , C and T or A , C and electrophoresis, UV absorbance, or analytical HPLC .
T ) or at least four different bases. As a non - limiting example , In another embodiment, the mRNA or mmRNA may be
the guanine base upstream of the coding region in the 25 sequenced
primary construct may be substituted with adenine, cytosine , transcriptaseby-PCR
methods including , but not limited to reverse
.
thymine, or any of the nucleotides described herein . In
another non - limiting example the substitution of guanine In one embodiment, the mRNA or mmRNA may be
bases in the primary construct may be designed so as to 30 quantified using methods such as, but not limited to , ultra
leave one guanine base in the region downstream of the violet visible spectroscopy (UV /Vis). A non -limiting
transcription start site and before the start codon (see Esvelt example of a UV /Vis spectrometer is a NANODROP®
et al .Nature (2011 ) 472 ( 7344 ):499-503; herein incorporated spectrometer ( ThermoFisher, Waltham , Mass.). The quanti
by reference in its entirety ). As a non -limiting example, at 35
fied mRNA or mmRNA may be analyzed in order to
least 5 nucleotides may be inserted at 1 location downstream determine if the mRNA or mmRNA may be of proper size ,
of the transcription start site but upstream of the start codon check that no degradation of the mRNA or mmRNA has
and the at least 5 nucleotides may be the samebase type . occurred . Degradation of the mRNA and /or mmRNA may
cDNA Template Removal and Clean -Up be checked by methods such as, but not limited to , agarose
The cDNA template may be removed using methods 40 gel electrophoresis, HPLC based purification methods such
as , but not limited to , strong anion exchange HPLC , weak
known in the art such as , but not limited to , treatment with
Deoxyribonuclease I (DNase I). RNA clean - up may also anion exchange HPLC , reverse phase HPLC (RP -HPLC ),
include a purification method such as, but not limited to , and hydrophobic interaction HPLC (HIC -HPLC ), liquid
chromatography -mass spectrometry (LCMS ), capillary elec
AGENCOURT® CLEANSEQ® system from Beckman 45 trophoresis (CE ) and capillary gel electrophoresis (CGE).
Coulter (Danvers, Mass .), HPLC based purification methods
such as , but not limited to , strong anion exchange HPLC , Signal Sequences
weak anion exchange HPLC , reverse phase HPLC (RP The primary constructs or mmRNA may also encode
HPLC ), and hydrophobic interaction HPLC (HIC -HPLC ). additional features which facilitate trafficking of the poly
Capping and/or Tailing Reactions 50 peptides to therapeutically relevant sites. One such feature
The primary construct or mmRNA may also undergo which used
aids in protein trafficking is the signal sequence . As
herein , a " signal sequence " or " signal peptide" is a
capping and /or tailing reactions. A capping reaction may be
performed by methods known in the art to add a 5 ' cap to the polynucleotide or polypeptide, respectively, which is from
5' end of the primary construct. Methods for capping 55 about is9 toincorporated 200 nucleotides (3-60 amino acids) in length
include, but are not limited to , using a Vaccinia Capping which at the 5 ' (or N - terminus) of the coding
region or polypeptide encoded , respectively . Addition of
enzyme (New England Biolabs, Ipswich , Mass.).
A poly - A tailing reaction may be performed by methods these sequences result in trafficking of the encoded poly
known in the art, such as, but not limited to , 2' O -methyl peptide to the endoplasmic reticulum through one or more
transferase and by methods as described herein . If the 60 secretory pathways. Some signal peptides are cleaved from
primary construct generated from cDNA does not include a ported the protein by signal peptidase after the proteins are trans
poly - T, it may be beneficial to perform the poly - A - tailing .
reaction before the primary construct is cleaned . Table 5 is a representative listing of protein signal
mRNA Purification 65 sequences which may be incorporated for encoding by the
Primary construct or mmRNA purification may include, polynucleotides, primary constructs or mmRNA of the
but is not limited to , mRNA or mmRNA clean - up , quality invention .
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TABLE 5
Signal Sequences
NUCLEOTIDE SEO SEO
SEQUENCE ID ENCODED ID
ID Description (5 ' - 3 ') NO . PEPTIDE NO .
SS a - 1 ATGATGCCATCCTCAGTCT 32 MMP SSVSW 94
001 antitrypsin CATGGGGTATTTTGCTCTT GILLAGLCCL
GGCGGGTCTGTGCTGTCTC VPVSLA
GTGCCGGTGTCGCTCGCA
SS G - CSF ATGGCCGGACCGGCGACT 33 MAGPATOSP 95
002 CAGTCGCCCATGAAACTC MKLMALQL
ATGGCCCTGCAGTTGTTGC LLWHSALWT
TTTGGCACTCAGCCCTCTG VQEA
GACCGTCCAAGAGGCG
SS Factor IX ATGCAGAGAGTGAACATG 34 MQRVNMIM 96
003 ATTATGGCCGAGTCCCCAT AESPSLITICL
CGCTCATCACAATCTGCCT LGYLLSAEC
GCTTGGTACCTGCTTTCCG TVFLDHENA
CCGAATGCACTGTCTTTCT NKILNRPKR
GGATCACGAGAATGCGAA
TAAGATCTTGAACCGACC
CAAACGG
SS Prolactin ATGAAAGGATCATTGCTG 35 MKGSLLLLL 97

004 TTGCTCCTCGTGTCGAACC VSNLLLCQS
TTCTGCTTTGCCAGTCCGT VAP
AGCCCCC
SS Albumin ATGAAATGGGTGACGTTC 36 MKWVTFISL 98

005 ATCTCACTGTTGTTTTTGT LFLFSSAYSR
TCTCGTCCGCCTACTCCAG GVFRR
GGGAGTATTCCGCCGA
SS HMMSP38 ATGTGGTGGCGGCTCTGGT 37 MWWRLWW 99

006 GGCTGCTCCTGTTGCTCCT LLLLLLLLPM
CTTGCTGTGGCCCATGGTG WA
TGGGCA
MLS ornithine TGCTCTTTAACCTCCGCAT 38 MLFNLRILLN 100
001 carbamoyl CCTGTTGAATAACGCTGCG NAAFRNGHN
transferase TTCCGAAATGGGCATAAC FMVRNFRCG
TTCATGGTACGCAACTTCA QPLQ
GATGCGGCCAGCCACTCC
AG
MLS Cytochrome ATGTCCGTCTTGACACCCC 39 MSVLTPLLL 101
002 C Oxidase TGCTCTTGAGAGGGCTGA RGL TGSARR
subunit SA CGGGGTCCGCTAGACGCC LPV PRAKIHS
TGCCGGTACCGCGAGCGA L
AGATCCACTCCCTG
MLS Cytochrome ATGAGCGTGCTCACTCCGT 40 MSVLTPLLL 102

003 C Oxidase TGCTTCTTCGAGGGCTTAC RGL TGSARR
subunit SA GGGATCGGCTCGGAGGTT LPVPRAKIHS
GCCCGTCCCGAGAGCGAA L
GATCCATTCGTTG
SS Type III , TGACAAAAATAACTTTATC 41 MVTKITLSPO 103

007 bacterial TCCCCAGAATTTTAGAATC NFRIQKQETT
CAAAAACAGGAAACCACA LLKEKSTEK
CTACTAAAAGAAAAATCA NSLAKSILAV
ACCGAGAAAAATTCTTTA KNHFIELRSK
GCAAAAAGTATTCTCGCA LSERFISHKN
GTAAAAATCACTTCATCG T
AATTAAGGTCAAAATTAT
CGGAACGTTTTATTTCGCA
TAAGAACACT
SS Viral ATGCTGAGCTTTGTGGATA 42 MLSFVDTRT 104

008 CCCGCACCCTGCTGCTGCT LLLLAVTSCL
GGCGGTGACCAGCTGCCT ATCQ
GGCGACCTGCCAG
SS viral ATGGGCAGCAGCCAGGCG 43 MGS SQAPRM 105

009 CCGCGCATGGGCAGCGTG GSVGGHGL
GGCGGCCATGGCCTGATG MALLMAGLI
GCGCTGCTGATGGCGGGC LPGILA
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TABLE 5 - continued
Signal Sequences
NUCLEOTIDE SEO SEO
ID Description (5 ' - 3 ') NO . PEPTIDE NO .
CTGATTCTGCCGGGCATTC
TGGGG
SS Viral ATGGCGGGCATTTTTTATT 44 MAGIFYFLFS 106

010 TTCTGTTTAGCTTTCTGTTT FLFGICD
GGCATTTGCGAT
SS Viral ATGGAAAACCGCCTGCTG 45 MENRLLRVF 107

011 CGCGTGTTTCTGGTGTGGG LVWAALTM
CGGCGCTGACCATGGATG DGASA
GCGCGAGCGCG
SS Viral ATGGCGCGCCAGGGCTGC 46 MARQGCFGS 108

012 TTTGGCAGCTATCAGGTGA YQVISLFTFA
TTAGCCTGTTTACCTTTGC IGVNLCLG
GATTGGCGTGAACCTGTG
CCTGGGC
SS Bacillus ATGAGCCGCCTGCCGGTG 47 MSRLPVLLL 109

013 CTGCTGCTGCTGCAGCTGC LQLLVRPGL
TGGTGCGCCCGGGCCTGC
AG
SS Bacillus ATGAAACAGCAGAAACGC 48 MKQQKRLY 110

014 CTGTATGCGCGCCTGCTGA ARLLTLLFAL
CCCTGCTGTTTGCGCTGAT IFLLPHSSAS
TTTTCTGCTGCCGCATAGC A
AGCGCGAGCGCG
SS Secretion ATGGCGACGCCGCTGCCT 49 MATPLPPPSP 111

015 signal CCGCCCTCCCCGCGGCACC RHLRLLRLL
TGCGGCTGCTGCGGCTGCT LSG
GCTCTCCGCCCTCGTCCTC
GGC
SS Secretion ATGAAGGCTCCGGGTCGG 50 MKAPGRLVL 112

016 signal CTCGTGCTCATCATCCTGT IILCSVVFS
GCTCCGTGGTCTTCTCT
SS Secretion ATGCTTCAGCTTTGGAAAC 51 MLQLWKLL 113

017 signal TTGTTCTCCTGTGCGGCGT CGVLT
GCTCACT
SS Secretion ATGCTTTATCTCCAGGGTT 52 MLYLQGWS 114

018 signal GGAGCATGCCTGCTGTGG MPAVA
CA
SS Secretion ATGGATAACGTGCAGCCG 53 MDNVQPKIK 115

019 signal AAAATAAAACATCGCCCC HRPFCFSVK
TTCTGCTTCAGTGTGAAAG GHV KMLRL
GCCACGTGAAGATGCTGC DIINSLVTTV
GGCTGGATATTATCAACTC FMLIVSVLAL
ACTGGTAACAACAGTATT IP
CATGCTCATCGTATCTGTG
TTGGCACTGATACCA
SS Secretion ATGCCCTGCCTAGACCAA 54 MPCLDOQLT 116

020 signal CAGCTCACTGTTCATGCCC VHALPCPAQ
TACCCTGCCCTGCCCAGCC PSSLAFCQV
CTCCTCTCTGGCCTTCTGC GFLTA
CAAGTGGGGTTCTTAACA
GCA
SS Secretion ATGAAAACCTTGTTCAATC 55 MKTLFNPAP 117

021 signal CAGCCCCTGCCATTGCTGA AIADLDPQF
CCTGGATCCCCAGTTCTAC YTL SDVFCC
ACCCTCTCAGATGTGTTCT NESEAEILTG
GCTGCAATGAAAGTGAGG LTVGSAADA
CTGAGATTTTAACTGGCCT
CACGGTGGGCAGCGCTGC
AGATGCT
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TABLE 5 - continued
Signal Sequences
NUCLEOTIDE SEO SEQ
ID Description (5 ' - 3 ' ) NO . PEPTIDE NO .
SS Secretion ATGAAGCCTCTCCTTGTTG 56 MKP LLVVFV 118

022 signal TGTTTGTCTTTCTTTTCCTT FLFLWDPVL
TGGGATCCAGTGCTGGCA A
SS Secretion ATGTCCTGTTCCCTAAAGT 57 MSCSLKFTLI 119

023 signal TTACTTTGATTGTAATTTT VIFFTCTLSSS
TTTTTACTGTTGGCTTTCA
TCCAGC
SS Secretion ATGGTTCTTACTAAACCTC 58 MVLTKPLOR 120

024 signal TTCAAAGAAATGGCAGCA NGSMMSFEN
TGATGAGCTTTGAAAATGT VKEKSREGG
GAAAGAAAAGAGCAGAG PHAHTPEEEL
AAGGAGGGCCCCATGCAC CFVVTHTPQ
ACACACCCGAAGAAGAAT VQTTLNLFF
TGTGTTTCGTGGTAACACA HIFKVLTQPL
CTACCCTCAGGTTCAGACC SLLWG
ACACTCAACCTGTTTTTCC
ATATATTCAAGGTTCTTAC
TCAACCACTTTCCCTTCTG
TGGGGT
SS Secretion ATGGCCACCCCGCCATTCC 59 MAT PPFRLIR 121

025 signal GGCTGATAAGGAAGATGT KMFSFKVSR
TTTCCTTCAAGGTGAGCAG WMGLACFRS
ATGGATGGGGCTTGCCTG LAAS
CTTCCGGTCCCTGGCGGCA
TCC
SS Secretion ATGAGCTTTTTCCAACTCC 60 MSFFQLLMK 122

026 signal TGATGAAAAGGAAGGAAC RKELIPLVVF
TCATTCCCTTGGTGGTGTT MTVAAGGA
CATGACTGTGGCGGCGGG SS
TGGAGCCTCATCT
SS Secretion ATGGTCTCAGCTCTGCGGG 61 MVSALRGAP 123

027 signal GAGCACCCCTGATCAGGG LIRVHSSPVS
TGCACTCAAGCCCTGTTTC SPSVSGPAAL
TTCTCCTTCTGTGAGTGGA VSCLSSQSSA
CCACGGAGGCTGGTGAGC LS
TGCCTGTCATCCCAAAGCT
CAGCTCTGAGC
SS Secretion ATGATGGGGTCCCCAGTG 62 MMGSPVSHL 124

028 signal AGTCATCTGCTGGCCGGCT LAGFCVWV
TCTGTGTGTGGGTCGTCTT VLG
GGGC
SS Secretion ATGGCAAGCATGGCTGCC 63 MASMAAVL 125

029 signal GTGCTCACCTGGGCTCTGG TWALALLSA
CTCTTCTTTCAGCGTTTTC FSATOA
GGCCACCCAGGCA
SS Secretion ATGGTGCTCATGTGGACC 64 MVLMWTSG 126

030 signal AGTGGTGACGCCTTCAAG DAFKTAYFL
ACGGCCTACTTCCTGCTGA LKGAPLQFS
AGGGTGCCCCTCTGCAGTT VCGLLQVLV
CTCCGTGTGCGGCCTGCTG DLAILGQAT
CAGGTGCTGGTGGACCTG A
GCCATCCTGGGGCAGGCC
TACGCC
SS Secretion ATGGATTTTGTCGCTGGAG 65 MDFVAGAIG 127

031 signal CCATCGGAGGCGTCTGCG GVCGVAVG
GTGTTGCTGTGGGCTACCC YPLDTVKVR
CCTGGACACGGTGAAGGT IQTEPLYTGI
CAGGATCCAGACGGAGCC WHCVRDTY
AAAGTACACAGGCATCTG HRERVWGFY
GCACTGCGTCCGGGATAC RGL SLPVCT
GTATCACCGAGAGCGCGT VSLVSS
GTGGG
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TABLE 5 - continued
Signal Sequences
NUCLEOTIDE SEO SEO
GCTTCTACCGGGGCCTCTC
GCTGCCCGTGTGCACGGT
GTCCCTGGTATCTTCC
SS Secretion ATGGAGAAGCCCCTCTTCC 66 MEKPLFPLV 128

032 signal CATTAGTGCCTTTGCATTG PLHWFGFGY
GTTTGGCTTTGGCTACACA TALVVSGGI
GCACTGGTTGTTTCTGGTG VGYVKTGSV
GGATCGTTGGCTATGTAA PSLAAGLLF
AAACAGGCAGCGTGCCGT GSLA
CCCTGGCTGCAGGGCTGCT
CTTCGGCAGTCTAGCC
SS Secretion ATGGGTCTGCTCCTTCCCC 67 MGLLLPLAL 129
033 signal TGGCACTCTGCATCCTAGT CILVLC
CCTGTGC
SS Secretion ATGGGGATCCAGACGAGC 68 MGIQTSPVLL 130

034 signal CCCGTCCTGCTGGCCTCCC ASLGVGLVT
TGGGGGTGGGGCTGGTCA LLGLAVG
CTCTGCTCGGCCTGGCTGT
GGGC
SS Secretion ATGTCGGACCTGCTACTAC 69 MSDLLLLGLI 131

035 signal TGGGCCTGATTGGGGGCC GGLTLLLLLT
TGACTCTCTTACTGCTGCT LLAFA
GACGCTGCTAGCCTTTGCC
SS Secretion ATGGAGACTGTGGTGATT 70 METVVIVAI 132

036 signal GTTGCCATAGGTGTGCTGG GVLATIFLAS
CCACCATGTTTCTGGCTTC FAALVLVCR
GTTTGCAGCCTTGGTGCTG
GTTTGCAGGCAG
SS Secretion ATGCGCGGCTCTGTGGAG 71 MAGSVECT 133

037 signal TGCACCTGGGGTTGGGGG WGWGHCAP
CACTGTGCCCCCAGCCCCC SPLLLWTLLL
TGCTCCTTTGGACTCTACT FAAPFGLLG
TCTGTTTGCAGCCCCATTT
GGCCTGCTGGGG
SS Secretion ATGATGCCGTCCCGTACCA 72 MMP SRTNLA 134

038 signal ACCTGGCTACTGGAATCCC TGIPSSKVKY
CAGTAGTAAAGTGAAATA SRLSSTDDG
TTCAAGGCTCTCCAGCACA YIDLQFKKTP
GACGATGGCTACATTGAC PKI PYKAIAL
CTTCAGTTTAAGAAAACCC ATVLFLIGA
CTCCTAAGATCCCTTATAA
GGCCATCGCACTTGCCACT
GTGCTGTTTTTGATTGGCG
CC
SS Secretion ATGGCCCTGCCCCAGATGT 73 MALPQMCD 135

039 signal GTGACGGGAGCCACTTGG GSHLASTLR
CCTCCACCCTCCGCTATTG YCMTVSGTV
CATGACAGTCAGCGGCAC VLVAGTLCF
AGTGGTTCTGGTGGCCGG A
GACGCTCTGCTTCGCT
SS Vrg - 6 TGAAAAAGTGGTTCGTTG 74 MKKWFVAA 136
041 CTGCCGGCATCGGCGCTG GIGAGLLML
CCGGACTCATGCTCTCCAG SSAA
CGCCGCCA
SS PhoA ATGAAACAGAGCACCATT 75 MKQSTIALA 137

042 GCGCTGGCGCTGCTGCCG LLPLLFTPVT
CTGCTGTTTACCCCGGTGA KA
CCAAAGCG
SS OmpA ATGAAAAAAACCGCGATT 76 MKKTAIAIA 138

043 GCGATTGCGGTGGCGCTG VALAGFATV
GCGGGCTTTGCGACCGTG AQA
GCGCAGGCG
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TABLE 5 - continued
Signal Sequences
NUCLEOTIDE SEO SEO
SS STI ATGAAAAAACTGATGCTG 77 MKKLMLAIF 139
044 GCGATTTTTTTTAGCGTGC FSVLSFPSFS
TGAGCTTTCCGAGCTTTAG QS
CCAGAGC
SS STII ATGAAAAAAAACATTGCG 78 MKKNIAFLL 140

045 TTTCTGCTGGCGAGCATGT ASMFVFSIAT
TTGTGTTTAGCATTGCGAC NAYA
CAACGCGTATGCG
SS Amylase ATGTTTGCGAAACGCTTTA 79 MFAKRFKTS 141

046 AAACCAGCCTGCTGCCGC LLPLFAGFLL
TGTTTGCGGGCTTTCTGCT LFHLVLAGP
GCTGTTTCATCTGGTGCTG AAAS
GCGGGCCCGGCGGCGGCG
AGC
SS Alpha ATGCGCTTTCCGAGCATTT 80 MRFPSIFTAV 142
047 Factor TTACCGCGGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 81 MRFPSIFTTV 143

048 Factor TTACCACCGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 82 MRFPSIFTSV 144

049 Factor TTACCAGCGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 83 MRFPSIFTHV 145

050 Factor TTACCCATGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 84 MRFPSIFTIV 146

051 Factor TTACCATTGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 85 MRFPSIFTFV 147

052 Factor TTACCTTTGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 86 MRFPSIFTEV 148

053 Factor TTACCGAAGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Alpha ATGCGCTTTCCGAGCATTT 87 MRFPSIFTGV 149

054 Factor TTACCGGCGTGCTGTTTGC LFAASSALA
GGCGAGCAGCGCGCTGGC
G
SS Endoglucanase ATGCGTTCCTCCCCCCTCC 88 MRS SPLLRS 150

055 V TCCGCTCCGCCGTTGTGGC AVVAALPVL
CGCCCTGCCGGTGTTGGCC ALA
CTTGCC
SS Secretion ATGGGCGCGGCGGCCGTG 89 MGAAAVRW 151

056 signal CGCTGGCACTTGTGCGTGC HLCVLLALG
TGCTGGCCCTGGGCACAC TRGRL
GCGGGCGGCTG
SS Fungal ATGAGGAGCTCCCTTGTGC 90 MRSSLVLFF 152
057 TGTTCTTTGTCTCTGCGTG VSAWTALA
GACGGCCTTGGCCAG
SS Fibronectin ATGCTCAGGGGTCCGGGA 91 MLRGPGPGR 153

058 CCCGGGCGGCTGCTGCTG LLLLAVLCL
CTAGCAGTCCTGTGCCTGG GTSVRCTET
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TABLE 5 - continued
Signal Sequences
NUCLEOTIDE SEO SEO
GGACATCGGTGCGCTGCA GKS KR
CCGAAACCGGGAAGAGCA
AGAGG
SS Fibronectin ATGCTTAGGGGTCCGGGG 92 MLRGPGPGL 154

059 CCCGGGCTGCTGCTGCTGG LLLAVQCLG
CCGTCCAGCTGGGGACAG TAVPSTGA
CGGTGCCCTCCACG
SS Fibronectin ATGCGCCGGGGGGCCCTG 93 MRRGALTGL 155

060 ACCGGGCTGCTCCTGGTCC LLVLCLSVV
TGTGCCTGAGTGTTGTGCT LRAAPSATS
ACGTGCAGCCCCCTCTGCA KKRR
ACAAGCAAGAAGCGCAGG
20
In the table , SS is secretion signal and MLS is mitochon taining at least one protein cleavage site. The protein cleav
drial leader signal. The primary constructs ormmRNA of the age site may be located at the N -terminus , the C -terminus,
present invention may be designed to encode any of the at any space between the N- and the C -termini such as, but
signal sequences of SEQ ID NOS 94-155 , or fragments or not limited to , half-way between the N- and C -termini,
variants thereof. These sequences may be included at the 25 between the N -terminus and the half way point, between the
beginning of the polypeptide coding region , in the middle or half way point and the C -terminus, and combinations
at the terminus or alternatively into a flanking region . thereof.
Further , any of the polynucleotide primary constructs of the The polypeptides of the present invention may include,
present invention may also comprise one or more of the 30 but is not limited to , a proprotein convertase (or prohormone
sequences defined by SEQ ID NOs 32-93 . These may be in convertase ), thrombin or Factor Xa protein cleavage signal.
the first region or either flanking region . Proprotein convertases are a family of nine proteinases,
Additional signal sequences which may be utilized in the comprising seven basic amino acid -specific subtilisin -like
present invention include those taught in , for example , serine proteinases related to yeast kexin , known as prohor
databases such as those found at http : //www.signalpeptid- 35 mone convertase 1/3 (PC1/3), PC2 , furin , PC4 , PC5/6 ,
e.de/ or http://proline.bic.nus.edu.sg/spdb/. Those described paired basic amino -acid cleaving enzyme 4 (PACE4) and
in U.S. Pat . Nos. 8,124,379 ; 7,413,875 and 7,385,034 are PC7, and two other subtilases that cleave at non -basic
also within the scope of the invention and the contents of residues, called subtilisin kexin isozyme 1 (SKI- 1) and
each are incorporated herein by reference in their entirety . proprotein convertase subtilisin kexin 9 (PCSK9). Non
Target Selection 40 limiting examples of protein cleavage signal amino acid
According to the present invention, the primary constructs sequences are listing in Table 7. In Table 7 , “ X ” refers to any
comprise at least a first region of linked nucleosides encod amino acid , “ n ” may be 0 , 2 , 4 or 6 amino acids and " * "
ing at least one polypeptide of interest. The polypeptides of refers to the protein cleavage site . In Table 7 , SEQ ID NO :
interest or “ Targets ” of the present invention are listed in 16090 refers to when n = 4 and SEQ ID NO : 16091 refers to
Lengthy Table 6. Shown in Lengthy Table 6 , in addition to 45 when n = 6 .
the name and description of the gene encoding the polypep
tide of interest ( Target Description ) are the ENSEMBL TABLE 7
Transcript ID (ENST), the ENSEMBL Protein ID (ENSP) Protein Cleavage Site Sequences
and when available the optimized open reading frame
sequence ID (Optimized ORF SEQ ID ). For any particular 50 Protein Amino Acid SEQ
gene there may exist one or more variants or isoforms. Cleavage Signal Cleavage Sequence ID NO
Where these exist, they are shown in the table as well. It will Proprotein R -X -X -R * 16088
be appreciated by those of skill in the art that disclosed in the convertase R -X -K /R -R * 16089
Table are potential flanking regions. These are encoded in K / R - Xn - K / R * 16090 or
each ENST transcript either to the 5 ' ( upstream ) or 3' 55 16091
(downstream ) of the ORF or coding region . The coding Thrombin L -V -P - R -G - S 16092
region is definitively and specifically disclosed by teaching L -V -P - R * 16093
the ENSP sequence. Consequently, the sequences taught A / F /G / I / L / T /V /M - A / 16094
flanking that encoding the protein are considered flanking F /G / I / L / T / V / W - P - R *
regions. It is also possible to further characterize the 5' and 60 Factor Xa I -E -G -R * 16095
3' flanking regions by utilizing one or more available data I-D -G -R * 16096
bases or algorithms. Databases have annotated the features A -E -G - R * 16097
contained in the flanking regions of the ENST transcripts A /F /G / I / L / T /V /M - D /
E -G -R *
16098
and these are available in the art .
Protein Cleavage Signals and Sites 65
In one embodiment, the polypeptides of the present inven In one embodiment, the primary constructs and the
tion may include at least one protein cleavage signal con mmRNA of the present invention may be engineered such
US 10,703,789 B2
71 72
that the primary construct or mmRNA contains at least one and mmRNA can be released from a carrier region or a
encoded protein cleavage signal. The encoded protein cleav fusion partner by treatment with a specific protease for said
age signal may be located before the start codon , after the protein cleavage site .
start codon , before the coding region , within the coding In one embodiment, the polypeptides , primary constructs
region such as , but not limited to , half way in the coding 5 and mmRNA of the present invention may include a
region, between the start codon and the half way point, sequence encoding the 2A peptide. In one embodiment, this
between the half way point and the stop codon , after the sequence may be used to separate the coding region of two
coding region , before the stop codon , between two stop or more polypeptides of interest. As a non- limiting example,
the sequence encoding the 2A peptide may be between
codons, after the stop codon and combinations thereof. 10 coding
In one embodiment, the primary constructs or mmRNA of presenceregion A and coding region B (A -2Apep -B ). The
the present invention may include at least one encoded one long protein2A into
of the peptide would result in the cleavage of
protein A , protein B and the 2A
protein cleavage signal containing at least one protein cleav peptide . Protein A and protein B may be the same or different
age site . The encoded protein cleavage signal may include , polypeptides of interest. In another embodiment, the 2A
but is not limited to , a proprotein convertase (or prohormone 15 peptide may be used in the polynucleotides , primary con
convertase ), thrombin and /or Factor Xa protein cleavage structs and/or mmRNA of the present invention to produce
signal . One of skill in the art may use Table 1 above or other two , three , four, five , six , seven , eight, nine , ten or more
known methods to determine the appropriate encoded pro proteins.
tein cleavage signal to include in the primary constructs or Incorporating Post Transcriptional Control Modulators
mmRNA of the present invention . For example , starting with 20 In one embodiment, the polynucleotides , primary con
the signal of Table 7 and considering the codons of Table 1 structs and /ormmRNA of the present invention may include
one can design a signal for the primary construct which can at least one post transcriptional control modulator. These
produce a protein signal in the resulting polypeptide. post transcriptional control modulators may be, but are not
In one embodiment, the polypeptides of the present inven limited to , small molecules , compounds and regulatory
tion include at least one protein cleavage signal and / or site. 25 sequences. As a non -limiting example , post transcriptional
As a non - limiting example , U.S. Pat. No. 7,374,930 and controlmay be achieved using small molecules identified by
U.S. Pub . No. 20090227660, herein incorporated by refer PTC Therapeutics Inc. ( South Plainfield , N.J.) using their
ence in their entireties , use a furin cleavage site to cleave the GEMSTM (Gene Expression Modulation by Small-Mole
N -terminal methionine of GLP - 1 in the expression product 30 clues ) screening technology .
from the Golgi apparatus of the cells . In one embodiment, The post transcriptional control modulatormay be a gene
the polypeptides of the present invention include at least one expression modulator which is screened by the method
protein cleavage signal and /or site with the proviso that the detailed in or a gene expression modulator described in
polypeptide is not GLP - 1. International Publication No.WO2006022712 , herein incor
In one embodiment, the primary constructs ormmRNA of porated by reference in its entirety . Methods identifying
the present invention includes at least one encoded protein 35 RNA regulatory sequences involved in translational control
are described in International Publication No.
cleavage signal and/or site . WO2004067728 , herein incorporated by reference in its
In one embodiment, the primary constructs or mmRNA of entirety ; methods identifying compounds that modulate
the present invention includes at least one encoded protein untranslated region dependent expression of a gene are
cleavage signal and/or site with the proviso that the primary 40 described in International Publication No. WO2004065561,
construct or mmRNA does not encode GLP - 1 . herein incorporated by reference in its entirety .
In one embodiment, the primary constructs ormmRNA of In one embodiment, the polynucleotides , primary con
the present invention may include more than one coding structs and/or mmRNA of the present invention may include
region . Where multiple coding regions are present in the at least one post transcriptional controlmodulator is located
primary construct or mmRNA of the present invention , the 45 in the 5 ' and /or the 3 ' untranslated region of the polynucle
multiple coding regions may be separated by encoded pro otides, primary constructs and /or mmRNA of the present
tein cleavage sites. As a non -limiting example , the primary invention
construct or mmRNA may be signed in an ordered pattern . In another embodiment, the polynucleotides, primary
On such pattern follows AXBY form where A and B are constructs and/or mmRNA of the present invention may
coding regions which may be the same or different coding 50 include at least one post transcription control modulator to
regions and /or may encode the same or different polypep modulate premature translation termination . The post tran
tides, and X and Y are encoded protein cleavage signals scription control modulators may be compounds described
which may encode the same or different protein cleavage in or a compound found by methods outlined in International
signals . second such pattern follows the form AXYBZ Publication Nos. WO2004010106 , WO2006044456 ,
where A and B are coding regions which may be the same 55 WO2006044682, WO2006044503 and WO2006044505 ,
or different coding regions and /or may encode the same or each of which is herein incorporated by reference in its
different polypeptides, and X , Y and Z are encoded protein entirety.Asa non - limiting example , the compound may bind
cleavage signals which may encode the same or different to a region of the 28S ribosomal RNA in order to modulate
protein cleavage signals . A third pattern follows the form premature translation termination (See e.g.,
ABXCY where A , B and C are coding regions which may be 60 WO2004010106 , herein incorporated by reference in its
the same or different coding regions and /ormay encode the entirety ) .
same or different polypeptides, and X and Y are encoded In one embodiment, polynucleotides, primary constructs
protein cleavage signals which may encode the same or and /ormmRNA of the present invention may include at least
different protein cleavage signals . one post transcription control modulator to alter protein
In one embodiment, the polypeptides , primary constructs 65 expression . As a non -limiting example, the expression of
and mmRNA can also contain sequences that encode protein VEGF may be regulated using the compounds described in
cleavage sites so that the polypeptides, primary constructs or a compound found by the methods described in Interna
US 10,703,789 B2
73 74
tional Publication Nos . WO2005118857 , WO2006065480 , has decreased binding affinity to major groove interacting
WO2006065479 and WO2006058088, each of which is partner, as compared to an unmodified nucleotide ).
herein incorporated by reference in its entirety . The polynucleotides, primary constructs, and mmRNA
The polynucleotides, primary constructs and /or mmRNA can optionally include other agents (e.g., RNAi- inducing
of the present invention may include at least one post 5 agents , RNAi agents, siRNAs, shRNAs,miRNAs, antisense
transcription controlmodulator to control translation . In one RNAs, ribozymes, catalytic DNA, RNA , RNAs that induce
embodiment, the post transcription control modulator may triple helix formation , aptamers, vectors, etc. ). In some
be a RNA regulatory sequence. As a non - limiting example, embodiments , the polynucleotides, primary constructs, or
the RNA regulatory sequence may be identified by the mmRNA may include one or more messenger RNAs (mR
methods described in International Publication No. 10 NAs) and one or more modified nucleoside or nucleotides
WO2006071903 , herein incorporated by reference in its (e.g., mmRNA molecules). Details for these polynucle
entirety . otides, primary constructs , and mmRNA follow .
III. Modifications
Polynucleotides and Primary Constructs
15
The polynucleotides , primary constructs , and mmRNA of
Herein , in a polynucleotide (such as a primary construct the invention includes a first region of linked nucleosides
encoding a polypeptide of interest, a first flanking region
or an mRNA molecule), the terms “modification ” or, as located at the 5 ' terminus of the first region , and a second
appropriate, " modified ” refer to modification with respect to flanking region located at the 3' terminus of the first region .
A , G , U or C ribonucleotides. Generally, herein , these terms In some embodiments , the polynucleotide, primary con
are not intended to refer to the ribonucleotide modifications 20 struct, or mmRNA (e.g., the first region , first flanking region ,
in naturally occurring 5'- terminalmRNA cap moieties . In a or second flanking region ) includes n number of linked
polypeptide, the term “ modification ” refers to a modification
as compared to the canonical set of 20 amino acids,moiety ) nucleosides having Formula (la ) or Formula (Ia - 1 ):
The modifications may be various distinct modifications .
In some embodiments, the coding region , the flanking 25 (la )
regions and /or the terminal regionsmay contain one , two, or
more ( optionally different) nucleoside or nucleotide modi -Y1-75 B
fications . In some embodiments, a modified polynucleotide , 1111 R4
primary construct, or mmRNA introduced to a cell may
exhibit reduced degradation in the cell, as compared to an 30
R311111
RS. R!")
211
unmodified polynucleotide, primary construct, or mmRNA.
The polynucleotides, primary constructs, and mmRNA m '
can include any useful modifi on , such as to the sugar, the Y3 =
nucleobase , or the internucleoside linkage (e.g. to a linking P
phosphate /to a phosphodiester linkage/to the phosphodiester 35 Y4
backbone ). One or more atomsof a pyrimidine nucleobase (Ia - 1 )
may be replaced or substituted with optionally substituted ?.
amino , optionally substituted thiol, optionally substituted Yl - Y5 1111R4
alkyl (e.g., methyl or ethyl), or halo ( e.g., chloro or fluoro ).
In certain embodiments, modifications (e.g., one or more 40
modifications ) are present in each of the sugar and the
internucleoside linkage . Modifications according to the pres
RS.
De
RI,
RI:
'R21
ent invention may be modifications of ribonucleic acids R2

(RNAs) to deoxyribonucleic acids (DNAs), threose nucleic Y = P
acids ( TNAs ), glycol nucleic acids (GNAs), peptide nucleic 45
acids (PNAs), locked nucleic acids (LNAs) or hybrids
thereof). Additional modifications are described herein .
As described herein , the polynucleotides , primary con or a pharmaceutically acceptable saltor stereoisomer
structs , and mmRNA of the invention do not substantially thereof,
induce an innate immune response of a cell into which the 50 wherein
mRNA is introduced . Features of an induced innate immune U is O , S , N (R )nu , or C (R'nu, wherein nu is an integer
response include 1 ) increased expression of pro - inflamma from 0 to 2 and each Rº is , independently , H , halo , or
tory cytokines, 2 ) activation of intracellular PRRS (RIG -I, optionally substituted alkyl;
MDA5, etc , and /or 3 ) termination or reduction in protein is a single bond or absent;
translation . 55 each of R ', R ?', R ", R ?", R1, R2, R3, and R is, indepen
In certain embodiments , itmay desirable to intracellularly dently , if present, H , halo , hydroxy, thiol, optionally substi
degrade a modified nucleic acid molecule introduced into tuted alkyl, optionally substituted alkoxy , optionally substi
the cell. For example, degradation of a modified nucleic acid tuted alkenyloxy, optionally substituted alkynyloxy,
molecule may be preferable if precise timing of protein optionally substituted aminoalkoxy, optionally substituted
production is desired . Thus, in some embodiments, the 60 alkoxyalkoxy, optionally substituted hydroxyalkoxy , option
invention provides a modified nucleic acid molecule con ally substituted amino , azido , optionally substituted aryl,
taining a degradation domain , which is capable of being optionally substituted aminoalkyl, optionally substituted
acted on in a directed manner within a cell. In another aminoalkenyl, optionally substituted aminoalkynyl, or
aspect, the present disclosure provides polynucleotides com absent; wherein the combination of R3 with one or more of
prising a nucleoside or nucleotide that can disrupt the 65 R1', R1" , R2', R2", or R5 ( e.g., the combination ofRl' and
binding of a major groove interacting , e.g. binding , partner R3, the combination of R1" and R3, the combination of R2'
with the polynucleotide ( e.g., where the modified nucleotide and R3, the combination of R2 " and R3, or the combination
US 10,703,789 B2
75 76
of R5 and R3) can join together to form optionally substi -continued
tuted alkylene or optionally substituted heteroalkylene and , (Ia - 3)
taken together with the carbons to which they are attached ,
provide an optionally substituted heterocyclyl (e.g., a bicy Fyl - 45 B
clic , tricyclic, or tetracyclic heterocyclyl); wherein the com 5
bination of R5 with one or more of R1', R1" , R2', or R2" R31111 RI ' R4
RI:
(e.g., the combination ofRl' and R5, the combination ofR1" p2u ' R2"
and R5, the combination of R2' and R5, or the combination
of R2" and R5) can join together to form optionally substi m'
tuted alkylene or optionally substituted heteroalkylene and , 10
Y = P
taken together with the carbons to which they are attached ,
provide an optionally substituted heterocyclyl (e.g., a bicy Y4
clic, tricyclic , or tetracyclic heterocyclyl); and wherein the (Ia - 4 )
combination of R4 and one or more of R " , R ?", R ?', R ?", R , 15 Yl - Y5 B
or R $ can join together to form optionally substituted alky
lene or optionally substituted heteroalkylene and , taken RS
together with the carbons to which they are attached , provide
an optionally substituted heterocyclyl ( e.g., a bicyclic, tri
cyclic , or tetracyclic heterocyclyl); each of m ' and m " is, 20 m'
independently , an integer from 0 to 3 (e.g., from 0 to 2 , from
0 to 1 , from 1 to 3 , or from 1 to 2 ); Y = P
each of Y ?, Y ?, and Y >, is , independently, O , S , Se , Y4
-NRNI , optionally substituted alkylene, or optionally
substituted heteroalkylene, wherein RM is H , optionally 25 (Ia -5 )
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, or absent; -Y1-75 B
each Y4 is , independently , H , hydroxy, thiol, boranyl, ' R4
optionally substituted alkyl, optionally substituted alkenyl, RS. R!
optionally substituted alkynyl, optionally substituted alkoxy , 30 R2"
optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted thioalkoxy , optionally R2
substituted alkoxyalkoxy, or optionally substituted amino; Y = P
each Y is , independently, O , S , Se, optionally substituted
alkylene (e.g., methylene ), or optionally substituted het 35 Y4
eroalkylene ;
n is an integer from 1 to 100,000 ; and
B is a nucleobase ( e.g., a purine , a pyrimidine , or deriva In some embodiments , the polynucleotide, primary con
tives thereof), wherein the combination of B and R '', the struct, or mmRNA (e.g., the first region , the first flanking
combination of B and R2', the combination of B and R !", or region , or the second flanking region ) includes n number of
the combination of B and R2" can , taken together with the 40 linked nucleosides having Formula ( Ib ) or Formula ( Ib - 1):
carbons to which they are attached , optionally form a
bicyclic group (e.g., a bicyclic heterocyclyl) or wherein the
combination of B , RI", and R3 or the combination of B , R2", (Ib )
and R3 can optionally form a tricyclic or tetracyclic group 45 B
( e.g., a tricyclic or tetracyclic heterocyclyl, such as in
Formula (Ilo )-( IIp ) herein ). In some embodiments, the poly 71
nucleotide , primary construct, or mmRNA includes a modi R

R4
fied ribose . In some embodiments, the polynucleotide, pri
mary construct , or mmRNA ( e.g., the first region , the first 50 R5 72
flanking region , or the second flanking region ) includes n Y} = P
number of linked nucleosides having Formula (Ia - 2 )-( 1a -5 )
or a pharmaceutically acceptable saltor stereoisomer Y4
thereof. ( Ib - 1)
55 B
(Ia - 2) R3'
yl B R4
60 RS y2
R Y =
R2
Y = P
65
or a pharmaceutically acceptable saltor stereoisomer
thereof,
US 10,703,789 B2
77 78
wherein alkoxyalkoxy , optionally substituted hydroxyalkoxy , option
U is O , S , N (Rºnu , or C (R'nu, wherein nu is an integer ally substituted amino , azido , optionally substituted aryl,
from 0 to 2 and each Rº is , independently, H , halo , or optionally substituted aminoalkyl, optionally substituted
optionally substituted alkyl; aminoalkenyl, or optionally substituted aminoalkynyl ,
is a single bond or absent; 5 wherein one and only one of B ', B2, and B3 is a nucleobase ;
each of R ', R3, R3" , and R * is , independently, H , halo , each of Rbl, Rb2, Rb3, R3, and R? is, independently , H ,
hydroxy, optionally substituted alkyl, optionally substituted halo , hydroxy, thiol, optionally substituted alkyl, optionally
alkoxy , optionally substituted alkenyloxy, optionally substi substituted alkoxy , optionally substituted alkenyloxy,
tuted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkynyloxy, optionally substituted
optionally substituted alkoxyalkoxy , optionally substituted 10 aminoalkoxy, optionally substituted alkoxyalkoxy, option
hydroxyalkoxy, optionally substituted amino , azido , option ally substituted hydroxyalkoxy, optionally substituted
ally substituted aryl, optionally substituted aminoalkyl, amino, azido , optionally substituted aryl, optionally substi
optionally substituted aminoalkenyl, optionally substituted tuted aminoalkyl, optionally substituted aminoalkenyl or
aminoalkynyl, or absent; and wherein the combination of R 15 optionally substituted aminoalkynyl;
and R3' or the combination of R1 and R3" can be taken each of Yl, Y , and Y , is , independently, O , S , Se,
together to form optionally substituted alkylene or option -NRN1_ , optionally substituted alkylene , or optionally
ally substituted heteroalkylene ( e.g., to produce a locked substituted heteroalkylene, wherein RM is H , optionally
nucleic acid ); substituted alkyl, optionally substituted alkenyl, optionally
each RS is , independently , H , halo , hydroxy, optionally 20 substituted alkynyl, or optionally substituted aryl;
substituted alkyl, optionally substituted alkoxy, optionally each Y4 is , independently , H , hydroxy, thiol, boranyl,
substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted aminoalkoxy, optionally substituted optionally substituted alkynyl, optionally substituted alkoxy,
alkoxyalkoxy, or absent;
each of Y !, Y ?, and Y3 is , independently , O , S , Se , 25 alkynyloxy, optionally alkenyloxy
optionally substituted , optionally substituted
-NRMI , optionally substituted alkylene, or optionally substituted alkoxyalkoxy, or optionallythioalkoxy
substituted , optionally
substituted amino;
substituted heteroalkylene, wherein RM is H , optionally each Y is , independently , O , S , Se , optionally substituted
substituted alkyl, optionally substituted alkenyl , optionally alkylene (e.g., methylene), or optionally substituted het
substituted alkynyl, or optionally substituted aryl;
each Y4 is, independently, H , hydroxy, thiol, boranyl, eroalkylene;
optionally substituted alkyl, optionally substituted alkenyl, 30 n is an integer from 1 to 100,000 ; and
optionally substituted alkynyl, optionally substituted alkoxy , wherein the ring including U can include one or more
optionally substituted alkenyloxy, optionally substituted double bonds.
alkynyloxy, optionally substituted alkoxyalkoxy, or option In particular embodiments , the ring including U does not
ally substituted amino ; 35 have a double bond between UCBRE or between
n is an integer from 1 to 100,000 ; and CBRb3_CB2Rb2 .
B is a nucleobase . In some embodiments , the polynucleotide, primary con
In some embodiments, the polynucleotide, primary con struct , or mmRNA (e.g., the first region , first flanking region ,
struct, ormmRNA (e.g. ,the first region , first flanking region , or second flanking region ) includes n number of linked
or second flanking region ) includes n number of linked 40 nucleosides having Formula (Id ):
nucleosides having Formula (Ic ):
(Id )
(Ic ) B
Yl— YS B3 45 -Yl — Y
Rb3
R3 B1 B2
R3
RS
RO2
Y3 =
Roi 50 |
Y = P Y4
Y4 a pharmaceutically acceptable saltor stereoisomer
or
thereof,
or a pharmaceutically acceptable salt or stereoisomer 55 wherein
thereof, U is O , S , N (Rºnus or C (R'nu , wherein nu is an integer
wherein from 0 to 2 and each Rº is , independently, H , halo , or
U is O , S , N (Rºnu, or C (Rºnu,wherein nu is an integer optionally substituted alkyl;
from 0 to 2 and each R is , independently, H , halo , or each R? is, independently , H , halo , hydroxy, thiol, option
optionally substituted alkyl; 60 ally substituted alkyl, optionally substituted alkoxy, option
is a single bond or absent; ally substituted alkenyloxy , optionally substituted alkyny
each of B ', B2, and Bº is , independently , a nucleobase loxy , optionally substituted aminoalkoxy, optionally
(e.g., a purine, a pyrimidine, or derivatives thereof, as substituted alkoxyalkoxy, optionally substituted hydroxy
described herein ), H , halo , hydroxy, thiol, optionally sub alkoxy, optionally substituted amino , azido , optionally sub
stituted alkyl, optionally substituted alkoxy, optionally sub- 65 stituted aryl, optionally substituted aminoalkyl, optionally
stituted alkenyloxy , optionally substituted alkynyloxy, substituted aminoalkenyl, or optionally substituted amino
optionally substituted aminoalkoxy , optionally substituted alkynyl;
US 10,703,789 B2
79 80
each of Y ' , Y ?, and Y ?, is, independently , O , S , Se ,
-NRN1_ , optionally substituted alkylene , or optionally ( If)
substituted heteroalkylene, wherein RM is H , optionally Yl - Y5 B
substituted alkyl, optionally substituted alkenyl, optionally 5 "R4
substituted alkynyl, or optionally substituted aryl; R3 RI"
Rii
each Y4 is , independently, H , hydroxy, thiol, boranyl, R2. U" * R2"
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy, 10
optionally substituted alkenyloxy , optionally substituted Y =
alkynyloxy, optionally substituted thioalkoxy , optionally Y4
substituted alkoxyalkoxy , or optionally substituted amino ; (If- 1 )
each Ys is , independently, O , S, optionally substituted 15
Yl — Y B
alkylene (e.g. , methylene), or optionally substituted het *R4
eroalkylene ; R !! RII
n is an integer from 1 to 100,000 ; and R2 R2"
B is a nucleobase (e.g., a purine, a pyrimidine, or deriva 20
tives thereof) . Y = P
In some embodiments , the polynucleotide, primary con Y4
struct, or mmRNA ( e.g., the first region , first flanking region ,
or second flanking region ) includes n number of linked 25 or a pharmaceutically acceptable salt or stereoisomer
nucleosides having Formula (le ): thereof,
wherein
each of U 'and U " is , independently, O , S, N , N (Rºmu, or
(le ) N (Rumus or C (R ) , wherein nu is an integer from 0 to 2
30
and each R is, independently , H , halo , or optionally sub
Y5' B stituted alkyl ( e.g., U ' is O and U " is N );
'N is a single bond or absent;
each of R " , R2', R ?", R ?" , R ", and R * is , independently, H ,
U" halo , hydroxy, thiol, optionally substituted alkyl, optionally
substituted alkoxy , optionally substituted alkenyloxy,
RO - N 35 optionally substituted alkynyloxy, optionally substituted
aminoalkoxy, optionally substituted alkoxyalkoxy, option
ally substituted hydroxyalkoxy , optionally substituted
or a pharmaceutically acceptable saltor stereoisomer amino , azido , optionally substituted aryl, optionally substi
thereof,
tuted aminoalkyl, optionally substituted aminoalkenyl,
40 optionally substituted aminoalkynyl, or absent; and wherein
wherein the combination of Rl' and R3, the combination of Rl" and
each of U ' and U " is, independently , O , S, N (Rºnu or R3, the combination ofR2' and R3, or the combination of R2"
C (R ')mu, wherein nu is an integer from 0 to 2 and each R? and R? can be taken together to form optionally substituted
alkylene or optionally substituted heteroalkylene (e.g., to
is , independently , H , halo , or optionally substituted alkyl; 45 produce a locked nucleic acid ); each of m ' and m " is ,
each Róis, independently, H , halo , hydroxy, thiol, option independently , an integer from 0 to 3 (e.g., from 0 to 2 , from
ally substituted alkyl, optionally substituted alkoxy , option 0 to 1, from 1 to 3 , or from 1 to 2 );
ally substituted alkenyloxy , optionally substituted alkyny each of Yl, Y ?, and Y }, is , independently, O , S , Se ,
NRNI , optionally substituted alkylene, or optionally
loxy , optionally substituted aminoalkoxy, optionally 50
substituted heteroalkylene, wherein RM1 is H , optionally
substituted alkoxyalkoxy, optionally substituted hydroxy substituted alkyl, optionally substituted alkenyl, optionally
alkoxy , optionally substituted amino , azido , optionally sub substituted alkynyl, optionally substituted aryl, or absent;
each Y4 is , independently, H , hydroxy, thiol, boranyl,
stituted aryl, optionally substituted aminoalkyl, optionally optionally substituted alkyl, optionally substituted alkenyl,
substituted aminoalkenyl, or optionally substituted amino optionally substituted
alkynyl; alkynyl, optionally substituted alkoxy,
55 optionally substituted alkenyloxy, optionally substituted
each Ys' is , independently , O , S , optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally
alkylene (e.g., methylene or ethylene ), or optionally substi substituted alkoxyalkoxy , or optionally substituted amino ;
tuted heteroalkylene; each Y is , independently, O , S , Se , optionally substituted
alkylene (e.g., methylene), or optionally substituted het
n is an integer from 1 to 100,000 ; and 60 eroalkylene;
B is a nucleobase (e.g., a purine, a pyrimidine, or deriva n is an integer from 1 to 100,000 ; and
tives thereof). B is a nucleobase ( e.g., a purine, a pyrimidine , or deriva
In some embodiments, the polynucleotide , primary con tives thereof). In some embodiments of the polynucleotides, primary
struct, or mmRNA (e.g., the first region, first flanking region , 65 constructs , or mmRNA ( e.g., Formulas ( la ), ( Ia- 1)-(Ia-3 ),
or second flanking region ) includes n number of linked ( Ib )-(If), and ( IIa )-(IIp )), the ring including U has one or two
nucleosides having Formula (If) or (If -1 ): double bonds .
US 10,703,789 B2
81 82
In some embodiments of the polynucleotides , primary In some embodiments of the polynucleotides, primary
constructs , or mmRNA ( e.g., Formulas (la )-( Ia -5 ), ( b )- (If constructs, or mmRNA ( e.g., Formulas (la )-(Ia -5 ), ( Ib )-( If
1 ), ( IIa )-( IIp ), ( IIb - 1 ), (IIb - 2 ), (IIc - 1 )- (IIC -2 ), (IIn - 1) , (IIn -2 ), 1), ( IIa )-( IIp ), (IIb - 1), ( IIb - 2 ), ( IIc- 1 )-(IIc- 2 ), (IIn - 1), (IIn - 2 ),
(IVA )-(IV1), and ( IXa)-(IXr)), each of R ', R " , and R !", if (IVA)-(IV1), and (IXa )-(IXr)), RS and one or more of R " ,
present, is H. In further embodiments , each of R2, R2', and 5 R '', R2', or R ?" join together to form optionally substituted
R >", if present, is , independently, H , halo ( e.g., fluoro ), alkylene or optionally substituted heteroalkylene and , taken
hydroxy, optionally substituted alkoxy ( e.g., methoxy or together with the carbons to which they are attached , provide
ethoxy), or optionally substituted alkoxyalkoxy . In particu an optionally substituted heterocyclyl (e.g., a bicyclic , tri
lar embodiments , alkoxyalkoxy is (CH2) (OCH_CH2)s1 cyclic , or tetracyclic heterocyclyl, R? and one or more of R " ,
(CH2),3OR',wherein s1 is an integer from 1 to 10 ( e.g.,from 10 R "(,CH2 R ”,51orO R(CH2
" join) 62O together
(CH2) 63—to, form heteroalkylene
each of bl(, e.g. b2,,
1 to 6 or from 1 to 4 ), each of s2 and s3, independently , is and b3 )are wherein
, independently, an integer from 0 to 3 ).
an integer from 0 to 10 ( e.g., from 0 to 4 , from 0 to 6 , from
1 to 4 , from 1 to 6 , or from 1 to 10 ), and R ' is H or constructs, orembodiments In some of the polynucleotides , primary
C1-20alkyl). In some embodiments , s2 is 0, sl is 1 or 2, s3 15 1), (IIa ) -(IIp ), ( IIb - 1 ), (IIb - 2 ),, (Formulas
mmRNA ( e.g. (la )-(Ia - 5), ( Ib )-( If
IIC - 1 )-( IIc -2 ), (IIn - 1 ), ( IIn - 2 ),
is 0 or 1 , and R ' is C1-6 alkyl. (IVA)-(IV1), and (IXa)-(IXr)), each Y² is , independently, O ,
In some embodiments of the polynucleotides , primary S , or - NRM—, wherein RM is H , optionally substituted
constructs , or mmRNA (e.g., Formulas (la )-(Ia - 5 ), ( Ib )-(If alkyl, optionally substituted alkenyl, optionally substituted
1 ), ( Ila )- ( llp ), ( IIB - 1 ), (IIb - 2 ), (IIC- 1 )- ( IIC - 2 ), ( IIn - 1 ), ( IIn -2 ), alkynyl, or optionally substituted aryl. In particular embodi
(IVa )-(IV1), and (IXa )-(IXr)), each of R ?, R ', and R ?", if 20 ments, Y2 is NRNI wherein RM is H or optionally
present, is H. In further embodiments , each of R1, R1, and substituted alkyl ( e.g., C1-6 alkyl, such as methyl, ethyl,
R !", if present, is , independently , H , halo (e.g., fluoro ), isopropyl, or n - propyl).
hydroxy, optionally substituted alkoxy (e.g. , methoxy or In some embodiments of the polynucleotides , primary
ethoxy ), or optionally substituted alkoxyalkoxy. In particu constructs, or mmRNA ( e.g., Formulas (Ia )-( Ia - 5 ), ( Ib )-( If
lar embodiments, alkoxyalkoxy is (CH2)sz(OCH2CH2)s? 25 1 ), (IIa )-(IIp ), (IIb -1), ( IIb - 2), (IIc- 1)-(IIc- 2), (IIn - 1), ( IIn - 2 ),
(CH2) 53OR ', wherein sl is an integer from 1 to 10 (e.g., from (IVA )-(IV1), and (IXa )-( IXr)), each Y3 is , independently , O
1 to 6 or from 1 to 4 ), each of s2 and s3, independently , is or S.
an integer from 0 to 10 (e.g., from 0 to 4 , from 0 to 6 , from In some embodiments of the polynucleotides, primary
1 to 4 , from 1 to 6 , or from 1 to 10 ), and R ' is H or C1-20 constructs, or mmRNA (e.g., Formulas (la )-([ a-5 ), ( Ib )-( If
alkyl). In some embodiments , s2 is 0 , sl is 1 or 2 , s3 is 0 or 30 1), ( Ila )-(IIp ), (IIb - 1), ( IIb - 2 ), (IIC- 1)-(IIC -2 ), (IIn - 1), (IIn - 2 ),
1 , and R ' is C1-6 alkyl. (IVA)- (IV1), and (IXa)-(IXr) ), R is H ; each R2 is, indepen
In some embodiments of the polynucleotides, primary dently , H , halo (e.g., fluoro ), hydroxy, optionally substituted
constructs, or mmRNA (e.g., Formulas (la )-(la -5 ), (lb )-(If alkoxy ( e.g., methoxy or ethoxy ), or optionally substituted
1 ), (IIa )-( IIp ), (IIb - 1), (IIb - 2 ), ( IIC - 1 )-(IIC - 2 ), (IIn - 1), (IIn - 2 ), alkoxyalkoxy (e.g., (CH2), (OCH2CH2)s? (CH2) s3OR ',
(IVa )-( IV1), and (IXa)-(IXr)), each of R3, R4, and RS is, 35 wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or
independently , H , halo ( e.g., fluoro ), hydroxy, optionally from 1 to 4 ), each of s2 and s3 , independently , is an integer
substituted alkyl, optionally substituted alkoxy (e.g. , from 0 to 10 (e.g., from 0 to 4 , from 0 to 6 , from 1 to 4 , from
methoxy or ethoxy ), or optionally substituted alkoxyalkoxy. 1 to 6 , or from 1 to 10 ), and R ' is H or C1-20 alkyl, such as
In particular embodiments, R3 is H , R4 is H , R is H , or R3, wherein s2 is 0 , sl is 1 or 2 , s3 is 0 or 1 , and R ' is C1-6 alkyl);
R *, and Rø are all H. In particular embodiments , R² is C1-6 40 each Y2 is , independently , 0 or -NRAN1 wherein RNi is
alkyl, R * is C1-6 alkyl,R? is C1-6 alkyl, or R3, R4, and Rs are H , optionally substituted alkyl, optionally substituted alk
all C1-6 alkyl. In particular embodiments ,R3 and R4 are both enyl , optionally substituted alkynyl, or optionally substi
H , andRis C1-6 alkyl. tuted aryl (e.g., wherein RM is H or optionally substituted
In some embodiments of the polynucleotides, primary alkyl (e.g., C-, alkyl, such as methyl, ethyl, isopropyl, or
constructs , or mmRNA (e.g., Formulas (la )-(Ia -5), (lb )-(If- 45 n -propyl)); and each Y is, independently , O or S (e.g., S ).
1 ), (IIa )-( IIp ), (IIb - 1), (IIb - 2 ), ( IIC - 1 )-(IIC - 2 ), (IIn - 1), (IIn - 2 ), In further embodiments, R3 is H , halo (e.g. , fluoro ),hydroxy,
(IVa )-(IV1), and (IXa )-(IXr)), R3 and RS join together to optionally substituted alkyl, optionally substituted alkoxy
form optionally substituted alkylene or optionally substi (e.g.,methoxy or ethoxy ), or optionally substituted alkoxy
tuted heteroalkylene and , taken together with the carbons to alkoxy. In yet further embodiments , each Yl is , indepen
which they are attached , provide an optionally substituted 50 dently , O or NRM—, wherein RM is H , optionally sub
heterocyclyl (e.g., a bicyclic , tricyclic , or tetracyclic hetero stituted alkyl, optionally substituted alkenyl, optionally
cyclyl, such as trans -3 % 4' analogs,wherein R3 and RS join substituted alkynyl, or optionally substituted aryl (e.g.,
together to form heteroalkylene (e.g., ( CH2) .O (CH2) 620 wherein RM is H or optionally substituted alkyl (e.g., C1-6
( CH2)63 , wherein each of bi, b2 , and b3 are, indepen alkyl, such as methyl, ethyl, isopropyl, or n -propyl)); and
dently , an integer from 0 to 3 ). 55 each Y4 is, independently , H , hydroxy, thiol, optionally
In some embodiments of the polynucleotides, primary substituted alkyl, optionally substituted alkoxy, optionally
constructs, or mmRNA ( e.g., Formulas (la)-(Ia -5 ), ( Ib )-( If substituted thioalkoxy , optionally substituted alkoxyalkoxy,
1 ), (IIa )-( IIp ), (IIb - 1), (IIb - 2 ), (IIC - 1) -(IIC - 2 ), (IIn - 1 ), (IIn -2 ), or optionally substituted amino .
(IVA)-(IV1), and (IXa)-( IXr)), R3 and one or more of R '', In some embodiments of the polynucleotides, primary
R ?", R2, R ?", or RS join together to form optionally substi- 60 constructs, or mmRNA (e.g., Formulas ( la )-(1a-5), ( Ib )-(If
tuted alkylene or optionally substituted heteroalkylene and , 1 ), ( IIa )-( IIp ), ( IIb - 1 ), (11b - 2 ), (IIc - 1 )- (IIC -2 ), (IIn - 1) , ( IIn - 2 ),
taken together with the carbons to which they are attached , (IVa )-(IV1), and (IXa )-(IXr)), each R ' is , independently, H ,
provide an optionally substituted heterocyclyl ( e.g., a bicy halo (e.g., fluoro ), hydroxy , optionally substituted alkoxy
clic ,tricyclic , or tetracyclic heterocyclyl,R3 and one or more ( e.g., methoxy or ethoxy ), or optionally substituted alkoxy
of R '', R ", R ', R ?", or R $ join together to form heteroalky- 65 alkoxy (e.g., (CH2),2(OCH CH2)s?(CH2),ZOR', wherein
lene ( e.g., - (CH2)61O (CH2) 62O (CH2) 63— , wherein each of sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to
b1, b2 , and b3 are, independently , an integer from 0 to 3). 4 ), each of s2 and s3 , independently , is an integer from 0 to
US 10,703,789 B2
83 84
10 ( e.g., from 0 to 4 , from 0 to 6 , from 1 to 4 , from 1 to 6 ,
or from 1 to 10 ), and R ' is H or C1-20 alkyl, such as wherein (IIa )
s2 is 0 , sl is 1 or 2, s3 is 0 or 1, and R ' is C1-6 alkyl); R2 is Yl - Y5 B
H ; each Y is, independently , O or - NRW wherein R ^ N1 U
is H , optionally substituted alkyl, optionally substituted 5

alkenyl, optionally substituted alkynyl, or optionally substi R4
tuted aryl (e.g., wherein RM is H or optionally substituted R3
alkyl (e.g. , C1-6 alkyl, such as methyl, ethyl, isopropyl, or R2
n -propyl)); and each Yº is , independently, 0 or S (e.g., S ). 10 Y = P
In further embodiments, R3 is H , halo (e.g., fluoro ),hydroxy, -
optionally substituted alkyl, optionally substituted alkoxy Y4

(e.g., methoxy or ethoxy), or optionally substituted alkoxy
alkoxy . In yet further embodiments, each Y ' is, indepen
dently , O or --NRNI—
NRM_ , wherein RM1 is H , optionally sub 15
stituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, or optionally substituted aryl ( e.g.,
wherein RM1 is H or optionally substituted alkyl (e.g., C1-6 ( IIb )
alkyl, such as methyl, ethyl, isopropyl, or n - propyl)); and Y5 B
each Y4 is , independently, H , hydroxy, thiol, optionally 20
substituted alkyl, optionally substituted alkoxy, optionally R4
substituted thioalkoxy, optionally substituted alkoxyalkoxy,
or optionally substituted amino . R2
In some embodiments of the polynucleotides, primary
constructs , or mmRNA (e.g., Formulas (la )-(Ia -5 ), ( Ib ) -(If- 25 Y = P or
1), ( IIa)-(IIp ), (IIb - 1), (IIb -2 ), ( IIc - 1)-( IIc -2 ), ( IIn -1), (IIn - 2),
(IVa )-(IV1), and (IXa)-(IXr )), the ring including U is in the Y4
B -D ( e.g., B - D - ribo ) configuration .
In some embodiments of the polynucleotides, primary 30
constructs , or mmRNA (e.g., Formulas (la )-(Ia - 5 ), ( b )-(If
1 ), ( Ila )-(IIp ), (IIb - 1 ), ( IIb -2 ), ( IIc - 1 )-(IIc -2 ), (IIn - 1 ), (IIn - 2 ),
( IVA)- (IV1), and (IXa)-(IXr) ), the ring including U is in the (IIC )
C - L (e.g., a -L -ribo ) configuration . B
In some embodiments of the polynucleotides, primary 35
constructs , or mmRNA (e.g., Formulas (la )-(Ia - 5 ), ( Ib )-(If
1 ), (Ila )-( IIp ), (IIb - 1 ), ( IIb -2 ), ( IIc - 1)- (IIc- 2 ), ( IIn - 1 ), (IIn -2 ), R3
R4
(IVA )-(IV1), and (IXa)-(IXr)), one or more B is not pseudou
ridine (W ) or 5 -methyl-cytidine (mC). In some embodi 40
y2 R2
ments, about 10 % to about 100 % of n number of B nucle
obases is not y ormC (e.g. , from 10 % to 20 % , from 10 % Y = P
to 35 % , from 10 % to 50 % , from 10 % to 60 % , from 10 % to
75 % , from 10 % to 90 % , from 10 % to 95 % , from 10 % to
98 % , from 10 % to 99 % , from 20 % to 35 % , from 20 % to 45 a pharmaceutically acceptable saltor stereoisomer
50 % , from 20 % to 60 % , from 20 % to 75 % , from 20 % to orthereof
90 % , from 20 % to 95 % , from 20 % to 98 % , from 20 % to wherein. Innu particular is an
embodiments, U is O or C (Rºnus
integer from 0 to 2 and each Rº is ,
99 % , from 20 % to 100 % , from 50 % to 60 % , from 50 % to
75 % , from 50 % to 90 % , from 50 % to 95 % , from 50 % to 50 independently , H , halo , or optionally substituted alkyl ( e.g.,
U is CH2 or CH- ). In other embodiments, each of
98 % , from 50 % to 99 % , from 50 % to 100 % , from 75 % to R ', R2, R3, R4, and R is, independently , H , halo , hydroxy ,
90 % , from 75 % to 95 % , from 75 % to 98 % , from 75 % to thiol, optionally substituted alkyl, optionally substituted
99 % , and from 75 % to 100 % of n number of B is not y or alkoxy, optionally substituted alkenyloxy, optionally substi
mC). In some embodiments , B is not y or m®C . tuted alkynyloxy, optionally substituted aminoalkoxy,
In some embodiments of the polynucleotides, primary 55 optionally substituted alkoxyalkoxy, optionally substituted
constructs, or mmRNA ( e.g., Formulas (la)-(Ia -5 ), ( Ib )-( If hydroxyalkoxy, optionally substituted amino , azido, option
1 ), ( Ila )-(IIp ), (IIb - 1), (IIb - 2 ), (IIc- 1)- (IIC - 2 ), ( IIn -1 ), (IIn -2 ), ally substituted aryl, optionally substituted aminoalkyl,
(IV )-( IV1), and ( IXa)- (IXr )), when B is an unmodified optionally substituted aminoalkenyl, optionally substituted
nucleobase selected from cytosine, guanine , uracil and 60 aminoalkynyl, or absent ( e.g., each R and R2 is , indepen
adenine, then at least one of Y ?, Y ?, or Y3 is not 0 . dently , H , halo , hydroxy , optionally substituted alkyl, or
In some embodiments, the polynucleotide , primary con optionally substituted alkoxy ; each R3 and R * is, indepen
struct, or mmRNA includes a modified ribose. In some dently, H or optionally substituted alkyl; and R is H or
embodiments, the polynucleotide , primary construct, or hydroxy ), and --- is a single bond or double bond .
mmRNA (e.g. , the first region , the first flanking region , or 65 In particular embodiments, the polynucleotidesor
the second flanking region ) includes n number of linked mmRNA includes n number of linked nucleosides having
nucleosides having Formula (IIa )-(IIc ): Formula (IIb - 1) -(IIb - 2 ):
US 10,703,789 B2
85 86
-continued
(IIb - 1 ) (IIC - 3)
B
-Yl Y5 B
5
RS
R2' y2 R2
Y} = P or 10 Y = P or
-
|
Y4 Y4
(IIb - 2 ) (IIC - 4 )
B -yl - y5 B
Yl — YS
15
RS
R2 R2
Y = P 20 Y = P
Y4 Y4
or a pharmaceutically acceptable salt or stereoisomer or a pharmaceutically acceptable salt or stereoisomer

thereof. In some embodiments, U is O or C (R'nu, wherein 25 thereof
nu is an integer from 0 to 2 and each Rºis, independently , nu is an. Ininteger some embodiments, U is O or C (R'nu, wherein
from 0 to 2 and each Rºis , independently ,
H , halo , or optionally substituted alkyl ( e.g., U is CH , H , halo , or optionally substituted alkyl (e.g., U is CH2
orindependently
CH- ). ,InH ,other embodiments , each of R1 and R ? is ,
halo , hydroxy, thiol, optionally substituted or CH— ). In some embodiments, each of R ', R ?, and R3
alkyl, optionally substituted alkoxy, optionally substituted 30 is, independently , H , halo , hydroxy, thiol, optionally substi
alkenyloxy, optionally substituted alkynyloxy, optionally tuted alkyl, optionally substituted alkoxy, optionally substi
substituted aminoalkoxy, optionally substituted alkoxy tuted alkenyloxy, optionally substituted alkynyloxy, option
alkoxy, optionally substituted hydroxyalkoxy, optionally ally substituted aminoalkoxy, optionally substituted
substituted amino , azido, optionally substituted aryl, option alkoxyalkoxy, optionally substituted hydroxyalkoxy, option
ally substituted
alkenyl , optionallyaminoalkyl
substituted, optionally substituted
aminoalkynyl , or absentamino-
(e.g., 35 optionally
ally substituted amino , azido , optionally substituted aryl,
each Rl and R2 is, independently, H , halo , hydroxy, option aminoalkenyl substituted aminoalkyl, optionally substituted
ally substituted alkyl, or optionally substituted alkoxy, e.g., absent (e.g., ,eachoptionally substituted aminoalkynyl, or
H , halo , hydroxy, alkyl, or alkoxy). In particular embodi hydroxy, optionally R substituted ' and R2 is , independently , H , halo ,
alkyl, or optionally substi
ments, R2, ethoxy
methoxy is hydroxy or optionally
, or any substituted
described herein ). alkoxy (e.g.s 40 tuted alkoxy, e.g., H , halo , hydroxy , alkyl, or alkoxy; and
In particular embodiments, the polynucleotide, primary each R is , independently, H or optionally substituted
construct, or mmRNA includes n number of linked nucleo alkyl)) . In particular embodiments, R2 is optionally substi
sides having Formula ( IIc -1)-( IIc -4 ): tuted alkoxy ( e.g., methoxy or ethoxy, or any described
45 herein ). In particular embodiments, Rl is optionally substi
tuted alkyl, and R² is hydroxy. In other embodiments, R ' is
( IIC - 1) hydroxy, and R2 is optionally substituted alkyl. In further
-Yl- Y B embodiments, R is optionally substituted alkyl.
In some embodiments , the polynucleotide, primary con
50 struct, or mmRNA includes an acyclic modified ribose . In
some embodiments, the polynucleotide, primary construct,
R2 ormmRNA (e.g. , the first region , the first flanking region , or
the second flanking region ) includes n number of linked
Y = P nucleosides having Formula (IId )- (IIf ):
| 55
(IIc -2 ) (Ild )
Yl. B
-Yl— Y5 B
R 60
-R4
R!
R31
R2 RS R2
Y} = P Y = P
-
65 |
Y4 Y4
US 10,703,789 B2
87 88
-continued -continued
( IIe ) ( II )
yl — y B3
-yl - Y .
? B
R?
-R4 5 R311111
RS
'Rb3
B2
RS Rb2
R2 ROL
Y = P
Y = or
10
—
|
Y4
-Y5 B or a pharmaceutically acceptable salt or stereoisomer
-R4 thereof.
R 15
R3 In some embodiments , the polynucleotide , primary con
RS struct, or mmRNA includes a sugar moiety having a con
R2 tracted or an expanded ribose ring . In some embodiments ,
Y = 1
the polynucleotide, primary construct, or mmRNA (e.g., the
20
first region , the first flanking region , or the second flanking
Y4 region ) includes n number of linked nucleosides having
Formula
(IIk )- (Ilm ):
thereof. 25
( IIK )
In some embodiments , the polynucleotide , primary con -yl - y5 B
struct, or mmRNA includes an acyclic modified hexitol. In
some embodiments , the polynucleotide, primary construct, R5 R
or mmRNA ( e.g., the first region , the first flanking region , or R3 R4
the second flanking region ) includes n number of linked 30 R2
nucleosides Formula (Ilg )- (Ilj ):
Y = P
|
(IIg ) Y4
Fyl - ys B 35
(III )
-Yl - ys B
R3311
RS
"R4
R1"
22 R2" R3 R4
R?
40 y2
Y = P
Y4 Y = P or
-
( IIH ) Y4
Yl - Y5 B 45 ( IIm )
-yl - Y
R311111
R$ R ?' R4
R 1!" R3111011 R4
RI' R1"
R5
R ?"
50 y? R2"
Y =
Y = P
R?
Y4 |
Y4
55
or a pharmaceutically acceptable salt or stereoisomer
( III) thereof, wherein each of R " , Rl", R2', and R2" is, indepen
-yl— Y : B3 dently , H , halo , hydroxy, optionally substituted alkyl,
optionally substituted alkoxy , optionally substituted alkeny
R3111111
RS B2
60 loxy, optionally substituted alkynyloxy , optionally substi
tuted aminoalkoxy, optionally substituted alkoxyalkoxy, or
Rb2 absent; and wherein the combination of R2 and R3 or the
combination of R2" and R3 can be taken together to form
Y = P or
optionally substituted alkylene or optionally substituted het
1 65 eroalkylene.
Y4 In some embodiments , the polynucleotide, primary con
struct, or mmRNA includes a locked modified ribose . In
US 10,703,789 B2
89 90
some embodiments, the polynucleotide, primary construct, forms a tetracyclic heterocyclyl. In some embodiments , the
or mmRNA (e.g., the first region , the first flanking region , or polynucleotide , primary construct, or mmRNA (e.g., the first
the second flanking region ) includes n number of linked region , the first flanking region , or the second flanking
nucleosides having Formula (IIn ): region ) includes n number of linked nucleosides having
5 Formula ( Ilo ) :
( IIn )
yl — y5 B
( IIO )
-yl Y5
10 R4
R4 -T2"
R3")
12 R3' R3 •R12a
* R ?'
Y = P 15
Y4 Il
Y = P or
|
thereof, wherein R3' is O , S , or NRMNi wherein RN1 is 20
H , optionally substituted alkyl, optionally substituted alk
enyl, optionally substituted alkynyl, or optionally substi
tuted aryl and R3" is optionally substituted alkylene (e.g.,
-CH2- , -CH2CH2- , or CH2CH2CH2– ) or option
ally substituted heteroalkylene (e.g. , CHÁNH , 25
CH , CH NH— , -CH OCH or CH ,CH OCH - )
(e.g., R3' is O and R3" is optionally substituted alkylene (e.g.,
CH , — , CH ,CH , — , or CH CH CH , - )). (IIp )
In some embodiments, the polynucleotide , primary con yl — Y
struct, or mmRNA includes n number of linked nucleosides 30 R4 T2"
having Formula (IIn - 1 )- (II-n2 ):
R3* -R12a
(IIn - 1)
yl - YS U
B 35 R 120
Y} = P
R3" ) Y4
R3' 40
Y = P
or
thereof, wherein R122, R12e , T '', Tl", T?', T ?" , V +, and V3 are
Y4 as described herein .
yl - 45 B
(IIn - 2 )
45 Any of the formulas for the polynucleotides , primary
constructs , or mmRNA can include one or more nucleobases
described herein (e.g., Formulas (b1)-(b43)).
R3" ]
In one embodiment, the present invention provides meth
ods of preparing a polynucleotide, primary construct, or
50 mmRNA, wherein the polynucleotide comprises n number
Y = P
of nucleosides having Formula ( Ia ), as defined herein :
|
Y4
(la )
55 -yl- y } B
or a pharmaceutically acceptable saltor stereoisomer R3111111 " R4
thereof, wherein R3' is O , S , or —NRMN1 wherein RN1 is RS
O
R!
H , optionally substituted alkyl, optionally substituted alk
enyl, optionally substituted alkynyl, or optionally substi R2"
tuted aryl and R. " is optionally substituted alkylene (e.g., 60 m '
-CH2CH2- , or CH2CH2CH2– ) or option
allyCH2substituted heteroalkylene (e.g., -CHINH-, Y = P
CH ,CH NH? , CH ,OCH , — , or CH , CH ,OCH , – ) Y4
(e.g., Rºis O and R *" is optionally substituted alkylene (e.g.,
CH2 -CH2CH2- , or CH2CH2CH2- )). 65
In some embodiments , the polynucleotide, primary con the method comprising reacting a compound of Formula
struct, or mmRNA includes a locked modified ribose that (IIIa ), as defined herein :
US 10,703,789 B2
91 92
nucleotide (e.g.,mmRNA molecule),wherein the polynucle
(IIIa ) otide comprises n number of nucleosides having Formula
(la -2 ), as defined herein :
Y . -Yl. B
U. 5
Y4 R392371
RS R ' 'R4
Yl — Y5
(Ia- 2 )
R2"
10 R3 R4
Y} = P -Y7 R?
Y4
Y = P
15 Y4
with an RNA polymerase , and a cDNA template .
In a further embodiment, the present invention provides
methods of amplifying a polynucleotide, primary construct, the method comprising reacting a compound of Formula
or mmRNA comprising at least one nucleotide (e.g.,
mmRNA molecule ), the method comprising : reacting a 20 (Illa- 2 ), as defined herein :
compound of Formula ( IIIa ), as defined herein , with a
primer, a cDNA template , and an RNA polymerase.
In one embodiment, the present invention provides meth
ods of preparing a polynucleotide, primary construct, or
mmRNA comprising at least one nucleotide (e.g., mmRNA
molecule), wherein the polynucleotide comprises n number 25 (IIIa - 2)
of nucleosides having Formula (la ), as defined herein :
?6. -Yl. B
(Ia - 1 )
-Yl - Y5 B 30 Y4
R!
R? R4,
??
R5
Y =
(116 ) 35 Y = P
A
R2
-Y7
172
Y4
40
the method comprising reacting a compound of Formula
(IIIa - 1 ), as defined herein :
with an RNA polymerase , and a cDNA template.
(IIIa- 1 ) 45 In a further embodiment, the present invention provides
methods of amplifying a modified mRNA comprising at
Y6
( ?? RI
B
" R4
least one nucleotide (e.g., mmRNA molecule), the method
comprising :
reacting a compound of Formula (IIIa - 2 ), as defined
Y =
RS
Of
Y7
50 herein , with a primer, a cDNA template , and an RNA
55
polymerase .
In some embodiments , the reaction may be repeated from
1 to about 7,000 times. In any of the embodiments herein , B
may be a nucleobase of Formula (b1)-(b43).
The polynucleotides, primary constructs , and mmRNA
can optionally include 5 ' and/or 3 ' flanking regions, which
with an RNA polymerase , and a cDNA template . are described herein .
In a further embodiment, the present invention provides Modified RNA (mmRNA ) Molecules
methods of amplifying a polynucleotide, primary construct, 60 The present invention also includes building blocks, e.g.,
or mmRNA comprising at least one nucleotide ( e.g., modified ribonucleosides , modified ribonucleotides, of
mmRNA molecule), the method comprising :
reacting a compound of Formula (IIIa -1 ), as defined modified RNA (mmRNA ) molecules . For example , these
herein , with a primer, a cDNA template , and an RNA building blocks can be useful for preparing the polynucle
polymerase . 65 otides, primary constructs, or mmRNA of the invention .
In one embodiment, the present invention provides meth In some embodiments , the building block molecule has
ods of preparing a modified mRNA comprising at least one Formula (IIIa ) or ( IIIa - 1 ):
US 10,703,789 B2
93 94
(b24 ), (b25 ), and (b32 )-(b36 ), such as formula (b10 ) or
(IIIa ) (b32)). In particular embodiments , Formula ( IVa) or (IVb ) is
combined with a modified guanine (e.g., any one of formulas
Y6. -Yl. ?. (b15 )-(b17 ) and (b37)-(640 )). In particular embodiments ,
—
Y4
5 Formula (IVA ) or (IVb ) is combined with a modified adenine
R311 " R4 ( e.g., any one of formulas (b18 )-(b20) and (b41)-(b43 )).
RS
In some embodiments , the building block molecule ,
which may be incorporated into a polynucleotide, primary
m ' 10 construct, or mmRNA , has Formula ( IVC)- (IVk ):
Y = P -Y7
Y4 (IVC)
that
4
(IIIa - 1 )
15 ?6.
?6. B
O
RS
20
titud
( IVd)
Y = P Y7
Y6
25

thereof, wherein the substituents are as described herein
(e.g., for Formula (la ) and (Ia -1 )), and wherein when B is an 30
Onur (IVe)
unmodified nucleobase selected from cytosine , guanine,
uracil and adenine, then at least one of Y !, Y ?, or Y? is not
0.
In some embodiments , the building block molecule,
which may be incorporated into a polynucleotide, primary 35
Y.
f Y4
R300
75 B,
construct , or mmRNA , has Formula ( IVA )-(IVb ):

HO R2
(IV ) (IV )
40
??.
Y4
B
or
45
Y6
Y4
7 R31117
Y5
HO R2
B.
( IVg )
(IVb) 50 ?6. Y5 B
to
?6. Y4
Y4
B
R311111
55
HO
HO ?? ,
(IVh)
or a pharmaceutically acceptable salt or stereoisomer 60 ?6.
thereof, wherein B is as described herein (e.g., any one of B
(61)-(b43 )). In particular embodiments , Formula (IVa ) or Y4
(IVb ) is combined with a modified uracil ( e.g., any one of Rum R !,
formulas (b1)-(69 ), (b21 )-(b23), and (b28 )-(631), such as
formula (b1), (b8), (b28 ), (b29), or (b30 )). In particular 65 HO OCH3
embodiments, Formula (IVA ) or (IVb) is combined with a
modified cytosine ( e.g., any one of formulas (b10 )- (b14 ) ,
US 10,703,789 B2
95 96
(IVA) (Vb )
R29
YO. -Yl ys B
N
5
y4 -R27,
R30 R !,
?6 .
HO
10
1
Y4
(IV ;) R31114
Y6 -Yl. B R?
Y4
R311001 R !,
15
thereof, wherein B is as described herein ( e.g., any one of
HO OCH3 (61)- (643 ) .
( IVK) In other embodiments, the building block molecule ,
20 which may be incorporated into a polynucleotide , primary
??. Y construct, or mmRNA , has Formula (IXa )- ( IXd ):
Y5 B
Y4
R !, or (IXa )
25
HO Y.
Y5 B.
(IVI)
Yl. Y5 B 30
fitry
Y4 HO
R31110 R! (IXb )
HO 35 ?6.

(61)-(643)). In particular embodiments, one of Formulas 40 HO Br,
(IVC)-(IVk ) is combined with a modified uracil (e.g., any ( IXC)
one of formulas (b1)-(69 ), (b21 )-(b23 ), and (b28 )-(b31),
such as formula (b1 ), (b8 ), (b28 ), (b29 ), or (b30 )). In
particular embodiments, one of Formulas (IVc)-(IVk ) is
combined with a modified cytosine (e.g., any one of formu- 45
las (b10 )-(b14 ), (b24 ), (b25 ), and (b32 )-(636 ), such as
formula (b10 ) or (b32 )). In particular embodiments, one of
Y6
17
Y4
B
Formulas (IVC)- (IVk ) is combined with a modified guanine

(e.g., any one of formulas (b15 )-(b17 ) and (b37 )-(b40 )). In
HON beim or
( IXd )
particular embodiments, one of Formulas (IVC )-(IVk ) is 50
combined with a modified adenine (e.g. , any one of formulas ??. YL
(618 )- (b20 ) and (641 ) -(643)) . Y5 B,
In other embodiments , the building block molecule , Y4
construct , or mmRNA , has Formula (Va ) or (Vb ):
HO
(Va ) or a pharmaceutically acceptable saltor stereoisomer
?6.
fi 3OY4
Yl
B
or
60 thereof, wherein B is as described herein (e.g., any one of
(b1)-(b43)). In particular embodiments , one of Formulas
(IXa)-( IXd) is combined with a modified uracil (e.g., any
one of formulas (b1)-(69 ), (b21 )-(b23 ), and (b28 )- (b31 ),
such as formula (b1 ), (b8), (b28 ), (b29 ), or (b30 )). In
65 particular embodiments , one of Formulas (IXa)-(IXd) is
combined with a modified cytosine (e.g., any one of formu
las (b10 )-(b14 ), (b24 ), (b25 ), and (b32 )- (636 ), such as
US 10,703,789 B2
97 98
formula (b10 ) or (b32 )). In particular embodiments, one of -continued
Formulas ( IXa)-( IXd) is combined with a modified guanine ( IXi)
(e.g., any one of formulas (b15 )-(b17 ) and (b37)-(640 )). In
4744
particular embodiments, one of Formulas (IXa)-(IXd ) is yo.
combined with a modified adenine (e.g., any one of formulas 5
(618 )- (620 ) and (b41)- (643 ) ).
In other embodiments, the building block molecule,
which may be incorporated into a polynucleotide, primary ??,
construct, or mmRNA , has Formula (IXe)-(IXg):
CH3
hty
10
( IXj)
( IXe)
Y6.
BH2
-Y ! ys B 15
of
Y6
CH3, or
HOME
thly
R ?, (IXk)
ty
(IXf) 20
Y6. -Y
Y6. B
Y4
25
OH ,
R ?, or
(IXg)
Se 30
??. -Y ! (b1)- (b43)). In particular embodiments, one of Formulas
B
Y4 (IXH )- (IXk ) is combined with a modified uracil (e.g., any
one of formulas (b1 )-(69 ), (b21 )-(b23 ), and (b28 )-(b31),
such as formula (b1), (b8 ), (b28 ), (b29 ), or (b30 )). In
35
particular embodiments, one of Formulas (IXH )-( IXk) is
HO R ?, combined with a modified cytosine (e.g., any one of formu
las (b10 ) -(b14 ), (b24 ), (b25 ), and (b32 )-(b36 ), such as
or a pharmaceutically acceptable salt or stereoisomer formula (b10 ) or (b32 )). In particular embodiments , one of
thereof,wherein B is as described herein (e.g., any one of 40 (Formulas (IXh )of-(IXk
e.g. , any one ) is combined
formulas with) anda modified
(b15 )-(b17 guanine
(b37)-(b40 )). In
(b1)-(b43 )). In particular embodiments , one of Formulas particular embodiments, one of Formulas (IXh)-(IXk ) is
( IXe)-(IXg) is combined with a modified uracil (e.g., any combined with a modified adenine ( e.g., any one of formulas
one of formulas (b1 )-(b9), (b21 )-(b23 ), and (b28 ) (b31 ),
such as formula (b1), (b8 ), (b28 ), (b29), or (b30 )). In (b18 )-(b20 ) and (b41 )-(643 )).
particular embodiments, one of Formulas ( IXe)-(IXg) is 45 In other embodiments, the building block molecule,
combined with a modified cytosine (e.g., any one of formu which may be incorporated into a polynucleotide, primary
las (b10)-(b14 ), (b24 ), (b25 ), and (b32 )-(636 ), such as construct, or mmRNA , has Formula ( IXI) -(IXr ):
formula (b10 ) or (b32 )). In particular embodiments, one of
Formulas (IXe)-(IXg ) is combined with a modified guanine
( e.g., any one of formulas (b15 )-(b17 ) and (b37)-(b40 )). In 50 ( IXI)
particular embodiments, one of Formulas (IXe)-(IXg) is
combined with a modified adenine (e.g. , any one of formulas
(618 )- (b20 ) and (b41 ) -(643 )).
In other embodiments, the building block molecule,
construct, or mmRNA , has Formula (IXh)-(IXk):
HO
OH r2 BH2 H
tillityy
?? ??,
ty (IXh) (IXm )
60
??. ?? .
B
Y4
R !,
65
HO
US 10,703,789 B2
99 100
-Adly
-continued
(IXn ) (BB - 1)
NH
B 5 N
HO
NH2,
OH , OH
they the end

10
( IXo)
HO OH
HO (BB - 2 )
B
ui
N,
thly th
HO
(IXp )
20
HO
B
HO OH
(BB - 3)
25 NH
tyy
'N ,
(IXq)
30 HO
HO
OH
OH
HO OH
35 (BB -4 )
tity tet
HO Br, or CI
( IXr)
'N
?? .
B 40 ?? .
OH
HO OCH3 45 HO OH
NH
(BB -5 )
thereof, wherein each rl and r2 is , independently, an integer CH3,
from 0 to 5 ( e.g., from 0 to 3, from 1 to 3 , or from 1 to 5 )
and B is as described herein (e.g., any one of (b1)-(b43)). In 50
particular embodiments , one of Formulas (IXI) -( IXr ) is HO
1
combined with a modified uracil (e.g., any one of formulas OH
(b1)-(b9), (b21)-(b23), and (b28 )- (631), such as formula
(bn), (b8 ), (b28 ), (b29), or (b30 )). In particular embodi
ments, one of Formulas ( IX1)-( IXr ) is combined with a 55 HO OH
modified cytosine (e.g., any one of formulas (b10 )-(b14 ), (BB - 6 )
(b24 ), (b25 ), and (b32)-(136 ), such as formula (b10 ) or
(632 )). In particular embodiments, one of Formulas (IXI)
(IXr) is combined with a modified guanine ( e.g., any one of NH ,
formulas (b15) -(b17) and (b37 )-(b40 )). In particular 60
embodiments, one of Formulas (IXI)-(IXr) is combined with
a modified adenine (e.g., any one of formulas (b18 )-(620 )
and (b41)-(b43)).
In some embodiments, the building block molecule,
construct, or mmRNA , can be selected from the group
?? .
fit
OH
HO OH
consisting of:
US 10,703,789 B2
101 102
(BB - 7) (BB -12 )
NH2
City
5 NH
'N ,
?? . HO
NH ,
OH
10
HO OH

(BB - 8 ) 15 thereof, wherein each r is , independently , an integer from 0
H3C to 5 ( e.g., from 0 to 3 , from 1 to 3 , or from 1 to 5 ) .
In some embodiments , the building block molecule ,
which may be incorporated into a polynucleotide, primary
construct, or mmRNA , can be selected from the group
20 consisting of:
HO NH ,
OH (BB -13)
25
HO OH
N,
??.
(BB - 9 )
30 OH
CH2
OH
HO (BB - 14 )
NH2, 35 H?N
OH N
HO OH
40 ?? .
OH
(BB - 10 )
45 HO OH
NH (BB - 15 )
?? .
ht
OH
HO OH
'N NH2,
50
HO
OH
N,
55 HO OH
(BB -11) (BB - 16 )
60
N,
HO HO
NH , and
OH OH
65
HO OH HO POH
US 10,703,789 B2
103 104
-continued
(BB - 17) (BB -21)
NH2
5 H3C .
NH
?? . ?6.
OH
Y4
10
HO OH
OH
(BB - 22 )
15
HO .
(BB - 18 ) NH
H
N s1 NH , ?6.
20
HO
it OH
HO OH 25
HOW OH
(BB -23)
NH
yo -Y !
(BB -19 ) 30
Y4
NH
QUI! OH
HO 35
and
OH
40
(BB -24)
H2N NH
73
45
( BB -20 ) yo Y1
Y4
NH 50
HO
OH
OH
55
OH (BB -25)
or a pharmaceutically acceptable saltor stereoisomer NH

thereof, wherein each r is , independently, an integer from 0 60
to 5 ( e.g., from 0 to 3 , from 1 to 3, or from 1 to 5 ) and sl
is as described herein .
In some embodiments, the building block molecule ,
which may be incorporated into a nucleic acid (e.g., RNA ,
mRNA , polynucleotide, primary construct, or mmRNA), is 65
Y6
fi Y4
a modified uridine (e.g., selected from the group consisting HOW OH

of:
US 10,703,789 B2
105 106
(BB -26 ) (BB -31)
5 CH3,
NH HN
re 0,
10
Yo .
Y4
-Y
HO OH OH
(BB - 27 ) (BB - 32)
15
F3C N
NH NH
y6 . - Yl Y6 YL
20
Y4
HO OH HO "OH
25 (BB - 33 )
HN OCH3,
(BB -28)
30
Y6 Yl
HN NH Y4
-
y6
Y4
Yl
35
HO OH
HOW OH
40 (BB - 34 )
OCH3,
HN
(BB - 29 ) 45
?6.
NH Y4
73
N
Y6 Y! 50
OH
Y4
OH 55
(BB - 35 )
(BB - 30 )
H3C . N NH
HN NH2,
?6.
Y4
HON OH
60
65
yo
€ Y4
-Y
OH
US 10,703,789 B2
107 108
(BB - 36 ) (BB -41)
5
*CF3, HN N
Y6
th Y4 10
Y6
Y4
-YL
HOW
N
"OH
CF3
OH
(BB - 37 ) 15 (BB -42)
OH ,
HN NH , HN
41
73
20
?6 . ??.
Y4 Y4
25
HO OH HO OH
(BB -38 ) (BB -43)
HN CF3, 30
ZT HN
OH ,
76 -YL
-
?6 S
Y4 -
35 Y4
HO
HO OH
40 (BB -44 )
(BB -39)
OH ,
CH3, HN
HN
45
Y6
f Y4
YI
50
?6.
Y4
Yl
OCH3
OH
55 (BB -45)
(BB -40 )
CH3, OFmoc,
HN HN
?6.
f Y4
CF3
60
65
Y6
Y4
N
HO POH HOS OH
US 10,703,789 B2
109 110
(BB -46 ) (BB -51)
5
OFmoc , HN ?? ,
HN
Y6.
f Y4 10
Y6.
Y4
N
OH
HOW OH
OFmoc
(BB - 52)
(BB -47) 15
OFmoc ,
HN
OFmoc ,
HN
76.
f
20 -
??. -Y ! Y4
Y4
HO H ???3
CO2Fmoc
(BB -48 )
25
30
HON
HN
OH
OH
?? ,
(BB -53)
YU N
N NHFmoc , Y6 -
Y6
th Y4
-Y !
35
Y4
POH
(BB -49) 40
CO2H (BB - 54 )
OCOCF3
NH2, OMe,
HN
??.
f Y4
-Y !
45
50
y6.
ti
Y4
HO OH
HOY OH
(BB -50) 55
(BB -55)
OH
OMe,
HN OFmoc, HN
?6.
fi Y4
YL
HO OH
60
65
Y6
Y4
HO
N
OH
US 10,703,789 B2
111 112
(BB -56 ) (BB -61)
OMe, 5 CH3,
HN HN
NH
?6. Yl Se
Y4 10
POH ?? POH
(BB -57) (BB -62)
15
LOMe, NH ,
HN HN
Y6 20 ?6.
Y4 Y4
OCH3 25 HO OH
(BB -58) (BB -63)
COMe, NH ,
HN
HN
f
30
?6. -YI N
| ?? . -P - Y !
Y4
35
OH HO OCHZ
(BB -64)
(BB -59) 40 CO2Fmoc
H3C
CH3, N NHFmoc,
HN
YO.
f Y4
Y! N
45 Y6.
f Y4
50 OH
HOY OH
55 (BB -65)
(BB -60 )
CO2H
CH3, H3C
HN N NH ,
?6.
fL -YL
HO OH
60
65
yo
Y4
HO POH
US 10,703,789 B2
113 114
(BB -66 ) (BB -71)
??, 5 H3C CH3,

HN 'N
Yo. -Y N ?6. -Y !
Y4 10
Y4
HO POH
(BB -67) (BB -72)
15
OFmoc ,
HN HN
YÓ
th N
20
yo.
Y4
OH
OH 25
(BB -68) (BB -73 )
HN N
HN 30
??.
YO YL N
Y4
Y4
35
HO OH
HO OH
(BB -69)
40 (BB -74)
HN
HN
Y6 -Y N
POH
45
50
??.
f
Y4
-Y
HO "OH
(BB -70 ) 55
(BB -75)
HN HN
60
Y6. N
-Y ! YO.
Y4
HO
H OCH3
65
Y4
HO OH
US 10,703,789 B2
115 116
(BB -76 ) (BB -81)
5
HN HN
- Y4
Y
10
Yo. YU
HO POH OH
(BB -77) (BB - 82 )
15
HN HN
yo -YU 20 Y6.
Y4 Y4
HO 25 OH
(BB - 78 ) (BB -83)
OH ,
HN HN
f
30
yo YL ?6.
Y4 Y4
35
HO OH POH
(BB -79 ) 40 (BB - 84 )
„OH ,
HN HN
'N 45
YoA
. ??. YU
Y4 Y4
50
OH OH
55 (BB -85)
(BB -80)
HN NH
?6.
f Y4
Yl
60
65
Y6
Y4
yl
CH3
OH
OH
US 10,703,789 B2
117 118
(BB -86 ) (BB - 91)
NH 5 NH
?6. YL Y6 Yl
Y4 10
Y4
OH
HO OH HO
(BB -87) (BB -92)
15
NH NH
Y6 20
yo YI
Y4 Y4
H?c'
OH 25
(BB -88) (BB - 93 )
NH NH
30
Y. YU Y6
Y4 Y4
35
HOW CH3
(BB -89) 40 (BB - 94 )
NH NH
45
?6. Yl 0, YO YL
Y4
50
HO" OCH
55
(BB - 90 ) (BB -95)
CH3,
NH N
60
Y6 -Y ! Y6
Y4 Y4
65
HO HO POH
US 10,703,789 B2
119 120
(BB - 96 ) (BB - 101)
S
H3C . 5 H3C
NH N NH
YÓ. Y6 YL
Y4 10
OH HO OH
(BB -97) (BB - 102)
H3C 15
NH SO3H ,
HN
?6. O, 20
Y6. -YI
Y4
Y4
OH
25
(BB -98 ) OH
(BB - 103)
HN NH
SO3Fmoc ,
f
30
HN
??. -YU NH
Y4
HO OH
35 of
??.
Y4
(BB -99) 40
Pune OH
(BB - 104 )
HN NH SO3H ,
HN
NH
Yo. -
Y4
HO OH
45
50
YO
f Y4
-Y
HOP OH
f Y4
H3C
NH
's ,
(BB - 100 ) 55
60
fY4
-Y !
jo
HN
N
(BB - 105 )
SO3Fmoc,
65
HO POH HOS OH
US 10,703,789 B2
121 122.
( BB- 106 ) ( BB - 111 )
5 HC.
SOH, NH
HN
y6. N 'S ,
Y6. -Y
10 Y4
Y4
HO) OH
HO OH
( BB - 112 )
( BB- 107) 15
SO3Fmoc, HN OCH3,
HN
20,
6 -Y
V6. P -Y ,
|
y4
Y4
25
OH OH
( BB - 113 )
( BB - 108 )
30 CH3,
' NH HN
Y6. - y6.
Y4 35 y4
HO OH OH
( BB - 109 ) ( BB - 114 )
40
CH3,
HN OCH3,
y6.
45
50
ff )
y6.
Y4
N
OCH3
HO OH
55. ( BB -115 )
( BB - 110 )
HN NH , ' NH
73
Y6.
4
60
65
Y6.
Y4
ya
) N S,
OH HO OCH ;
US 10,703,789 B2
123 124
(BB - 116 ) (BB - 121)
HN NH 5
HN NH
yo.
f Y4
- Yl
10
y6
f Y4
HO HO OCH3
(BB - 117 ) (BB - 122)
15
HN NH
HN NH
- Y4
YL 20 YO.
-
Y4
YL
CH3
(BB - 118 )
25 OH
(BB - 123 )
HN NH
HN NH
30 73
??. -YI
YO Y1 0,
Y4
Y4
35
HO OCH
HO OCH3
(BB -124 )
40
(BB - 119 ) HN NH
HN NH yo YL O, and
45
Y4
yo. YL 0,
H?c
Y4
50
HOY
(BB -125 )
HN NH
Y6 Yl
HN NH
(BB -120) 55
60
- Y4
??
Y4
65 thereof,wherein Y !, Y ), Y4 , Y?, and r are as described herein
CH3 (e.g., each r is , independently , an integer from 0 to 5 , such
as from 0 to 3 , from 1 to 3 , or from 1 to 5 )) .
US 10,703,789 B2
125 126
In some embodiments, the building block molecule , -continued
which may be incorporated into a polynucleotide, primary (BB -131 )
construct, or mmRNA , is a modified cytidine (e.g., selected H3C
from the group consisting of: 5 NH
(BB - 126 )
NH2
YO N
10
Y4
yo. -Y !
Y4 OH
15 (BB - 132)
NH
HOY OH CH3,
(BB - 127 )
NH2 20 y6.
H3C
Y4
YO
thY4 25 Oum is POH
(BB - 133)
CH3
HN
HOY POH
ty
(BB -128 ) 30
NH2 N
Y6
Y4
-YL
HN
35
?6.
ti
OH
(BB -134 )
OH 40 CH3
HN
(BB - 129 )
NH2
Y6
Y4
45
50
Y6.
f Y4
?? OCH ;
HOY OH
55 (BB - 135)
(BB -130 ) H3C CH3
NH2
N N
t 60
??. Y6.
Y4 Y4
65
HO OH POH
US 10,703,789 B2
127 128
(BB- 136 ) (BB - 141)
H3C . CH3 NH2
N
5 N
?6.
f Y4 10
Y6.
?4
OH
OCH3 CH3
(BB - 137) 15 (BB - 142)
NH2 NH2
HO 'N
0, 20
Y6 -Y ! Y6.
Y4 Y4
CH3
25
HO OH
(BB - 138 ) (BB - 143)
NHAC NH2
ACO 30
N
YO.
ti Y4
-YI
35
y6.
f
Y4
H3C
How OH HO OH
NH2
(BB-139 ) 40 (BB - 144 )
NH2
TBDMS
N
Y6.
Y4
-Y !
HO OH
45
50
YO .
+ HO
(BB - 140 ) 55 (BB -145)

NH2 NH2
F3C . N 'N
ft
Y6
Y4
60
65
?6.
Y4
-Y !
N
HO POH HO
US 10,703,789 B2
129 130
(BB - 146 ) (BB - 151)
NH2 NH2
5 OHC , N
Y6
that HO
N
BI
10
Y6.
fb N
POH
(BB - 147) 15 (BB - 152)
NH2 NH2
???. N
20
N
Y6. Yl Y6
Y4
25
HO OH OCH3
(BB - 148 ) (BB - 153)
NH2 S
30 H3C N N
y6.
It
Y4
HO "CH3
35
Y!
NH ,
HO OH
NHAC
(BB - 149) 40 (BB - 154 )
NH2
Br
N
45
Y6
Y4
-Y !
OH
50
Y6.
fi YI
HC
(BB - 150 ) (BB - 155)

NHAC 55 NH2
Br
60
?6 . -YL Y6. -Y !
Y4 Y4
65
HO OCH3 HOU POH
US 10,703,789 B2
131 132
-continued
(BB - 156 ) (BB - 160 )
NH2
?? .
5 H3C . ??
Y6
Y4
"OH
0,
10
HO
OH
ht HO OH
or
ui
(BB -157)
H3C NH2 (BB - 161)
20
NH
Y6
f Y4 25
?? .
OH
0,
HO OH OH
30

(BB - 158 ) thereof, wherein each r is , independently, an integer from 0
NH
to 5 (e.g., from 0 to 3 , from 1 to 3 , or from 1 to 5 ).
35 In some embodiments, the building block molecule,
CO2Fmoc which may be incorporated into a polynucleotide , primary
construct, or mmRNA , is a modified adenosine ( e.g.,
yo -P - Y ! N NHFmoc, and selected from the group consisting of:
I
40
(BB - 162)
HO OH NH2
'N ,
45
??
(BB - 159 ) N
Y4
NH CH3
50
COH HO OH
?6. N NH2.
Y4 55
(BB - 163)
NH2
HO OH
N.
N,
60
or a pharmaceutically acceptable saltor stereoisomer Y6 . YU
N
thereof,wherein Y !, Y , Y4 , Yº , and r are as described herein
(e.g. , each r is , independently , an integer from 0 to 5, such OH
as from 0 to 3, from 1 to 3 , or from 1 to 5)). For example ,
the building block molecule , which may be incorporated 65
into a polynucleotide, primary construct, or mmRNA , can HO CH3
be:
US 10,703,789 B2
133 134
ffyou
(BB - 164) (BB - 170)
NH2 NH2
5 N,
yo. ?? -Y
X
Y4
10
H3C
HO OH HO ??3
(BB - 165)
NH2 (BB - 171)
NH2
15
N,
N,
?6.
N Y6 Y
Y4
20
HO
(BB - 166 ) HO OCH3
NH2 25 (BB - 172 )
'N , NH2
Yo -YL
Y4 30 Y6.
N
Y4
HO
(BB - 167) 35
NH2 HO OH
N,
Y6. Yl N
Y4
40 (BB - 173)
NH2
N
HO
45 Y6 .
(BB - 168)
NH2
Y4
N,
Yo YL 50
HO OH
Y4
HO 55
(BB - 174 )
(BB - 169)
the
NH2 NH2
-1
'N
60
Y6
Y4
65
?? HO OH
US 10,703,789 B2
135 136
(BB - 175 ) (BB -179)
NH2
??,
N 5
?6. HN
OCH3,
H
HO OH
10
15
?6.
€ Y4
HO OH
N
(BB - 180)
(BB - 176 ) NH2
NH2
20
'N ,
H3C
f
N
N
Yo YL N
SCH3, Y4
Y4 25
HO OH
OH
(BB - 181)
NH2
30
N,
(BB - 177 ) Y6 P YL
N
OH , 35
Y4 1
HN
HO OH
40
Y6# (BB - 182)
Y4
SCH3, NH2
45 N,
HO OH
50
?6.
ti N
N
(BB - 178 ) HO OH
OH ,
55
(BB - 183)
HN
NH2
Y6
41
Y4
OCH3,
60
65
??.
f N
N,
HO OH
HO OH
US 10,703,789 B2
137 138
(BB -184 ) (BB -190 )
NH2
theo
NH
5
S
Y6. YU ??.
N
N
Y4 Y4
10
HO OH HO OH
(BB - 185)
NH2 (BB - 191)
15 NH2
N,
Br
?6. N,
HO OH
20
Y6
f y4
-Y !
**
'N
(BB - 186 ) HO OH
NH2 25
(BB - 192)
N,
NH2
Y4 30
S
?6. YL
HO OH Y4
NH2
(BB- 187) 35
HO OH
N,
YO.
40
Y4 (BB -193)
NH2
HO OH
45
(BB - 188 ) N,
Y4
Yl
HS
NH2
N,
50
YO
(1 )Y4
HO OH
HO OH 55
(BB - 189 ) (BB - 194 )
NH2 NH2
N
f
N, N,
YO.
ft
Y4
YU
60
65
Y6
Y4
-Y !
H2N
HO OH HO OH
US 10,703,789 B2
139 140
(BB - 195) (BB - 200 )
NH2 HN NH2
5
yo
th Y4
-Y !
N CH3,
10
yo
f YU
HO OH
HO OH
(BB - 196 ) or a pharmaceutically acceptable salt or stereoisomer
HN 15 thereof, wherein Y !, Y , Y4, Y?, and r are as described herein
(e.g., each r is , independently, an integer from 0 to 5 , such
'N ,
as from 0 to 3 , from 1 to 3 , or from 1 to 5 )).
In some embodiments, the building block molecule ,
Y6 which may be incorporated into a polynucleotide, primary
20 construct, or mmRNA , is a modified guanosine ( e.g.,
Y4 selected from the group consisting of:
HO OH
(BB - 201)
25
(BB - 197 ) 73
NH
HN
Y6 -Y !
-
N
30 Y4 NH2,
N
yo CH3
Yl
HO OH
35 (BB - 202)
HO OH
NH
HN
(BB - 198)
40
Y6
f Y4
-Y !
NH2,
flity
OH
Y6. HO CH3
N 45
(BB - 203)
NH
OH Y6
50
-
-Y !
N NH2,
olmay
(BB - 199 ) 55
OH
HN ( BB - 204)
-f14
N NH
and 60
Y6
N NH ,
Y4 Y4
65
HO OH HO
US 10,703,789 B2
141 142
(BB - 205 ) (BB -210 )
N. 5
NH NH
?6. ?6.
N NH2, N NH2,
Y4
10
HO OCHZ
(BB -211 )
15
(BB - 206 )
NH
yo
(
NH N
20
YO. -Y !
Y4 NH2,
POH
25
Quinn
(BB -212 )
(BB -207 ) 30
NH
flot
NH
?6.
Y6. -Y !
N
N NH , 35
H? OH
40
(BB -213)
(BB - 208)
45
NH NH
Y6 -Y YO. Y
Y4 NH ,
?4 H
50
1901 HOW OH
55
(BB - 209) ( BB - 214 )
OCH ;
f
NH N
60
YO -Y ! Y6
Y4 NH2, N NH ,
Y4
65
HON CH3 HOY OH
US 10,703,789 B2
143 144
(BB -215 ) (BB -220 )
f
5 NH
YO. -Y
Y6 -Y NH ,
NH2, Y4
Y4
10
HO
(BB -221)
(BB -216 )
15
NH
N Y6 . -Y
?6. NH2,
*Y 20
Y4
Y4 NH2
HO OH
OH (BB -222)
25
f
NH
(BB -217 ) yo -Y!
N
30 NH2,
YO.
th -Y !
NH2 35
HOY OH
(BB - 223)
f
NH
CI
HO OH ?6. -Y !
40 N NH ,
(BB -218 )
45 HO OH
NH (BB -224 )
?6. H3C
Y4 NH , NH
Br
50 Y6
Y4 NH ,
HO OH
55 HOY POH
(BB -219 ) (BB - 225 )
fL
NH NH
60
yo -Y Yo.
Y4 NH2, Y4
'N NH2
65
HO OH HO OH
US 10,703,789 B2
145 146
(BB - 226 ) (BB - 231)
NH 5
HS
-Y NH
N NH , S
Y4 Y6 -Y !
N NH2,
10 Y4
OH
POH
15
(BB- 227 )
(BB -232)
f
NH
S
Y6. -Y ! 20 NH
NH ,
Y4 Yo
'N
Y4
OH 25
HON OH
(BB -228)
30 (BB -233 )
NH
?6. -Y ! NH
Y4 NH , Y6. -Y
35
N N
Y4
OH
40 "OH
(BB - 229 )
(BB - 234)
NH 45
NH
Y6 -Y !
N
NH , y6.
Y4 N NH2,
Y4
50
OH
HOW OH
55
(BB -230 )
(BB -235 )
NH NH
60
YO -Y ! ??. -Y !
N -
NH , N NH ,
Y4
65
HO OH HO POH
US 10,703,789 B2
147 148
(BB - 236 ) (BB - 240 )
son
5 CH3
CH3 HN
yo -Y ! O or
N NH2, and ?? .
10
OH
HO OH
(BB - 237) HOY OH
15 (BB - 241)
N
f
NH H3C . CH3,
YO . H2N
N
Y4 NH , 20
HO
OH
OH
25
or a pharmaceutically acceptable salt or stereoisomer HOY ??

thereof,wherein Y !, Y ?, Y4, Yº, and r are as described herein
(e.g., each r is, independently , an integer from 0 to 5, such or a pharmaceutically acceptable salt or stereoisomer
as from 0 to 3 , from 1 to 3 , or from 1 to 5 )) . 30 thereof, wherein each r is, independently , an integer from 0
In some embodiments, the chemical modification can to 5 ( e.g., from 0 to 3 , from 1 to 3, or from 1 to 5 ).
include replacement of C group at C -5 of the ring (e.g., for In another embodiment, the chemical modification can
a pyrimidine nucleoside, such as cytosine or uracil ) with N1N include replacement of the hydrogen at C -5 of cytosine with
(e.g., replacement of the >CH group at C -5 with > NR 35 halo (e.g., Br, Cl, F, or I) or optionally substituted alkyl (e.g.,
group, wherein RN1 is H or optionally substituted alkyl). For methyl). For example , the building block molecule, which
example , the building block molecule , which may be incor may be incorporated into a polynucleotide , primary con
porated into a polynucleotide, primary construct, or struct, or mmRNA , can be:
mmRNA, can be :
40
(BB - 242)
(BB - 238 ) NH2
??.
OH
HN NH
or
45
50
HO
ht
OH
HO OH
0 or
OH
55
(BB - 239 )
(BB - 243)
NH2
H3C .
N NH H3C
N
HO
fi OH
or
60
65
HO
U
OH
or
OH HO OH
US 10,703,789 B2
149 150
-continued ally substituted C6-10 aryloxy ; optionally substituted C6-10
(BB - 244 ) aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl )
NH2 oxy ; a sugar (e.g., ribose , pentose, or any described herein );
TBDMS. a polyethyleneglycol (PEG ), O (CH2CH2O ), CH2CH ,OR ,
N 5 where R is H or optionally substituted alkyl, and n is an
integer from 0 to 20 ( e.g., from 0 to 4 , from 0 to 8 , from 0
or
to 10 , from 0 to 16 , from 1 to 4 , from 1 to 8 , from 1 to 10 ,
HO from 1 to 16 , from 1 to 20 , from 2 to 4 , from 2 to 8 , from
2 to 10 , from 2 to 16 , from 2 to 20 , from 4 to 8 , from 4 to
OH 10 10 , from 4 to 16 , and from 4 to 20 ); “ locked ” nucleic acids
(LNA ) in which the 2'-hydroxyl is connected by a C1-6
alkylene or C1. heteroalkylene bridge to the 4'-carbon of the
HO OH same ribose sugar, where exemplary bridges included meth
(BB - 245 ) ylene, propylene , ether, or amino bridges; aminoalkyl, as
NHAC 15 defined herein ; aminoalkoxy, as defined herein ; amino as
defined herein ; and amino acid , as defined herein
Aco Generally, RNA includes the sugar group ribose, which is a
5 -membered ring having an oxygen . Exemplary, non - limit
0 ing modified nucleotides include replacement of the oxygen
HO
0
OH
20 in ribose ( e.g., with S, Se , or alkylene , such asmethylene or
ethylene ); addition of a double bond (e.g., to replace ribose
with cyclopentenyl or cyclohexenyl); ring contraction of
ribose ( e.g., to form a 4 -membered ring of cyclobutane or
oxetane ); ring expansion of ribose (e.g., to form a 6- or
25 7 -membered ring having an additional carbon or heteroa
HO OH
tom , such as for anhydrohexitol, altritol, mannitol, cyclo
hexanyl, cyclohexenyl, and morpholino that also has a
or a pharmaceutically acceptable saltor stereoisomer phosphoramidate backbone); multicyclic forms (e.g., tricy
thereof, wherein each r is , independently, an integer from 0 clo ; and “ unlocked ” forms, such as glycol nucleic acid
to 5 ( e.g., from 0 to 3 , from 1 to 3, or from 1 to 5 ). 30 (GNA ) (e.g., R -GNA or S -GNA , where ribose is replaced by
In yet a further embodiment, the chemical modification glycol units attached to phosphodiester bonds ), threose
can include a fused ring that is formed by the NH , at the C - 4 nucleic acid (TNA , where ribose is replace with a -L
position and the carbon atom at the C -5 position . For threofuranosyl-(3' > 2')), and peptide nucleic acid (PNA ,
example , the building block molecule , which may be incor where 2 -amino -ethyl- glycine linkages replace the ribose and
porated into a polynucleotide, primary construct, or 35 phosphodiester backbone). The sugar group can also contain
mmRNA , can be : one or more carbons that possess the opposite stereochemi
cal configuration than that of the corresponding carbon in
ribose. Thus, a polynucleotide , primary construct, or
???
(BB - 246 ) mmRNA molecule can include nucleotides containing , e.g.,
40 arabinose, as the sugar.
NH Modifications on the Nucleobase
The present disclosure provides for modified nucleosides
and nucleotides . As described herein “ nucleoside” is defined
as a compound containing a sugar molecule (e.g., a pentose
HO
45 or ribose ) or a derivative thereof in combination with an
organic base (e.g., a purine or pyrimidine) or a derivative
OH thereof (also referred to herein as “ nucleobase ” ). As
described herein , “ nucleotide ” is defined as a nucleoside
including a phosphate group. The modified nucleotides may
HO OH
50 by synthesized by any useful method , as described herein
(e.g., chemically , enzymatically, or recombinantly to include
one or more modified or non -natural nucleosides ).
or a pharmaceutically acceptable saltor stereoisomer The modified nucleotide base pairing encompasses not
thereof, wherein each r is, independently, an integer from 0 only the standard adenosine- thymine, adenosine-uracil, or
to 5 (e.g., from 0 to 3 , from 1 to 3 , or from 1 to 5 ). 55 guanosine -cytosine base pairs, but also base pairs formed
Modifications on the Sugar between nucleotides and/or modified nucleotides compris
The modified nucleosides and nucleotides (e.g., building ing non -standard or modified bases, wherein the arrange
block molecules), which may be incorporated into a poly ment of hydrogen bond donors and hydrogen bond acceptors
nucleotide, primary construct, or mmRNA (e.g., RNA or permits hydrogen bonding between a non-standard base and
mRNA , as described herein ), can be modified on the sugar 60 a standard base or between two complementary non - stan
of the ribonucleic acid . For example , the 2' hydroxyl group dard base structures. One example of such non -standard base
(OH ) can be modified or replaced with a number of different pairing is the base pairing between the modified nucleotide
substituents . Exemplary substitutions at the 2 '-position inosine and adenine, cytosine or uracil.
include, but are not limited to , H ,halo , optionally substituted The modified nucleosides and nucleotides can include a
C1-6 alkyl; optionally substituted C1-6 alkoxy ; optionally 65 modified nucleobase. Examples of nucleobases found in
substituted Co-10 aryloxy ; optionally substituted C3-8 RNA include, but are not limited to , adenine, guanine ,
cycloalkyl; optionally substituted C3-8 cycloalkoxy; option cytosine, and uracil. Examples of nucleobase found in DNA
US 10,703,789 B2
151 152
include, but are not limited to , adenine , guanine, cytosine , wherein
and thymine . These nucleobases can be modified or wholly is a single or double bond;
replaced to provide polynucleotides, primary constructs , or each of T '', T ", T2 , T ?" , is , independently , H , optionally
mmRNA molecules having enhanced properties, e.g., resis substituted alkyl, optionally substituted alkoxy, or optionally
tance to nucleases through disruption of the binding of a 5 substituted thioalkoxy, or the combination of T ' and Tl" or
major groove binding partner. Table 8 , shown in FIG . 13, the combination of T ?' and T ?" join together (e.g., as in T ?)
identifies the chemical faces of each canonical nucleotide. to form O (oxo ), S ( thio ), or Se ( seleno);
Circles identify the atoms comprising the respective chemi C (RVD each of V and V2 is , independently, O , S , N (R )ny, or
cal regions. )mw, wherein nv is an integer from 0 to 2 and each R ”
10 is , independently , H , halo , optionally substituted amino acid ,
In some embodiments, B is a modified uracil. Exemplary optionally substituted alkyl, optionally substituted haloalkyl,
modified uracils include those having Formula (b1)-(b5) : optionally substituted alkenyl, optionally substituted alky
nyl, optionally substituted alkoxy, optionally substituted
alkenyloxy, optionally substituted alkynyloxy, optionally
substituted hydroxyalkyl, optionally substituted hydroxyalk
T!
(b1) 15 enyl, optionally substituted hydroxyalkynyl, optionally sub
stituted aminoalkyl (e.g., substituted with an N -protecting
x N
-R 12a group , such as any described herein , e.g., trifluoroacetyl ),
optionally substituted aminoalkenyl, optionally substituted
aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,
T?" 20 substituted with an N -protecting group , such as any
T2 described herein , e.g., trifluoroacetyl), optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbo
muhun R 120
(62)
nylalkenyl, optionally substituted alkoxycarbonylalkynyl, or
optionally substituted alkynyloxy (e.g., optionally substi
tuted with any substituent described herein , such as those
25 selected from ( 1) -(21) for alkyl) ;
R1° is H , halo , optionally substituted amino acid ,hydroxy,
R122, optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted amino
alkyl, optionally substituted hydroxyalkyl, optionally sub
R111 30
stituted hydroxyalkenyl, optionally substituted hydroxy
alkynyl, optionally substituted aminoalkenyl, optionally
substituted aminoalkynyl, optionally substituted alkoxy ,
optionally substituted alkoxycarbonylalkyl, optionally sub
stituted alkoxycarbonylalkenyl, optionally substituted
(63) alkoxycarbonylalkynyl, optionally substituted alkoxycarbo
R12C 35 nylalkoxy, optionally substituted carboxyalkoxy, optionally
R 10
substituted
oylalkyl;
carboxyalkyl, or optionally substituted carbam
Rilis H or optionally substituted alkyl;
R12a is H , optionally substituted alkyl, optionally substi
R11 N tuted hydroxyalkyl, optionally substituted hydroxyalkenyl,
mpum T2
(14)
40
optionally substituted hydroxyalkynyl, optionally substi
tuted aminoalkyl, optionally substituted aminoalkenyl, or
optionally substituted aminoalkynyl, optionally substituted
carboxyalkyl ( e.g., optionally substituted with hydroxy),
optionally substituted carboxyalkoxy, optionally substituted
R12C 45 carboxyaminoalkyl, or optionally substituted carbamoylal
R 10 kyl; and
N R12e is H , halo , optionally substituted alkyl, optionally
substituted alkoxy, optionally substituted thioalkoxy, option
ally substituted amino , optionally substituted hydroxyalkyl,
50 optionally substituted hydroxyalkenyl , optionally substi
N or
tuted hydroxyalkynyl, optionally substituted aminoalkyl,

optionally substituted aminoalkenyl, or optionally substi
tuted aminoalkynyl.
Other exemplary modified uracils include those having
(b5 ) 55 Formula (b6 )-(59):
RIO -R120 (66 )
R 120
60
13
R124
W ] -T2"
W2
T2
65
thereof,
US 10,703,789 B2
153 154
-continued carboxyalkyl ( e.g., optionally substituted with hydroxy and /
(b7) or an O -protecting group ), optionally substituted carboxy
R12C alkoxy ,optionally substituted carboxyaminoalkyl, or option
ally substituted carbamoylalkyl ( e.g., optionally substituted
-R120 5 with any substituent described herein , suchVa as those selected
from (1 )-(21 ) for alkyl), and wherein R and R12c taken
W together with the carbon atoms to which they are attached
W2 T2 can form optionally substituted cycloalkyl, optionally sub
stituted aryl, or optionally substituted heterocyclyl (e.g., a 5
10 or 6 -membered ring);
R129 is H , optionally substituted alkyl, optionally substi
(68) tuted hydroxyalkyl , optionally substituted hydroxyalkenyl,
Il! T !" optionally substituted hydroxyalkynyl, optionally substi
R 126 R12a, or tuted aminoalkyl, optionally substituted aminoalkenyl,
N optionally substituted aminoalkynyl, optionally substituted
15 carboxyalkyl (e.g., optionally substituted with hydroxy and /
or an O -protecting group ), optionally substituted carboxy
T2 alkoxy, optionally substituted carboxyaminoalkyl, option
ally substituted carbamoylalkyl, or absent;
R125 is H , optionally substituted alkyl, optionally substi
20 tuted alkenyl, optionally substituted alkynyl, optionally sub
12c (69) stituted hydroxyalkyl, optionally substituted hydroxyalk
enyl, optionally substituted hydroxyalkynyl, optionally
R 120 substituted aminoalkyl, optionally substituted aminoalkenyl,
N optionally substituted aminoalkynyl, optionally substituted
25 alkaryl, optionally substituted heterocyclyl, optionally sub
T ?", stituted alkheterocyclyl , optionally substituted amino acid ,
T2 optionally substituted alkoxycarbonylacyl, optionally sub
stituted alkoxycarbonylalkoxy, optionally substituted
alkoxycarbonylalkyl, optionally substituted alkoxycarbo
30 nylalkenyl, optionally substituted alkoxycarbonylalkynyl,
optionally substituted alkoxycarbonylalkoxy, optionally
or a pharmaceutically acceptable saltor stereoisomer substituted carboxyalkyl (e.g., optionally substituted with
thereof, hydroxy and/or an O -protecting group ), optionally substi
wherein tuted carboxyalkoxy, optionally substituted carboxyamino
is a single or double bond ; 35 alkyl , or optionally substituted carbamoylalkyl
12b
,
each of T '', Tl", T² , T ?" , is , independently, H , optionally tionwherein the combination of R and T ?' or the combina
of R125 and R 12c can join together to form optionally
substituted alkyl, optionally substituted alkoxy, or optionally substituted heterocyclyl; and
substituted thioalkoxy, or the combination ofTand Tl" join R12€ is H , halo , optionally substituted alkyl, optionally
together ( e.g., as in Tl) or the combination of T ?' and T ?" join 40 substituted alkoxy, optionally substituted thioalkoxy, option
together (e.g., as in T ?) to form O (oxo ), S (thio ), or Se ally substituted amino , optionally substituted aminoalkyl,
( seleno ), or each T and T2 is, independently, O (oxo ), S optionally substituted aminoalkenyl, or optionally substi
(thio ), or Se ( seleno ); tuted aminoalkynyl .
each of wl and W2 is, independently, N (RW )mw or Further exemplary modified uracils include those having
Cis(,RWindependently
)nw ,wherein, Hnw, optionally
is an integer from 0 to 2 and each R "
substituted alkyl, or option
a 45 Formula (b28 )-(131):
ally substituted alkoxy ;
each V? is , independently, O , S, N (RVA)nv, or C (RV )nv, (628)
wherein nv is an integer from 0 to 2 and each RVa is ,
independently , H , halo , optionally substituted amino acid , 50 Rb -R 12a
optionally substituted alkyl, optionally substituted hydroxy
alkyl, optionally substituted hydroxyalkenyl, optionally sub
stituted hydroxyalkynyl, optionally substituted alkenyl, RZ T2
optionally substituted alkynyl, optionally substituted hetero
cyclyl, optionally substituted alkheterocyclyl, optionally 55
substituted alkoxy, optionally substituted alkenyloxy, or
optionally substituted alkynyloxy, optionally substituted (529)
aminoalkyl ( e.g., substituted with an N -protecting group ,
such as any described herein , e.g., trifluoroacetyl, or sul
foalkyl), optionally substituted aminoalkenyl, optionally 60 RB N
R12a,
substituted aminoalkynyl, optionally substituted acylamino
alkyl ( e.g., substituted with an N -protecting group, such as
any described herein , e.g., trifluoroacetyl), optionally sub N * T2
stituted alkoxycarbonylalkyl, optionally substituted alkoxy
carbonylalkenyl, optionally substituted alkoxycarbonylalky- 65
nyl, optionally substituted alkoxycarbonylacyl, optionally
substituted alkoxycarbonylalkoxy, optionally substituted
US 10,703,789 B2
157 158
ally substituted alkynyloxy ( e.g. , optionally substituted with
(610) any substituentdescribed herein , such as those selected from
R13a R135 ( 1) -(21) for alkyl), wherein the combination of R136 and RVC
can be taken together to form optionally substituted hetero
R14 5 cyclyl;
N
each Vs is ,independently , N (RV ) , or C (RV ) ,wherein
nv is an integer from 0 to 2 and each R is, independently ,
R 15 N
H , halo , optionally substituted amino acid , optionally sub
73
min 10 stituted alkyl, optionally substituted alkenyl, optionally sub
stituted alkynyl, optionally substituted alkoxy, optionally
(b11)
substituted alkenyloxy, optionally substituted heterocyclyl,
-R136 optionally substituted alkheterocyclyl, or optionally substi
tuted alkynyloxy (e.g., optionally substituted with any sub
R 14 -RIO 15 stituent described herein , such as those selected from ( 1)
N (21) for alkyl) ( e.g., V5 is CH or N );
each of R13a and R136 is, independently, H , optionally
RIS N *T3" substituted acyl, optionally substituted acyloxyalkyl, option
T3' ally substituted alkyl, or optionally substituted alkoxy ,
20
m
wherein the combination of R136 and R14 can be taken
together to form optionally substituted heterocyclyl;
R 13a
(b12) each R14 is , independently , H , halo , hydroxy, thiol,
R 136 optionally substituted acyl, optionally substituted amino
25 acid , optionally substituted alkyl, optionally substituted
haloalkyl, optionally substituted alkenyl , optionally substi
N tuted alkynyl, optionally substituted hydroxyalkyl ( e.g., sub
stituted with an O -protecting group ), optionally substituted
RIS N hydroxyalkenyl, optionally substituted hydroxyalkynyl,
T'3' 30 optionally substituted alkoxy, optionally substituted alkeny
loxy, optionally substituted alkynyloxy , optionally substi
tuted aminoalkoxy , optionally substituted alkoxyalkoxy,
(613) optionally substituted acyloxyalkyl, optionally substituted
R13a R138 amino ( e.g., —NHR , wherein R is H , alkyl , aryl, or phos
35 phoryl), azido , optionally substituted aryl, optionally sub
stituted heterocyclyl, optionally substituted alkheterocyclyl,
'N optionally substituted aminoalkyl, optionally substituted
aminoalkenyl, or optionally substituted aminoalkyl; and
R 151 -T3", or each of R15 and R16 is , independently , H , optionally
T3' 40 substituted alkyl, optionally substituted alkenyl, or option
min
ally substituted alkynyl.
Further exemplary modified cytosines include those hav
(b14 ) ing Formula (b32 ) -(635):
45
R15 N T3". (632 )

T3' R 13a R 136
nan
50 R14 N
or a pharmaceutically acceptable saltor stereoisomer R15 N T},

thereof,
wherein
each of T3' and T3" is, independently , H , optionally
substituted alkyl, optionally substituted alkoxy, or optionally
substituted thioalkoxy , or the combination of T ' and T3" join
55
mhm R 136
(633)
together ( e.g., as in T ) to form (oxo ), S ( thio ), or Se
(seleno ); 60 R 14 -R 16
each V4 is , independently , O , S, N (R ) or C (R )
wherein ny is an integer from 0 to 2 and each RV is ,
independently , H , halo , optionally substituted amino acid , R15 N T3
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted alkoxy, 65
optionally substituted alkenyloxy , optionally substituted het
erocyclyl, optionally substituted alkheterocyclyl , or option
wiform
US 10,703,789 B2
159 160
-continued
(634 ) (b36 )
-R 136
R 14 5
N R 14a
R132 or
R 136 RIS N R 145
R 13a R 136
(635 )
10
montare
R 14 15 thereof,
wherein
each R136 is , independently , H , optionally substituted
RIS N acyl, optionally substituted acyloxyalkyl, optionally substi
tuted alkyl, or optionally
13b
substituted alkoxy, wherein the
20
combination of R and R 146 can be taken together to form
optionally substituted heterocyclyl;
each R14a and R 14h is , independently, H , halo , hydroxy,
thiol, optionally substituted acyl, optionally substituted
or a pharmaceutically acceptable saltor stereoisomer amino acid , optionally substituted alkyl, optionally substi
thereof, tuted haloalkyl, optionally substituted alkenyl, optionally
25 substituted alkynyl, optionally substituted hydroxyalkyl
wherein ( e.g., substituted with an O -protecting group ), optionally
each of T1 and T is , independently, O (oxo ), S (thio ), or substituted hydroxyalkenyl, optionally substituted alkoxy ,
Se (seleno ); optionally substituted alkenyloxy, optionally substituted
alkynyloxy, optionally substituted aminoalkoxy , optionally
each of R13a and R136 is , independently, H , optionally 30 substituted alkoxyalkoxy , optionally substituted acyloxy
substituted acyl, optionally substituted acyloxyalkyl, option alkyl, optionally substituted amino (e.g., -NHR , wherein R
ally substituted alkyl, or optionally
13b
substituted alkoxy, is H , alkyl, aryl, phosphoryl, optionally substituted amino
wherein the combination of R13 and R14 can be taken alkyl, or optionally substituted carboxyaminoalkyl), azido,
together to form optionally substituted heterocyclyl; optionally substituted aryl, optionally substituted heterocy
each R14 is, independently, H , halo, hydroxy, thiol, 35 clyl, optionally substituted alkheterocyclyl, optionally sub
stituted aminoalkyl, optionally substituted aminoalkenyl, or
optionally substituted acyl, optionally substituted amino optionally
acid , optionally substituted alkyl, optionally substituted substituted aminoalkynyl; and
haloalkyl, optionally substituted alkenyl, optionally substi alkyl, optionallyis ,substituted
each of R15 independently, H , optionally substituted
alkenyl, or optionally substi
tuted alkynyl, optionally substituted hydroxyalkyl ( e.g., sub 40 tuted alkynyl.
stituted with an O - protecting group), optionally substituted In particular embodiments, Rlub is an optionally substi
hydroxyalkenyl, optionally substituted hydroxyalkynyl , tuted amino acid (e.g., optionally substituted lysine ). In
optionally substituted alkoxy, optionally substituted alkeny some embodiments , R14a is H.
loxy , optionally substituted alkynyloxy, optionally substi In some embodiments , B is a modified guanine. Exem
tuted aminoalkoxy, optionally substituted alkoxyalkoxy, 45 plary modified guanines include compounds of Formula
optionally substituted acyloxyalkyl, optionally substituted (b15 )-(b17 ):
amino (e.g., NHR , wherein R is H , alkyl, aryl, or phos
phoryl), azido , optionally substituted aryl, optionally sub
stituted heterocyclyl, optionally substituted alkheterocyclyl, (b15)
optionally substituted aminoalkyl ( e.g., hydroxyalkyl, alkyl, 50 T4
alkenyl, or alkynyl ), optionally substituted aminoalkenyl, or •R18
optionally substituted aminoalkynyl; and vo
each of R15 and R16 is , independently , H , optionally N R 192
substituted alkyl, optionally substituted alkenyl, or option 55
N
ally substituted alkynyl (e.g., R15 is H , and R16 is H or |
R 196
optionally substituted alkyl).
In some embodiments, R15 is H , and R16 is Hor optionally (616 )
substituted alkyl. In particular embodiments, R14 is H , acyl, 75" R23
orhydroxyalkyl. In someembodiments , R14 is halo . In some 60
embodiments, both R 14 and R 15 are H. In some embodi
ments, both R15 and R16 are H. In some embodiments , each R21. -R24, or
of R14 and R 15 and R16 is H. In further embodiments , each N
of R13a and R136 is independently, H or optionally substi
tuted alkyl.
Further non -limiting examples of modified cytosines
include compounds of Formula (b36 ):
65
wonton R22
US 10,703,789 B2
161 162
(617 ) (639)
752 75"
-R18 5
N
R18
R17.
16 R 190 or
mafor R22
T6
10
mufton R 195
(640 )
thereof, 15
R18
wherein N
R21.
each of T4 , T4" , T5 , T5", T “ , and TÓ" is, independently , H , N
R 190
optionally substituted alkyl, or optionally substituted alkoxy ,
and wherein the combination of T4' and T4" (e.g., as in T4)
or the combination of Ts' and T5 " (e.g. , as in T™) or the 20
combination ofTand T6" (e.g., as in TC) join together form
O (oxo ), S ( thio ), or Se ( seleno ) ;
mafor R 196
each of V5 and Vó is , independently , O , S, N (R )nv, or or a pharmaceutically acceptable saltor stereoisomer

C (R )n ,wherein nv is an integer from 0 to 2 and each Rºd 25 thereof,
is , independently, H , halo , thiol, optionally substituted wherein
amino acid , cyano , amidine , optionally substituted amino each of T4 is , independently, H , optionally substituted
alkyl, optionally substituted aminoalkenyl, optionally sub alkyl, or optionally substituted alkoxy, and each T4 is ,
stituted aminoalkynyl, optionally substituted alkyl, option independently , O (oxo ), S (thio ), or Se (seleno );
ally substituted alkenyl, optionally substituted alkynyl, 30 each of R18, R19 , R196 , and R21 is, independently, H ,
>
optionally substituted alkoxy, optionally substituted alkeny halo , thiol, optionally substituted alkyl, optionally substi
loxy , or optionally substituted alkynyloxy (e.g., optionally tuted alkenyl, optionally substituted alkynyl, optionally sub
substituted with any sub tuent described herein , such as stituted thioalkoxy , optionally substituted amino , or option
those selected from (1 )-(21) for alkyl), optionally substituted 35 ally substituted amino acid .
thioalkoxy, or optionally substituted amino ; and In some embodiments , R 18 is H or optionally substituted
each of R17 , R18, R19 , R19 , R21, R22, R23 , and R24 is , alkyl
independently , H , halo , thiol, optionally substituted alkyl, ments.,Ineachfurther embodiments , T4 is oxo . In some embodi
of R 19a and R195 is , independently, H or option
optionally substituted alkenyl, optionally substituted alky ally substituted alkyl.
nyl, optionally substituted thioalkoxy, optionally substituted 40 In some embodiments , B is a modified adenine. Exem
amino, or optionally substituted amino acid .
Exemplary modified guanosines include compounds of plary modified adenines include compounds of Formula
(618 ) -(b20 ):
Formula (b37 )- (640 ):
45
pa (637) (518 )
R26a . -R 260
•R18
50
N
R 25
N
R 192
N
R27,
R 196
55 wofon
ga
(619)
(638) -R260
60
R28
R25
N „ R 19a, N R27 or
anton R 195 65
wafura

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US010703789B2

( 12 ) United States Patent ( 10 ) Patent No.: US 10,703,789 B2

Related U.S. Application Data CO7K 14/56 (2006.01)

( 56 ) References Cited EP 2113247 A2 11/2009

( 56 ) References Cited Qiu et al., “ Creating a flexible multiple microRNA expression

DLIN - K -DMA ( 2,2 -DILINOLEYL--DIMETHYLAMINOMETHYL - 11,3 )-DIOXOLANE )

DLIN - KC2- DMA

Only single outors are showi? tho

ANNI(2724) As:$ } (383 )

Batch B Deform Unform

93.75 375 750

- '. '. '. '.

nd ndani L2000 Untreated

and detectable tags and may include multiple cloning sites

3 UTR Albumin CATCACATTTAAAAGCATCTCAGCCTACCATG 8

3 UTR G - CSF GCCAAGCCCTCCCCATCCCATGTATTTATCTC 10

3UTR Col6a2 ; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTC 12

3 UTR RPN1 ; GGGGCTAGAGCCCTCTCCGCACAGCGTGGAG 13

3 UTR LRP1 ; low GGCCCTGCCCCGTCGGACTGCCCCCAGAAAG 14

3 UTR Nnt1 ; ATATTAAGGATCAAGCTGTTAGCTAATAATGC 15

3UTR Collal ; CTCCCTCCATCCCAACCTGGCTCCCTCCCACC 18

3UTR Nucbl; TCCTCCGGGACCCCAGCCCTCAGGATTCCTGA 20

3 UTR a - globin GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCC 21

TGA and one additional stop codon . In a further embodi

SS Prolactin ATGAAAGGATCATTGCTG 35 MKGSLLLLL 97

SS Albumin ATGAAATGGGTGACGTTC 36 MKWVTFISL 98

SS HMMSP38 ATGTGGTGGCGGCTCTGGT 37 MWWRLWW 99

MLS Cytochrome ATGAGCGTGCTCACTCCGT 40 MSVLTPLLL 102

SS Type III , TGACAAAAATAACTTTATC 41 MVTKITLSPO 103

SS Viral ATGCTGAGCTTTGTGGATA 42 MLSFVDTRT 104

SS viral ATGGGCAGCAGCCAGGCG 43 MGS SQAPRM 105

SS Viral ATGGCGGGCATTTTTTATT 44 MAGIFYFLFS 106

SS Viral ATGGAAAACCGCCTGCTG 45 MENRLLRVF 107

SS Viral ATGGCGCGCCAGGGCTGC 46 MARQGCFGS 108

SS Bacillus ATGAGCCGCCTGCCGGTG 47 MSRLPVLLL 109

SS Bacillus ATGAAACAGCAGAAACGC 48 MKQQKRLY 110

SS Secretion ATGGCGACGCCGCTGCCT 49 MATPLPPPSP 111

SS Secretion ATGAAGGCTCCGGGTCGG 50 MKAPGRLVL 112

SS Secretion ATGCTTCAGCTTTGGAAAC 51 MLQLWKLL 113

SS Secretion ATGCTTTATCTCCAGGGTT 52 MLYLQGWS 114

SS Secretion ATGGATAACGTGCAGCCG 53 MDNVQPKIK 115

SS Secretion ATGCCCTGCCTAGACCAA 54 MPCLDOQLT 116

SS Secretion ATGAAAACCTTGTTCAATC 55 MKTLFNPAP 117

SS Secretion ATGAAGCCTCTCCTTGTTG 56 MKP LLVVFV 118

SS Secretion ATGTCCTGTTCCCTAAAGT 57 MSCSLKFTLI 119

SS Secretion ATGGTTCTTACTAAACCTC 58 MVLTKPLOR 120

SS Secretion ATGGCCACCCCGCCATTCC 59 MAT PPFRLIR 121

SS Secretion ATGAGCTTTTTCCAACTCC 60 MSFFQLLMK 122

SS Secretion ATGGTCTCAGCTCTGCGGG 61 MVSALRGAP 123

SS Secretion ATGATGGGGTCCCCAGTG 62 MMGSPVSHL 124

SS Secretion ATGGCAAGCATGGCTGCC 63 MASMAAVL 125

SS Secretion ATGGTGCTCATGTGGACC 64 MVLMWTSG 126

SS Secretion ATGGATTTTGTCGCTGGAG 65 MDFVAGAIG 127

SS Secretion ATGGAGAAGCCCCTCTTCC 66 MEKPLFPLV 128

SS Secretion ATGGGGATCCAGACGAGC 68 MGIQTSPVLL 130

SS Secretion ATGTCGGACCTGCTACTAC 69 MSDLLLLGLI 131

SS Secretion ATGGAGACTGTGGTGATT 70 METVVIVAI 132

SS Secretion ATGCGCGGCTCTGTGGAG 71 MAGSVECT 133

SS Secretion ATGATGCCGTCCCGTACCA 72 MMP SRTNLA 134