WO2018232355A1

WO2018232355A1 - Rna antibodies

Info

Publication number: WO2018232355A1
Application number: PCT/US2018/037918
Authority: WO
Inventors: Sunny HIMANSU; Elisabeth NARAYANAN; Giuseppe Ciaramella
Original assignee: ModernaTx Inc
Current assignee: ModernaTx Inc
Priority date: 2017-06-15
Filing date: 2018-06-15
Publication date: 2018-12-20
Anticipated expiration: 2019-12-15

Abstract

Aspects of the disclosure relate to compositions and methods of treating and/or preventing disease by delivering antibodies to a subject. Compositions and treatments provided herein include one or more RNA polynucleotides having an open reading frame encoding one or more antibodies or fragments thereof. Methods for preparing and using such treatments are also provided.

Description

RNA ANTIBODIES RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional applications numbers 62/520,474, filed June 15, 2017, and 62/593,790, filed December 1, 2017, the entire contents of which are incorporated by reference herein in their entirety. BACKGROUND

Infectious diseases kill over 10 million people per year globally and are the fourth leading cause of death in the United States. They also represent a $68 billion dollar pharmaceutical market. Antibiotics and anti-virals overlook millennia of nature’s learnings by failing to utilize the immune system. And while intravenous immunoglobulin heeds nature’s example, its applications in infectious disease are few and its limitations numerous (e.g., cost, cumbersome manufacturing, risk of infection).

Efforts to develop monoclonal antibodies for infectious disease are underway, but are limited to individual targets, given the high cost of existing approaches to antibody generation. For example, seasonal influenza is a devastating illness that kills over 250,000 people per year, and in the U.S., accounts for more hospitalizations than lung cancer or kidney stones. SUMMARY

Aspects of the application relate to compositions and methods of preventing and/or treating conditions, diseases, and disorders related to infectious disease and cancer. In some aspects, a ribonucleic acid (RNA) composition is provided herein that builds on the knowledge that RNA (e.g., messenger RNA (mRNA)) can safely direct the body’s cellular machinery to produce nearly any protein of interest, from native proteins to antibodies and other entirely novel protein constructs that can have therapeutic activity inside and outside of cells.

The RNA (e.g., mRNA) compositions of the disclosure may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. The RNA compositions may be utilized to treat and/or prevent an infection by bacteria or virus of various genotypes, strains, and isolates. In some aspects, the disclosure relates to RNA compositions that may be utilized to provide passive immunization against an infectious disease. In some aspects, the disclosure relates to methods of treating and/or preventing infectious disease or cancer in a subject.

In some aspects the invention is a composition, comprising: a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or antigen-binding fragment thereof, wherein the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to (i) maintain antibody levels at a normal

physiological level or a supraphysiological level for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or (ii) maintain antibody levels at 50% or more of the normal antibody level for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours post-administration. In some embodiments the composition includes a delivery agent.

The present disclosure further provides a method of expressing antibodies in a human subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition or a polynucleotide, e.g. an mRNA, described herein, wherein the pharmaceutical composition or polynucleotide is suitable for administrating as a single dose or as a plurality of single unit doses to the subject. The drug may be administered in a clinical setting, e.g., hospital or clinical site, in an IV infusion over a few hours. For instance, it may be administered as a bolus IV injection, or as a procedure carried out in a day for a patient in the clinic/hospital. The single dose may be followed up by subsequent treatments, at a certain frequency, every week, two weeks, three weeks, four weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, every month, two months, three months, four months, five months, six months, or every year.

In other aspects the invention is a pharmaceutical composition, comprising a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or antigen-binding fragment thereof and a lipid nanocarrier.

In some embodiments, the polynucleotide is single stranded.

In some embodiments, the polynucleotide is double stranded.

In some embodiments, the polynucleotide is RNA.

In some embodiments, the polynucleotide is mRNA.

In some embodiments, the polynucleotide comprises at least one chemically modified nucleobase, sugar, backbone, or any combination thereof.

In some embodiments, the at least one chemically modified nucleobase is selected from the group consisting of pseudouracil (ψ), N1-methylpseudouracil

2-thiouracil (s2U), 4’-thiouracil, 5-methylcytosine, 5-methyluracil, and any combination thereof. In some embodiments, the at least one chemically modified nucleobase is a modified uracil such as 5-methoxyuracil.

In some embodiments, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the uracils are 5-methoxyuracils.

In some embodiments, the polynucleotide further comprises a miRNA binding site. In some embodiments, the miRNA binding site binds to miR-142.

In some embodiments, the miRNA binding site binds to miR-142-3p or miR-142-5p. In some embodiments, the miR-142 comprises SEQ ID NO: 104.

In some embodiments, the polynucleotide further comprises a 5' UTR.

In some embodiments, the 5' UTR comprises a nucleic acid sequence at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID NOs:71-88.

In some embodiments, the polynucleotide further comprises a 3' UTR.

In some embodiments, the 3' UTR comprises a nucleic acid sequence at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of SEQ ID NOs:89-99.

In some embodiments, the miRNA binding site is located within the 3' UTR.

In some embodiments, the polynucleotide has a 5' terminal cap selected from a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5' methylG cap, or an analog thereof.

In some embodiments, the polynucleotide further comprises a poly-A region.

In some embodiments, the poly-A region is at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 nucleotides in length.

In some embodiments, the poly-A region has about 10 to about 200, about 20 to about 180, about 50 to about 160, about 70 to about 140, or about 80 to about 120 nucleotides in length.

In some embodiments, the polynucleotide has:

(i) a longer plasma half-life;

(ii) increased expression of an antibody encoded by the ORF; (iii) a lower frequency of arrested translation resulting in an expression fragment; (iv) greater structural stability; or

(v) any combination thereof,

relative to a corresponding polynucleotide comprising SEQ ID NO: 3.

In some embodiments, the polynucleotide comprises:

(i) a 5'-terminal cap;

(ii) a 5'-UTR;

(iii) an ORF encoding an antibody;

(iv) a 3'-UTR; and

(v) a poly-A region.

In some embodiments, the 3'-UTR comprises a miRNA binding site.

The present disclosure provides, in certain aspects, a method of producing a polynucleotide as described herein, the method comprising modifying an ORF encoding an antibody by substituting at least one uracil nucleobase with an adenine, guanine, or cytosine nucleobase, or by substituting at least one adenine, guanine, or cytosine nucleobase with a uracil nucleobase, wherein all the substitutions are synonymous substitutions.

In some embodiments, the method further comprises replacing at least about 90%, at least about 95%, at least about 99%, or about 100% of uracils with 5-methoxyuracils.

The present disclosure provides, in certain aspects, a composition comprising a polynucleotide as described herein; and

a delivery agent.

In some embodiments, the delivery agent comprises a lipid nanoparticle including an ionizable lipid. In some embodiments the ionizable lipid is compound 18 or compound 25.

In some embodiments, the delivery agent further comprises a structural lipid.

In some embodiments, the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and any mixtures thereof.

In some embodiments, the delivery agent further comprises a PEG lipid.

In some embodiments, the PEG lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, compound 428 and any mixtures thereof.

In aspects, the present disclosure provides a polynucleotide comprising an open reading frame (ORF) encoding an antibody, wherein the uracil or thymine content of the ORF relative to the theoretical minimum uracil or thymine content of a nucleotide sequence encoding the antibody (%U_TM or %T_TM) is between about 125% and about 150%.

In some embodiments, the %U_TM or %T_TM is between about 100% and about 220%, about 134% and about 140%, about 134% and about 145%, about 130% and about 145%, about 120% and about 140%, about 124% and about 130%, about 130% and about 140%, about 114% and about 150%, or about 134% and about 148%.

In some embodiments, the uracil or thymine content of the ORF relative to the uracil or thymine content of the corresponding wild-type ORF (%U_WT or %T_WT) is less than 100%.

In some embodiments, the %U_WT or %T_WT is less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, or less than 74%.

In some embodiments, the %U_WT or %T_WT is between 68% and 74%.

In some embodiments, the uracil or thymine content in the ORF relative to the total nucleotide content in the ORF (%U_TL or %T_TL) is less than about 50%, less than about 40%, less than about 30%, or less than about 21%.

In some embodiments, the %U_TL or %T_TL is less than about 21%.

In some embodiments, the %U_TL or %T_TL is between about 14% and about 16%. In some embodiments, the guanine content of the ORF with respect to the theoretical maximum guanine content of a nucleotide sequence encoding the antibody (%G_TMX) is at least 71%, at least 72%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.

In some embodiments, the %G_TMX is between about 72% and about 80%, between about 72% and about 79%, between about 73% and about 78%, or between about 74% and about 77%.

In some embodiments, the cytosine content of the ORF relative to the theoretical maximum cytosine content of a nucleotide sequence encoding the antibody (%C_TMX) is at least 63%, at least 64%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or about 100%.

In some embodiments, the %C_TMX is between about 65% and about 80%, between about 65% and about 79%, between about 65% and about 78%, or between about 72% and about 77%.

In some embodiments, the guanine and cytosine content (G/C) of the ORF relative to the theoretical maximum G/C content in a nucleotide sequence encoding the antibody (%G/C_TMX) is at least about 81%, at least about 82%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the %G/C_TMX is between about 80% and about 100%, between about 85% and about 99%, between about 90% and about 97%, or between about 90% and about 93%.

In some embodiments, the G/C content in the ORF relative to the G/C content in the corresponding wild-type ORF (%G/C_WT) is at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, or at least about 110%.

In some embodiments, the average G/C content in the 3^rd codon position in the ORF is at least 20%, at least 21%, at least 22%, at least 23%, or at least 24% higher than the average G/C content in the 3^rd codon position in the corresponding wild-type ORF.

In some embodiments, the ORF further comprises at least one low-frequency codon. In some embodiments, the composition is formulated for in vivo delivery.

In some embodiments, the composition is formulated for intramuscular, subcutaneous, or intradermal delivery.

In some aspects, the present disclosure provides a composition comprising an RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a single-domain antibody or a fragment thereof having specificity for Ebola virus (e.g., a fragment capable of binding to the Ebola virus). In some embodiments, the single-domain antibody or fragment thereof is a variable domain of a heavy-chain (V_HH) antibody.

In some embodiments, the single-domain antibody (e.g., a V_HH antibody) or fragment thereof has binding specificity for an Ebola virus (EBOV) protein. In some embodiments, the EBOV protein is an EBOV glycoprotein. In some embodiments, the EBOV glycoprotein is GP, secreted GP (sGP), small sGP (ssGP), surface GP, or shed GP. In some embodiments, the EBOV protein is an EBOV nucleoprotein. In some embodiments, the EBOV protein is an EBOV matrix protein. In some embodiments, the EBOV matrix protein is VP24, VP30, VP35, or VP30.

In some aspects, the methods and compositions of the present application are useful for preventing and/or treating infection by Ebola virus. In some embodiments, the Ebola virus is from Ebola virus species Zaire ebolavirus, species Sudan ebolavirus, species

Bundibugyo ebolavirus, species Taï Forest ebolavirus, species Reston ebolavirus, or a combination thereof.

In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 90% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 95% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 96% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 97% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 98% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 99% identity to an amino acid sequence of any one of Tables 1 and 2. In some embodiments, the RNA polynucleotide encodes a single-domain antibody having greater than 95-99% identity to an amino acid sequence of any one of Tables 1 and 2.

In some embodiments, the RNA polynucleotide encodes a single-domain antibody having an amino acid sequence of any one of Tables 1 and 2, and wherein the RNA polynucleotide is codon optimized mRNA.

Aspects of the application provide an Ebola virus treatment that includes at least one RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a single-domain antibody, at least one 5′ terminal cap, and at least one chemical modification.

In other aspects, the invention is a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or single-chain variable fragment (scFv) having specificity for a human immunodeficiency virus (HIV) and a pharmaceutically acceptable carrier or excipient. In some embodiments the scFv comprises variable regions of heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide of about 4 to about 30 amino acids. In some embodiments the scFv comprises variable regions of heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide of about 20 amino acids.

In some embodiments the antibody or scFv comprises a polypeptide sequence of any of SEQ ID NOs: 182-198; a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 182-198; a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 182-198; a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 182-198; a polypeptide sequence having at least 98% sequence identity to any one of SEQ ID NOs: 182-198; or a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 182-198.

In some embodiments the ORF comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 166-171. In some embodiments the RNA comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 174-179.

In other aspects the invention is a composition, of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an isolated antibody that specifically binds to human immunodeficiency virus (HIV), wherein the antibody has two or more of the following characteristics: (a) demonstrates neutralization of HIV, with an ID₅₀ of within 20% of a control neutralization activity, wherein the control is a corresponding protein antibody; (b) is an scFv that demonstrates protection, as measured by decreased plasma viremia relative to baseline prior to administration of the scFv in an animal model of HIV infection when administered either before or after virus challenge; (c) is an scFv having a (G4S)₄ linker; or (d) wherein the antibody or scFv comprises three complementarity determining regions (CDRs) contained within any one of the variable region sequences listed in SEQ ID NO.200- 217 and a pharmaceutically acceptable carrier or excipient. In some embodiments the antibody is a full length antibody or an antigen-binding fragment thereof.

A composition of three or more ribonucleic acid (RNA) polynucleotides each having an open reading frame (ORF), wherein a first ORF encodes a full length antibody or antigen binding fragment thereof that specifically binds to HIV, wherein a second ORF encodes an scFv that specifically binds to HIV, and wherein a third ORF encodes an scFv that specifically binds to HIV is provided in other aspects.

In some embodiments the first ORF encodes an antibody that specifically interacts with a CD4 binding site. In some embodiments the first ORF encodes an N6 IgG.

In some embodiments the composition includes four RNA polynucleotides. In some embodiments two of the four RNA polynucleotides comprise ORFs encoding a light chain and a heavy chain of an N6 IgG. In some embodiments the ORFs encoding a light chain and a heavy chain of an N6 IgG have a nucleic acid sequence of SEQ ID NO.171 and 170 respectively. In some embodiments the ORFs encoding a light chain and a heavy chain of an N6 IgG have a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO.171 and 170 respectively.

In some embodiments the first ORF encodes a polypeptide having at least 80%, 81%, 82%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.186-187.

In some embodiments the second ORF encodes an scFv that specifically interacts with a V1/V2 region of HIV. In some embodiments the second ORF encodes PDGM_1400 (scFV- FC_var7). In some embodiments the second ORF encodes a polypeptide having at least 80%, 81%, 82%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.185, 188-192. In other embodiments the second ORF has at least 80%, 81%, 82%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.169. In some embodiments the RNA polynucleotide having the second ORF has a nucleic acid sequence of SEQ ID NO.177. In some embodiments the RNA polynucleotide having the second ORF has at least 80%, 85%, 90%, 95%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO.177.

In other embodiments the scFv that specifically interacts with a V1/V2 region of HIV demonstrates at least 2 times higher neutralization of HIV than an scFv that specifically interacts with a V1/V2 region of HIV and includes a (GGGS)3 (SEQ ID NO: 199) linker. In some embodiments the third ORF encodes an scFv that specifically interacts with a V3 base region.

In some embodiments the third ORF encodes a variant of PGT_121. In some embodiments the third ORF encodes a polypeptide having at least 80%, 81%, 82%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.182-184, 193-194, 197-198. In other

embodiments the third ORF has at least 80%, 81%, 82%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.166-168. In some embodiments the third ORF has at least 90%, 95%, 985, or 100% sequence identity to SEQ ID NO.166-168. In other embodiments the RNA

polynucleotide having the third ORF has a nucleic acid sequence of SEQ ID NO.174-176. In some embodiments the RNA polynucleotide having the third ORF has at least 80%, 85%, 90%, 95%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO.174-176. In some embodiments the third ORF encodes a functional variant of a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO.195-196. In some embodiments the scFv that specifically interacts with a V3 region of HIV demonstrates at least 2 times higher neutralization of HIV than an scFv that specifically interacts with a V3 region of HIV and includes a (GGGS)3 (SEQ ID NO: 199) linker.

In some embodiments the composition is formulated in a lipid nanoparticle (LNP) comprising an ionizable amino lipid, a structural lipid, a PEG lipid and a non-cationic lipid. In some embodiments each RNA polynucleotide is formulated in a separate LNP. In other embodiments all the RNA polynucleotides are formulated in a separate LNP. In some aspects, the invention is a method of treating an HIV infection in a subject, by administering to a subject any of the compositions described herein in a therapeutically effective amount to treat the HIV infection. In some embodiments the subject is a human. In some embodiments the method of treating is a method of passively immunizing a mammalian subject against an HIV infection by administering to the subject the composition, wherein the subject is at risk of having or being exposed to an influenza virus infection.

The invention is some aspect is a method of treating an HIV infection in a subject, comprising administering to a subject the composition as described herein, wherein each of the RNA polynucleotides in the composition is administered separately to the subject or is administered together in a single formulation in a therapeutically effective amount to treat the HIV infection.

In some embodiments, a 5’ terminal cap is 7mG(5')ppp(5')NlmpNp.

In some embodiments, the RNA polynucleotide comprises at least one chemical modification is selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine,), 5-methoxyuridine and 2’-O-methyl uridine.

In some embodiments, the RNA polynucleotide is formulated in a lipid nanoparticle (LNP) carrier. In further embodiments, the lipid nanoparticle carrier comprises a molar ratio of about 20-60% cationic lipid: 5-25% non-cationic lipid: 25-55% sterol; and 0.5-15% PEG- modified lipid. In other embodiments, the cationic lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9- ((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, a lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments the cationic lipid is an ionizable cationic lipid and the non- cationic lipid is a neutral lipid, and the sterol is a cholesterol.

Some embodiments of the present disclosure provide an Ebola virus treatment that includes at least one RNA polynucleotide having an open reading frame encoding a single- domain antibody, wherein at least 80% of the uracil in the open reading frame have a chemical modification, optionally wherein the Ebola virus treatment is formulated in a lipid nanoparticle. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, a chemical modification is in the 5-position of the uracil. In some embodiments, a chemical modification is an N1-methyl pseudouridine.

Some embodiments of the present disclosure provide methods of treating an Ebola virus infection in a subject, the method comprising administering to a subject having an Ebola virus infection a composition described herein in a therapeutically effective amount to treat the Ebola virus infection. In some embodiments, the composition comprises a polynucleotide that encodes a polypeptide targeted against a viral protein.

In some embodiments, an Ebola virus treatment is administered to the subject subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

Further aspects of the application provide methods of passively immunizing a mammalian subject against Ebola virus comprising administering to the subject a

composition described herein, wherein the subject is at risk of having or being exposed to Ebola virus infection. In some embodiments, the mammalian subject is a human. In some embodiments, the subject is a non-human primate. Non-limiting examples of non-human primate subjects include macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

In other aspects, the present disclosure provides a composition comprising an RNA (e.g., mRNA) polynucleotide having an open reading frame encoding a heavy-chain-only antibody (HCAb) having specificity for an influenza virus (e.g., an HCAb or a fragment thereof capable of binding to the influenza virus) and a pharmaceutically acceptable carrier or excipient. In some embodiments, the HCAb comprises a fragment crystallizable (Fc) region.

In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence of any of SEQ ID NOs: 1-10. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence having at least 98% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 1-10.

In other aspects a composition, of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or antigen-binding fragment thereof that specifically binds to an influenza A hemagglutinin (HA) subtype is provided. In some embodiments the composition is formulated in a lipid nanocarrier. In other embodiments the antibody or antigen-binding fragment thereof comprises a polypeptide sequence of any of SEQ ID NOs: 1-10 or has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-10. In some embodiments the antibody or antigen- binding fragment thereof is a broadly neutralizing antibody that cross-protects against influenza strains having different HA subtypes. In other embodiments the antibody cross protects against influenza virus having one or more of H1, H2 or H3. In some embodiments the antibody or antigen-binding fragment thereof binds to an epitope in the HA stem. In some embodiments the antibody or antigen-binding fragment thereof binds to a hydrophobic groove on the HA stem. In other embodiments the antibody or antigen-binding fragment thereof binds to an epitope in HA that prevents the viral fusion process. In yet other embodiments the antibody or antigen-binding fragment thereof binds to an epitope in HA that prevents virus attachment. In some embodiments the epitope is THLKFKYPAL…TGN. In other embodiments the epitope is an H3 epitope having the following amino acid sequence: Xaa1 Xaa2 Leu Xaa3 Xaa4 Lys Tyr Pro Xaa5 Xaa6, wherein Xaa1 is Thr, His, or Tyr; Xaa2 is His, Lys, Asn or Gln; Xaa3 is Lys, Glu or Asn; Xaa4 is Phe or Tyr, Xaa5 is Ala of Glu; and Xaa6 is Leu or Gln. In other embodiments the epitope is an H3 epitope having the following amino acid sequence: Xaa1 Xaa2 Leu Xaa3 Xaa4 Lys Tyr Pro Xaa5 Xaa6 Xaa7 Gly Asn, wherein Xaa1 is Thr, His, or Tyr; Xaa2 is His, Lys, Asn or Gln; Xaa3 is Lys, Glu or Asn; Xaa4 is Phe or Tyr, Xaa5 is Ala of Glu; Xaa6 is Leu or Gln, and Xaa 7 is Thr or Lys.

In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence of SEQ ID NO: 11. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence of SEQ ID NO: 12. In some embodiments, the RNA polynucleotide encodes an HCAb comprising a polypeptide sequence of SEQ ID NO: 13. In some embodiments, the HCAb binds to a hemagglutinin (HA) protein of the influenza virus. In some embodiments, the HA protein is a type 3 HA (H3). In some embodiments, the HA protein is a type 1 HA (H1).

In some embodiments, the influenza virus is a Type A influenza virus. In some embodiments, the Type A influenza virus is of subtype H3N2. In some embodiments, the Type A influenza virus is of subtype H1N1.

In some embodiments, the composition comprising the RNA (e.g., mRNA) polynucleotide having the open reading frame further comprises an adjuvant. In some embodiments, the open reading frame is codon-optimized.

In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises at least one chemical modification. In some embodiments, the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine,), 5-methoxyuridine and 2′-O-methyl uridine.

In some embodiments, the RNA (e.g., mRNA) polynucleotide comprises at least one 5′ terminal cap. In some embodiments, the at least one 5′ terminal cap comprises

7mG(5′)ppp(5′)NlmpNp.

In some embodiments, the composition comprising the RNA (e.g., mRNA) polynucleotide is formulated in a nanoparticle. In some embodiments, the nanoparticle has a mean diameter of 50-200 nm. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol.

In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In some embodiments, the nanoparticle has a polydispersity value of less than 0.4. In some embodiments, the nanoparticle has a net neutral charge at a neutral pH value. In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid:5-25% non-cationic lipid:25-55% sterol:0.5-15% PEG-modified lipid.

In some embodiments, at least 80% of the uracil in the open reading frame have a chemical modification. In some embodiments, 100% of the uracil in the open reading frame have a chemical modification. In some embodiments, the chemical modification is in the 5- position of the uracil. In some embodiments, the chemical modification is a N1-methyl pseudouridine.

In other aspects the invention is a composition of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to influenza A hemagglutinin (HA), wherein the antibody has two or more of the following characteristics: (a) binds to influenza HA with a dissociation constant (K_D) of less than 5.5x10^-9 M as measured in a real-time bio- layer interferometer based biosensor (Octet HTX assay); (b) demonstrates neutralization of a single influenza A virus selected from H1N1 and H3N2, with an IC₅₀ of less than 15 µg/mL; (c) is a heavy chain variable domain antibody (VHH) that demonstrates protection, as measured by increased survival in an animal model of influenza virus infection when administered either before or after virus challenge in comparison to a multi-domain antibody; or (d) wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain variable region sequences listed in Table 1 and a pharmaceutically acceptable carrier or excipient. In some embodiments the recombinant antibody or antigen-binding fragment thereof binds to influenza HA with a K_D of less than 1.3x10^-9 M. In other embodiments the recombinant antibody or antigen-binding fragment thereof demonstrates neutralization of a single influenza A virus selected from H1N1 and H3N2, with an IC₅₀ of less than 7 µg/mL.

A composition of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a single influenza A hemagglutinin (HA) subtype, wherein the antibody or antigen-binding fragment thereof does not bind to other HA subtypes and a

pharmaceutically acceptable carrier or excipient is provided in other aspects of the invention. In some embodiments the antibody or antigen-binding fragment thereof does not cross neutralize other HA subtypes. IN other embodiments the single HA subtype is H1or H3.

In other aspects the invention is a composition of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to an influenza A hemagglutinin (HA) subtype, wherein the antibody or antigen-binding fragment thereof does not bind a BNInfAb-1 (Broadly Neutralizing Influenzan antibody-1) epitope or a BNInfAb-2 (Broadly Neutralizing Influenzan antibody-2) epitope, and a pharmaceutically acceptable carrier or excipient.

In some embodiments the antibody or antigen-binding fragment thereof is a heavy chain variable domain antibody (VHH).

In other aspects a composition of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding a neutralizing anti-influenza single heavy chain variable domain antibody (VHH) that neutralizes an influenza virus at least 10 times more effectively relative to a control is provided. In some embodiments the control is an BNINFAB-1 antibody. In other embodiments the VHH is non-competing and non-interfering with BNINFAB-1.

An isolated polypeptide of at least one single domain antibody that specifically binds to a hemagglutinin (HA) protein of an influenza virus and having a sequence represented by any of SEQ ID NOs: 1 to 18 or to a sequence having at least 70% sequence identity with a sequence of any of SEQ ID NOs: 1 to 18, wherein the polypeptide does not comprise a naturally occurring antibody is provided in other aspects of the invention. In some embodiments the at least one single domain antibody is a VHH domain. In other aspects a composition of a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding any of the isolated polypeptides described herein is provided.

In some aspects, the disclosure provides methods of treating an influenza virus infection in a subject, comprising administering to a subject having an influenza virus infection a composition provided herein in a therapeutically effective amount to treat the influenza virus infection. In some embodiments, the influenza virus infection is a Type A influenza virus infection. In some embodiments, the Type A influenza virus infection is of subtype H3N2. In some embodiments, the Type A influenza virus infection is of subtype H1N1.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a non-human primate. Non-limiting examples of non-human primate subjects include macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

In some embodiments, the composition is administered subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs.

In some aspects, the disclosure provides methods of passively immunizing a mammalian subject against an influenza virus infection comprising administering to the subject a composition provided herein, wherein the subject is at risk of having or being exposed to an influenza virus infection. In some embodiments, the influenza virus infection is a Type A influenza virus infection. In some embodiments, the Type A influenza virus infection is of subtype H3N2. In some embodiments, the Type A influenza virus infection is of subtype H1N1.

In some embodiments, the mammalian subject is a human. In some embodiments, the mammalian subject is a non-human primate. Non-limiting examples of non-human primate subjects include macaques, marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIG.1 depicts the results of an exemplary phage ELISA showing nine anti-H3 and one anti-H1 HCAbs selected from a phage display library.

FIG.2 shows the results of an exemplary set of binding experiments, in which the binding of non-limiting HCAbs to HA of different strains of H3N2 and H1N1 was assessed.

FIG.3 shows exemplary results of epitope binning using non-limiting HCAbs.

FIG.4A depicts kinetic traces for non-limiting HCAbs binding to hemagglutinin. FIG.4B reports the kinetic parameters obtained during the exemplary kinetic characterization depicted in FIG.4A.

FIG.5A depicts survival data when mice have been treated with mRNA encoding antibody of the invention and challenged with a lethal dose of Influenza H1N1.

FIG.5B depicts a graph showing changes in viral load in the same animals as treated in FIG.5A.

FIG.6 is a schematic of certain polynucleotide constructs of the present invention illustrating the modular design of the encoding polynucleotides.

FIGs.7A-7B depict graphs showing expression of AB 9114 in non-human primates at doses of 0.1 mg/kg (7A) or 0.3 mg/kg (7B).

FIGs.8A-8C depict graphs showing tolerability to expression of AB 9114 in non- human primates including body weight (8A), aspartate aminotransferase levels (8B) and alanine aminotransferase levels (8C).

FIGs.9A-9C depict graphs showing tolerability to expression of AB 9114 in non- human primates including complement activation (9A), IL6 levels (9B) and MCP-1 levels (9C).

FIGs.10A-10B depict bar graphs showing changes in expression levels of AB 9114 formulated in PEG stearate LNPs (10B) versus PEG-DMG LNPs (10A) in non-human primates.

FIG.11 is a graph depicting a polyclonal phage ELISA showing that EBOV binders and enriched with each round of panning.

FIG.12A-12B is a set of graphs showing multiple screens (12A) and a VHH phage screen yielding EBOV Zaire and Sudan strain cross-binders (12B) . FIG.13 shows the most prevelant CDR3s from Round 1 of Screen 1, which were determined using Next Generation Sequencing (NGS). The sequences, from top to bottom, correspond to SEQ ID NOs: 146-155.

FIG.14A-14B show the sequences and Western blots of two soluble purified VHHs, ZSZ-01 (SEQ ID NO: 156) (14A) and ZSZ-02 (SEQ ID NO: 157) (14B).

FIG.15 is a graph depicting hEPO expression (ng/mL) at predose, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours following once weekly IV administration of hEPO mRNA–LNP formulations.

FIG.16 is a graph depicting levels of anti-PEG IgM (U/mL) following once weekly IV administration of the hEPO mRNA–LNP formulations.

FIG.17 shows in vitro biophysical characterization of IgG and scFV-FC. Single chains showed heterogeneity. A longer linker reduces dimer peaks.

FIG.18 shows that tolerance to conversion to scFV-FC format varied among three bnAbs.

FIG.19 shows antibody titers, neutralization data, meting temperature and aggregation properties for PGDM1400 scFv variants in comparison to IgG. Sequences at the bottom correspond from left to right to SEQ ID NOs: 158 and 159.

FIG.20 shows antibody titers, neutralization data, meting temperature and aggregation properties for PGT-121 scFv variants in comparison to IgG. Sequences at the bottom correspond from left to right to SEQ ID NOs: 158 and 159.

FIG.21 shows antibody titers, neutralization data, meting temperature and aggregation properties for 10-1074 scFv variants in comparison to IgG. The sequence at the bottom corresponds to SEQ ID NO: 158.

FIG.22 shows IgG expression. DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics, reagents and for biological assays to be able design, synthesize and deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo, in situ or ex vivo, such as to effect physiologic outcomes which are beneficial to the cell, tissue or organ and ultimately to an organism. One beneficial outcome is to cause intracellular translation of the nucleic acid and production of at least one encoded peptide or polypeptide of interest.

Antibodies, also known as immunoglobulins, are glycoproteins produced by B cells. Using a unique and highly evolved system of recognintion, antibodies can recognize a target and tag a target epitope, foreign entity or invading microbe for attack by the immune system thereby neutralizing its effect. The production of antibodies is the main function of the humoral immune system. Antibodies are secreted by a plasma cell which is a type of white blood cell.

Antibodies occur in two physical forms, a soluble form that is secreted from the cell, and a membrane-bound form that is attached to the surface of a B cell and is referred to as the B cell receptor (BCR). Soluble antibodies are released into the blood and tissue fluids, as well as many secretions to continue to survey for invading microorganisms.

The majority of antibodies comprise two heavy chains and two light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes isotypes (IgA, IgD, IgE, IgG and IgM) are known in mammals and trigger a different immune response for each different type of foreign object, epitope or microbe they encounter.

Frequently the binding of an antibody to an antigen has no direct biological effect. Rather, the significant biological effects are a consequence of secondary "effector functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. These functions include fixation of complement, binding of phagocytic cells, lymphocytes, platelets, mast cells, and basophils which have immunoglobulin receptors. This binding can activate the cells to perform some function. Some antibodies or immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

Currently, the majority of antibodies are generated using recombinant or cloning strategies and product heterogeneity is common to monoclonal antibody and other

recombinant biological production. Such heterogeneity is typically introduced either upstream during expression or downstream during manufacturing. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice which are used in cloning strategies. All of these however, suffer from drawbacks associated with the systems used for generation including degree of purity, speed of development, cross reactivity, low affinity and variable specificity.

In other aspects, the invention is a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or single-chain variable fragment (scFv) (i.e., one or more antibodies or scFv fragments) having specificity for a human immunodeficiency virus (HIV) and a pharmaceutically acceptable carrier or excipient. In some embodiments the scFv comprises variable regions of heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide of about 4 to about 30 amino acids.

Described herein are compositions (including pharmaceutical compositions) and methods for the design, preparation, manufacture and/or formulation of antibodies where at least one component of the antibody is encoded by a polynucleotide. As such the present invention is directed, in part, to polynucleotides, specifically IVT polynucleotides, chimeric polynucleotides and/or circular polynucleotides encoding one or more antibodies and/or components thereof.

Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the antibodies described herein.

According to the present invention, the polynucleotides are preferably modified in a manner as to avoid the deficiencies of or provide improvements over other antibody molecules of the art.

Provided herein, therefore, are antibodies or portions thereof encoded by

polynucleotide(s) and antibody compositions comprising at least one polynucleotide which have been designed to produce a therapeutic outcome and optionally improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access, engagement with translational machinery, mRNA half-life, translation efficiency, protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and/or modulation of a cell’s status, antibody target affinity and/or specificity, reduction of antibody cross reactivity, increase of antibody purity, increase or alteration of antibody effector function and/or antibody activity.

The methods of the present invention are and can be utilized to engineer novel polynucleotides for the in vivo production of antibodies in such a manner as to provide improvements over standard antibody technology.

In some embodiments, the polynucleotides are designed to produce one or more antibodies, or combinations of antibodies selected from the group consisting of IgA, IgG, IgM, IgE, and IgD.

An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The antibodies described herein can be derived from murine, rat, human, or any other origin. As used herein, the term“antibody” encompasses not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), single domain antibodies such as heavy-chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody’s amino acid sequence of the constant domain of its heavy chains (if applicable), immunoglobulins can be assigned to different classes. There are five major classes of naturally-occurring immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

An antibody such as anti-viral antibody described herein may comprise a heavy chain variable region (V_H), a light chain variable region (V_L), or a combination thereof. Optionally, the antibody may further comprise an antibody constant region or a portion thereof (e.g., C_H1, C_H2, C_H3, or a combination thereof). The heavy chain constant region can be of any suitable class as described herein and of any suitable origin, e.g., human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is derived from a human IgG (a gamma heavy chain). The light chain constant region can be a kappa chain or a lambda chain from a suitable origin. Antibody heavy and light chain constant regions are well known in the art, e.g., those provided in the IMGT database (www.imgt.org) or at www.vbase2.org/vbstat.php., both of which are incorporated by reference herein.

When needed, the antibody as described herein may comprise a modified constant region. For example, it may comprise a modified constant region that is immunologically inert, e.g., does not trigger complement mediated lysis, or does not stimulate antibody- dependent cell mediated cytotoxicity (ADCC). ADCC activity can be assessed using methods disclosed in U.S. Pat. No.5,500,362. Alternatively, the constant region may be modieifed such that it has an elevated effort activity, for example, enhanced ADCC activity. In some embodiments, the constant region can be modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8.

In some embodiments, the heavy chain constant region used in the antibodies described herein may comprise mutations (e.g., amino acid residue substitutions) to enhance a desired characteristic of the antibody, for example, increasing the binding activity to the neonatal Fc receptor (FcRn) and thus the serum half-life of the antibodies. It was known that binding to FcRn is critical for maintaining antibody homeostasis and regulating the serum half-life of antibodies. One or more (e.g., 1, 2, 3, 4, 5, or more) mutations (e.g., amino acid residue substitutions) may be introduced into the constant region at suitable positions (e.g., in C_H2 region) to enhance FcRn binding and enhance the half-life of the antibody. See, e.g., Dall’Acqua et al., J.B.C., 2006, 281:23514-23524; Robbie et al., Antimicrob. Agents Chemother, 2013, 57(12):6147; and Dall’Acqua et al., J. Immunol.2002169:5171-5180.

In some embodiments, the antibodies described herein specifically bind to the corresponding target antigen or an epitope thereof. An antibody that“specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets. An antibody“specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., HA of a specific influenza virus strain) or an antigenic epitope therein is an antibody that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes in the same antigen. It is also understood with this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such,“specific binding” or“preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that“specifically binds” to a target antigen or an epitope thereof may not bind to other antigens or other epitopes in the same antigen.

In some embodiments, an antibody as described herein has a suitable binding affinity for the target antigen or antigenic epitopes thereof. As used herein,“binding affinity” refers to the apparent association constant or K_A. The K_A is the reciprocal of the dissociation constant (K_D). The antibody described herein may have a binding affinity (K_D) of at least 10^-5, 10^-6, 10^-7, 10^-8, 10^-9, 10^-10 M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased K_D. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher K_A (or a smaller numerical value K_D) for binding the first antigen than the K_A (or numerical value K_D) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein).

For example, in some embodiments, the anti-influenza virus antibodies described herein have a higher binding affinity (a higher K_A or smaller K_D) to a first influenza virus strain or the HA antigen of a first influenza virus strain as compared to the binding affinity to second influenza virus strain or the HA antigen of the second influenza virus strain.

Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. In some embodiments, any of the anti-influenza virus antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free]/(Kd+[Free]) It is not always necessary to make an exact determination of K_A or K_D though, since sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., determined using a method such as ELISA or FACS analysis, is proportional to K_A or K_D, and thus can be used for comparisons, such as determining whether a higher affinity is, e.g., 2-fold higher, to obtain a qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

In one example, the antibody described herein is a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity and/or affinity. In some instances, one or more Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence (e.g., a germline sequence or a consensus sequence). The humanized antibody optimally may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have Fc regions modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, and/or six) which are altered with respect to the original antibody (termed one or more CDRs“derived from” one or more CDRs from the original antibody). Humanized antibodies may also involve optimized antibodies derived from affinity maturation.

In another example, the antibody as described herein is a chimeric antibody, which can include a heavy constant region and optionally a light constant region from a human antibody. Chimeric antibodies refer to antibodies having a variable region or part of variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals (e.g., a non-human mammal such as mouse, rabbit, and rat), while the constant portions are homologous to the sequences in antibodies derived from another mammal such as human. In some embodiments, amino acid modifications can be made in the variable region and/or the constant region.

In yet another example, the antibody described herein can be a single-domain antibody, which interacts with the target antigen via only one single variable domain such as a single heavy chain domain (as opposed to traditional antibodies, which interact with the target antigen via heavy chain and light chain variable domains). A single-domain antibody can be a heavy-chain antibody (VHH) which contains only an antibody heavy chain and is devoid of light chain. In additional to a variable region (for example, a V_H), a single-domain antibody may further comprise a contant region, for example, C_H1, C_H2, C_H3, C_H4, or a combination thereof.

In some embodiments, the antibodies and antigen binding fragments thereof comprises a heavy chain variable region having an amino acid sequence sharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity with any of the amino acid sequences provided herein (e.g. HA in Table 1). In some embodiments, the amino acid sequence of the antibody comprises an amino acid sequence provided herein.

In some examples, the antibody binds the same epitope as an antibody comprising any of the VH chains known in the art and/or exemplified herein and/or competes against such an antibody from binding to the antigen. Such an antibody may comprise the same heavy chain CDRs as those known in the art and/or exemplified herein. An antibody having the same CDR (e.g., CDR3) as a reference antibody means that the two antibodies have the same amino acid sequence in that CDR region as determined by the same methodology (e.g., the Kabat definition, the Chothia definition, the AbM definition, or the contact definition).

Alternatively, an antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions of one of the antibodies known in the art and/or exemplified herein and binds the same epitope of antigen with substantially similar affinity (e.g., having a K_D value in the same order). In one example, the amino acid residue variations are conservative amino acid residue substitutions. As used herein, a“conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some instances, the antibody may be a germlined variant of any of the exemplary antibodies disclosed herein. A germlined variant contains one or more mutations in the framework regions as relative to its parent antibody towards the corresponding germline sequence. To make a germline variant, the heavy or light chain variable region sequence of the parent antibody or a portion thereof (e.g., a framework sequence) can be used as a query against an antibody germline sequence database (e.g., www.bioinfo.org.uk/abs/,

www.vbase2.org, or www.imgt.org) to identify the corresponding germline sequence used by the parent antibody and amino acid residue variations in one or more of the framework regions between the germline sequence and the parent antibody. One or more amino acid substitutions can then be introduced into the parent antibody based on the germline sequence to produce a germlined variant.

In some examples, the antibody is a single chain antibody, which may comprise only one variable region (e.g., V_H) or comprise both a V_H and a V_L. Such an antibody can be encoded by a single RNA molecule. In other examples, the antibody described herein is a multi-chain antibody comprising an independent heavy chain and an independent light chain. Such a multi-chain antibody may be encoded by a single ribonucleic acid (RNA) molecule, which can be a bicistronic molecule encoding two separate polypeptide chains. Such an RNA molecule may contain a signal sequence between the two coding sequences such that two separate polypeptides would be produced in the translation process. Alternatively, the RNA molecule may include a sequence coding for a cleavage site (e.g., a protease cleavage site) inbetween the heavy and light chains such that it produces a single precursor polypeptide, which can be processed via cleavage at the cleavage site to produce the two separate heavy and light chains. Alternatively, the heavy and light antibody chains may be encoded by two separate RNA molecules.

The RNA (e.g., mRNA) treatments of the present disclosure comprise one or more polynucleotides, e.g., polynucleotide constructs, which encode one or more antibodies and antigen binding fragments thereof. In exemplary aspects, polynucleotides of the disclosure, e.g., antibody-encoding RNA polynucleotides, may include at least one chemical

modification. In exemplary aspects, polynucleotides of the disclosure, e.g., antibody- encoding RNA polynucleotides, may be fully modified (e.g., chemically modified) with respect to one or more nucleobases.

Aspects of the disclosure provide compositions comprising RNA polynucleotides encoding one or more antibodies and antigen binding fragments thereof. In some

embodiments, the antibodies and antigen binding fragments thereof encoded by an RNA polynucleotide of the present application comprises a heavy chain antibody comprising a variable (VH) domain. In some embodiments, the antibodies and antigen binding fragments thereof encoded by an RNA polynucleotide of the present application comprises a fragment crystallizable (Fc) region. The Fc region is the tail region of an antibodies and antigen binding fragments thereof which contains constant domains (e.g., CH₂ and CH₃); the other region of the antibodies and antigen binding fragments thereof being the Fab region which contains a variable domain (e.g., VH) and a constant domain (e.g., CH₁), the former of which defines binding specificity.

As described herein, antibodies can comprise a VH domain. In some embodiments, the VH domain further comprises one or more constant domains (e.g., CH₂ and/or CH₃) of an Fc region and/or one or more constant domains (e.g., CH₁) of a Fab region. In some embodiments, each of the one or more constant domains (e.g., CH₁, CH₂, and/or CH₃) can comprise or consist of portions of a constant domain. For example, in some embodiments, the constant domain comprises 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the corresponding full sequence. Targets and Compositions of the Invention

Targets of the Invention

The polynucleotides, constructs, and/or compositions of the present invention are useful in targeting or binding to polypeptides or proteins. In some embodiments

polynucleotides encode one or more antibodies or fragments thereof which bind to an infective agent such as a bacteria, virus or biomolecules thereof, cell surface molecules or cancer antigens. Examples of infectious agents which may be targeted or bound by the peptides or proteins encoded by the polynucleotides of the present invention include both bacteria and viruses.

Examples of viruses which may be targeted using the compositions or constructs of the present invention include, but are not limited to, adenovirus; chikungunya, Herpes simplex, type 1; Herpes simplex, type 2; Varicella-zoster virus; Epstein-barr virus; Human cytomegalovirus; Human herpesvirus, type 8; Human papillomavirus; BK virus; JC virus; Smallpox; Hepatitis B virus; Human bocavirus; Parvovirus B19; Human astrovirus;

Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinovirus; Severe acute respiratory syndrome virus; Hepatitis C virus; yellow fever virus; dengue virus; West Nile virus; Rubella virus; Hepatitis E virus; Human immunodeficiency virus (HIV); Influenza virus; Guanarito virus; Junin virus; Lassa virus; Machupo virus; Sabiá virus; Crimean- Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measles virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus; Human metapneumovirus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Coltivirus; or Banna virus.

Examples of pathogenic bacteria which may be targeted using the compositions or constructs of the present invention include, but are not limited to, Acinetobacter baumannii, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,

Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis,

Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, and/or Yersinia pseudotuberculosis.

In some embodiments the compositions and methods are useful for the treatment of chikungunya virus infection. Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to the Alphavirus genus of the Togaviridae family. Some embodiments of the present disclosure provide RNA encoding an anti-Chikungunya virus (CHIKV) antibody, which comprises one or more RNA polynucleotides having an open reading frame encoding at least one anti- CHIKV antibody, specific for a CHIKV antigenic polypeptide. In some embodiments, the RNA polynucleotide encodes two or more anti- CHIKV antibodies.

In some embodiments, the anti-CHIKV antibody is specific for an antigenic polypeptide which is a CHIKV structural protein or an antigenic fragment thereof. For example, a CHIKV structural protein may be an envelope protein (E), a 6K protein, or a capsid (C) protein. In some embodiments, the CHIKV structural protein is an envelope protein selected from E1, E2, and E3. In some embodiments, the CHIKV structural protein is E1 or E2. In some embodiments, the CHIKV structural protein is a capsid protein. In some embodiments, the antigenic polypeptide is a fragment or epitope of a CHIKV structural protein.

In some embodiments, the antibody is an antibody that binds to an epitope on the E2 protein of CHIKV. In some embodiments, the antibody binds to E2-A162, or an epitope formed by residues E2-G95, E2-A162, E2-A164, E2-E165, E2-E166 and/or E2-I167, or any combination thereof. In some embodiments, the antibody binds to an epitope formed by residues E2-Y69, E2-F84, E2-V113, E2-G114, E2-T116, and/or E2-D117, or any

combination thereof. In some embodiments, the epitope comprises E2-G95.

In some embodiments, the antibody is an antibody that binds to at least one of:

Subunit I-E2-E24 and Subunit I-E2-I121 and at least one of: Subunit II-E2-G55, Subunit II- E2-W64, Subunit II-E2-K66, Subunit II-E2-R80. In some embodiments, the antibody binds to Subunit I-E2-E24 and Subunit I-E2-I121 and at least one of: Subunit II-E2-G55, Subunit II-E2-W64, Subunit II-E2-K66, Subunit II-E2-R80. In some embodiments, the antibody binds to at least two of Subunit II-E2-G55, Subunit II-E2-W64, Subunit II-E2-K66, Subunit II-E2- R80. In some embodiments, the antibody binds to at least three of Subunit II-E2-G55, Subunit II-E2-W64, Subunit II-E2-K66, Subunit II-E2-R80. In some embodiments, the antibody binds to at least three of Subunit II-E2-G55, Subunit II-E2-W64, Subunit II-E2- K66, and Subunit II-E2-R80. In some embodiments, the antibody binds to Subunit II-E2- G55, Subunit II-E2-W64, Subunit II-E2-K66, and Subunit II-E2-R80.

In some embodiments, the antibody binds to the membrane distal region of a CHIKV E1/E2 trimer. In some embodiments, the antibody binds to the exterior face of the E1/E2 heterocomplex. The exterior face refers to the portion of the E1/E2 heterocomplex that is exposed when the E1/E2 hetero-protein is in its native form on the virion surface, such as in its trimeric form.

In some embodiments the compositions and methods are useful for the treatment of influenza virus infection. Influenza hemagglutinin (HA) is a glycoprotein found on the surface of influenza viruses. HA is responsible for binding the virus to cells with sialic acid on the membranes, such as cells in the upper respiratory tract or erythrocytes. HA is also responsible for the fusion of the viral envelope with the endosome membrane. As it is the major surface protein of the type A influenza virus and its function essential to the entry process, HA neutralization would provide an effective option for targeted therapeutics.

Accordingly, the present disclosure provides RNA (e.g., mRNA) polynucleotides encoding antibodies, for example, antibodies and antigen binding fragments thereof including heavy-chain-only antibodies (HCAbs), that bind influenza HA. Compositions and methods of treating influenza virus infection, as provided herein, may be used to induce a balanced immune response, comprising both cellular and humoral immunity.

Influenza HA has at least eighteen different HA subtypes, classified as subtypes H1 through H18. The first three HA subtypes, H1, H2, and H3, are found in human influenza viruses. The antibodies and antigen binding fragments thereof provided in the present disclosure can be useful in binding and/or neutralizing one or more of H1, H2, and H3. As such, compositions and methods provided herein can be useful in treating influenza subtypes characterized by H1, H2, and/or H3. For example, influenza strains of subtypes H1N1 and/or H3N2 can be targeted using compositions and methods described herein.

Examples of influenza strains of subtype H1N1 include A/swine/Iowa/15/1930, A/Solomon Island/3/2006, A/South Carolina/1/1918, A/New Caledonia/20/1999, A/Puerto Rico/08/1934, A/Ohio/1983, A/WSN/1933, or A/California/04/2009. Examples of influenza strains of subtype H3N2 include A/Moscow/10/1999, A/Sydney/5/1997, A/Hong

Kong/8/1968, A/Brisbane/10/2007, A/Victoria/3/1975, A/Johannesburg/33/1994, A/Los Angeles/2/1987, A/Fujian/411/2002, A/Switzerland/971593/2013, A/Christchurch/4/1985, A/California/7/2004, A/Perth/16/2009, and A/Wisconsin/67/2005. It should be appreciated that these exemplary strains are non-limiting, and are thus not all-encompassing of any conceivable existing or future influenza strain expressing the HA proteins (e.g., H1 and H3) targeted by the compositions and methods provided herein. In some embodiments, the VH domain comprises an amino acid sequence consisting of or essentially consisting of an amino acid sequence of Table 1 (SEQ ID NO: 1-13). Table 1: Amino acid VH sequences of HCAbs

The CDRs are presented below in Table 2 (Kabat method) and Table 3 (Chothia method)

Table 2

Table 3

In some embodiments, the antibodies and antigen binding fragments thereof encoded by an RNA polynucleotide of the present application comprises an amino acid sequence of the consensus sequence“CON-1” in Table 1 (SEQ ID NO: 11). CON-1 represents a consensus sequence for SEQ ID NOs: 1-10, where“X” is any amino acid and“Z” is optionally any amino acid or no amino acid at that position.

In some embodiments, the antibodies and antigen binding fragments thereof encoded by an RNA polynucleotide of the present application comprises an amino acid sequence of the consensus sequence“CON-2” in Table 1 (SEQ ID NO: 12). CON-2 represents a consensus sequence for SEQ ID NOs: 1-2 and 4-8, where“X” is any amino acid and“Z” is optionally any amino acid or no amino acid at that position. In some embodiments the consensus sequence provides a framework of VHH antibodies for an influenza VHH. In some embodiments, the antibodies and antigen binding fragments thereof encoded by an RNA polynucleotide of the present application comprises an amino acid sequence of the consensus sequence“CON-3” in Table 1 (SEQ ID NO: 13). CON-3 represents a consensus sequence for SEQ ID NOs: 1 and 4-5, where“X” is any amino acid and“Z” is optionally any amino acid or no amino acid at that position.

In other embodiments a polypeptide comrpsing any of the following sequences or nucelci acids encoding the peptides is provided.

VQLX₁EX₂GGG (SEQ ID NO: 56), wherein X₁ is L or V and X₂ is S or T

GGSLRLSCAASGF (SEQ ID NO: 57)

DSVKGRFTISRDN (SEQ ID NO: 58)

MNSLRAEDTAVYX₃CA (SEQ ID NO: 59); wherein X₃is Y or S

WGQGTLVTVSS (SEQ ID NO: 60)

The compositions may be designed as a single therapeutic that treats all strains of seasonal flu and pandemic flu where the polynucleotides encode IgG against hemaglutinins associated with emerging strains with pandemic potential.

According to the present invention, polynucleotides or constructs and their associated compositions may be designed to produce a commercially available antibody, a variant or a portion thereof in vivo.

In one embodiment the polynucleotide encodes CR6261. CR6261 is a monoclonal antibody that binds to a broad range of the influenza virus including the 1918 "Spanish flu" (SC1918/H1) and to a virus of the H5N1 class of avian influenza that jumped from chickens to a human in Vietnam in 2004 (Viet04/H5). CR6261 is reported to neutralize numerous strains from multiple subtypes. CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of hemagglutinin, the predominant protein on the surface of the influenza virus.

In some embodiments, the polynucleotides may encode a CR6261 single chain Fv (scFv) antibody fragments fused to the human IgG Fc moiety (scFv-Fc). In some

embodiments, CR6261 variable light and/or heavy chain domains are linked via a suitable linker known in the art and/or described herein, and the CR6261 variable light and/or heavy chain domains are further connected through a linker to a Fc moiety.

In some embodiments, the CR6261 variable light and/or heavy chain domains within a scFv-Fc construct are variants of the native CR6261 variable light and/or heavy chain domains. The native CR6261 variable light and/or heavy chain domains may comprise one or more single amino acid substitutions in order to optimize the CR6261 variable light and/or heavy chain domains for the scFv format and/or to ensure optimal stability and antigen binding.

In some embodiments, the CR6261 variable light and/or heavy chain domains in the scFv-Fc construct are arranged so that the variable heavy chain is 5’ to the variable light chain (V_H-V_L). In other embodiment, the CR6261 variable light and/or heavy chain domains in the scFv-Fc construct are arranged so that the variable light chain is 5’ to the variable heavy chain (V_L-V_H).

In some embodiments, the CR6261 scFv constructs comprise the Dall’Acqua half-life extending“YTE” Fc substitutions M265Y, S267T, and T269E (using the Kabat numbering).

In some embodiments, the CR6261 variable light domain may comprise at least one substitution. The CR6261 variable light domain may comprise one, two, three, four, five or more than five substitutions. As a non-limiting example, the CR6261 variable light domain may comprise the two substitutions A13S (where alanine (A) at position 13 is substituted with serine (S)) and L111T (where leucine (L) at position 111 is substituted with

threonine(T)). As another non-limiting example, the CR6261 variable light may comprise the four substitutions A13S (where alanine (A) at position 13 is substituted with serine (S)), L40K (wherein leucine (L) at position 40 is substituted with lysine (K)), G82E (where glycine (G) at position 82 is substituted with glutamic acid (E)) and L111T (where leucine (L) at position 111 is substituted with threonine (T)).

In some embodiments, the CR6261 variable heavy domain may comprise at least one substitution. The CR6261 variable heavy domain may comprise one, two, three, four, five or more than five substitutions. As a non-limiting example, the CR6261 variable heavy domain may comprise the two substitutions V11T (where valine (V) at position 11 is substituted with threonine (T)) and M93T (where methionine (M) at position 93 is substituted with threonine (T)). As another non-limiting example, the CR6261 variable heavy may comprise the three substitutions E6Q (where glutamic acid (E) at position 6 is substituted with glutamine (Q)), V11T (where valine (V) at position 11 is substituted with threonine (T) and M93T (where methionine (M) at position 93 is substituted with threonine (T)).

In some embodiments, the polynucleotides may encode CR9114. In some

embodiments, the polynucleotides may encode a CR9114 single chain Fv (scFv) antibody fragments fused to the human IgG Fc moiety (scFv-Fc). In some embodiments, CR9114 variable light and/or heavy chain domains are linked via a suitable linker known in the art and/or described herein, and the CR9114 variable light and/or heavy chain domains are further connected through a linker to a Fc moiety. In some embodiments, the CR9114 variable light and/or heavy chain domains within a scFv-Fc construct are variants of the native CR9114 variable light and/or heavy chain domains. The native CR9114 variable light and/or heavy chain domains may comprise one or more single amino acid substitutions in order to optimize the CR9114 variable light and/or heavy chain domains for the scFv format and/or to ensure optimal stability and antigen binding.

In some embodiments, the CR9114 variable light and/or heavy chain domains in the scFv-Fc construct are arranged so that the variable heavy chain is 5’ to the variable light chain (V_H-V_L). In other embodiment, the CR9114 variable light and/or heavy chain domains in the scFv-Fc construct are arranged so that the variable light chain is 5’ to the variable heavy chain (V_L-V_H).

In some embodiments, the CR9114 variable light domain may comprise at least one substitution. The CR9114 variable light domain may comprise one, two, three, four, five, six, seven or more than seven substitutions. As a non-limiting example, the CR9114 variable light domain may comprise the four substitutions S1Q (where serine (S) at position 1 is substituted with glutamine (Q)), Y2S (where tyrosine (Y) at position 2 is substituted with serine (S)), V3A (where valine (V) at position 3 is substituted with alanine (A)) and A9S (where alanine (A) at position 9 is substituted with serine (S)). As another non-limiting example, the CR9114 variable light may comprise the seven substitutions S1Q (where serine (S) at position 1 is substituted with glutamine (Q)), Y2S (where tyrosine (Y) at position 2 is substituted with serine (S)), V3A (where valine (V) at position 3 is substituted with alanine (A)), A9S (where alanine (A) at position 9 is substituted with serine (S)), F40K (where phenylalanine (F) at position 40 is substituted with lysine (K)), V58G (where valine (V) at position 58 is substituted with glycine (G)) and L110T (where leucine (L) at position 110 is substituted with threonine (T)).

In some embodiments, the CR9114 variable heavy domain may comprise at least one substitution. The CR9114 variable heavy domain may comprise one, two, three, four, five or more than five substitutions. As a non-limiting example, the CR9114 variable heavy domain may comprise the three substitutions V11T (where valine (V) at position 11 is substituted with threonine (T)), T87R (where threonine (T) at position 87 is substituted with arginine (R)) and V93T (valine (V) at position 93 is substituted with threonine (T)). As another non- limiting example, the CR9914 variable heavy domain may comprise the five substitutions V11T (where valine (V) at position 11 is substituted with threonine (T)), D46E (where aspartic acid (D) at position 46 is substituted with glutamic acid (E)), N84S (where asparagine (N) at position 84 is substituted with serine (S)), T87R (where threonine (T) at position 87 is substituted with arginine (R)) and V93T (valine (V) at position 93 is substituted with threonine (T)).

In some embodiments, the polynucleotides of the invention may be used to protect an infant against infection or disease, including but not limited to diseases caused by respiratory syncytial virus (RSV) alone or in combination with B. pertussis, by administering the polynucleotides to a pregnant female with a gestational infant. While not wishing to be bound by theory, the antibody encoded by the polynucleotide of interest may be transferred via the placenta to the gestational infant, protecting the infant against infection or disease. The polynucleotides of the invention may be administered alone or in combination with an immunogenic composition as described in WO2014/024024 and WO2014/024026, the contents of each which is herein incorporated by reference in its entirety.

Hepatitis C is a contagious liver disease that results from infection with the hepatitis C virus, one of the most common viral liver infections with approximately 150 million people have chronic infections with risk of liver cirrhosis and/or liver cancer, 3-4 million people infected yearly and about 350,000 deaths every year. It can range in severity from a mild illness lasting a few weeks to a serious, lifelong illness. HCV infection and associated liver cirrhosis is the most common indication for orthotopic liver transplantation among adults and HCV infection remains a problem after transplantation and recurrent hepatic infection is the leading cause of graft failure.

Chronic hepatitis C is characterized by a high turnover of infected cells and continuous de novo infection of target cells. Due to the vital role of de novo infection in maintenance of HCV infection, blocking of de novo infection is a potential target for antiviral therapy. The viral envelope glycoproteins, E1 and E2, are the major components of the HCV particle and hence play a pivotal role in the entry process and hypervariable region 1 (HVR1), consisting of the first 27 amino acids of E2 (aa 384–410), is a major target for neutralizing antibodies.

Another approach that is currently promoted for the treatment and prevention of HCV infection/re-infection is blocking a pathway preventing the immune system from recognizing and fighting cancer cells and pathogens. Current treatment involves a combination of IFN-α and ribavirin.

In one embodiment, the polynucleotides of the invention may be used in the treatment and/or prevention of hepatitis C virus (HCV) infection. As used herein“hepatitis C virus” or “HCV” means a viral disease that can lead to swelling of the liver. In one embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for prognosing, diagnosing, and/or treating of HCV in a subject. In another embodiment, the polynucleotides of the invention may be used to protect a subject from or inhibit HCV-mediated morbidity or mortality in a subject.

In one embodiment, the polynucleotides of the current invention may be used in combination with ribavirin, IFN-α and/or pegylated (peg) IFN-α to treat and/or prevent HCV.

Rabies is a wide spread viral disease that is transmitted from animals to humans. Around 60,000 people die annually from rabies, and the disease threatens over 3 billion people in rural areas of Asia and Africa where human vaccines and immunoglobulin are not readily available or accessible. Rabies is an RNA virus that belongs to the order

Mononegavirales. The viral genome encodes 5 proteins designated as N, P, M, G, and L. Currently, lethal rabies is prevented by administering a rabies virus vaccine and rabies virus immunoglobulin (RIG) post-exposure. Two types of RIG are employed, Human RIG (HRIG) and Equine RIG (ERIG).

In one embodiment, the polynucleotides of the invention may be used in the treatment or prevention of rabies virus infection. As used herein“rabies virus” is a virus normally spread to people from the saliva of infected animals and infects nerve cells. In one

embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for the treatment or prevention of rabies.

The human immunodeficiency virus (HIV) is a lentivirus that causes the acquired immunodeficiency syndrome (AIDS). HIV infects cells of the human immune system such as helper cells expressing the CD4 receptor on their surface, macrophages, and dendritic cells, compromising cell-mediated immunity and allowing opportunistic infections and cancers to thrive.

In one embodiment, the polynucleotides of the invention may be used in the treatment or prevention of human immunodeficiency virus (HIV). As used herein“human

immunodeficiency virus” or“HIV” means a variable retrovirus that invades and inactivates helper T cells of the immune system and is a cause of AIDS and AIDS-related complex. In one embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for the treatment or prevention of HIV.

In one embodiment, the polynucleotides may encode at least one neutralizing HIV antibody, which may target the HIV-1 viral spike.

It has been discovered herein that mRNA encoding antibodies can be used in the treatment of HIV infection. Although these complex proteins are difficult to engineer as mRNA therapeutics, optimal compositions, sequences and combinations having a broad spectrum of neutralization activities when expressed in vivo from mRNA have been developed according to the invention. The parameters involved in the creation and

optimization of full intact antibodies and single chain antibodies to enable co-expression in a therapeutically effective formulation are described herein.

Recently combinations of HIV antibodies have been developed for the prophylactic treatment of HIV infection. The antibodies are particularly useful in reducing the risk of HIV transmission for vulnerable populations and reducing the burden of disease globally. These antibodies are delivered as proteins. Although nucleic acid delivery of these antibodies would be desirable a number of challenges exist in the development of mRNA encoding antibodies. For instance, challenges exist in the expression of complex proteins such as antibodies and scFv. A number of factors can effect activity of scFv. Additionally optimal combinations of antibodies delivered as proteins don’t necessarily translate to optimal activity when the antibodies are delivered as mRNA.

Several effective combinations of HIV antibodies have been described in the art and include PGT12110-1074, PGDM1400, and 3BNC117 NIH 45-46 N6 (Scharf et al. Cell Reports 20147, 785-795). While combinations of these protein antibodies are useful as therapeutics, simply expressing the proteins as mRNAs and co-delivering the mRNAs does not result in an adequate therapeutic composition. For instance, co-expression of these proteins from mRNA as combinations of IgGs produces non-functional pairings. A therapeutically effective combination of these antibodies involves the optimal convergence of the spectrum of neutralizing activity across many HIV strains/clades, optimized level of expression, and protein stability contributing to half-life in vivo.

The methods described herein have resulted in the production of therapeutic compositions of mRNA that are able to achieve similar therapeutic results in comparison with the cocktails of protein antibodies, quite unexpectedly. Additionally, several of the scFv variants designed herein have significnatly higher expression and/or neutralization and/or thermostabiliity properties in comparison to scFv designed according to traditional rules/expections for designing scFv.

Thus, in some embodiments the invention is a composition of mRNA encoding a combination of HIV antibodies. For instance, the composition may include one or more intact intact antibodies such as IgGs, one or more mono- and bispecific scFvFcs, and IgG/sc “hybrids” and one or more antigen binding fragments. The composition may be any combination of mRNA/ lipid nanoparticle (LNP) carrier. For instance, multiple antibodies may be included on a single mRNA or separate mRNAs. In some embodiments each peptide chain of antibody is encoded by an individual mRNA. Additionally, each mRNA may be coformulated individually in an LNP. Alternatively one or more or all of the mRNAs may be formulated in an LNP.

The HIV compositions of the invention may be designed based on known HIV antibodies or newly developed HIV antibodies. In some embodiments the mRNA encodes known therapeutically effective HIV antibodies. In other embodiments the mRNA encodes variants of known HIV antibodies. In some embodiments the mRNA does not encode full intact PGT12110-1074, PGDM1400, and 3BNC117 NIH 45-46 N6. In other embodiments the mRNA does encode variants of one or more of PGT12110-1074, PGDM1400, and 3BNC117 NIH 45-46 N6, including antigen binding fragments thereof and scFv.

In some embodiments the composition comprises mRNA encoding a combination of an intact IgG and two scFvFcs. This product in some embodiments includes four mRNAs total, which produce an IgG (two mRNAs encoding light chain and heavy chain) and two scFv-Fcs (each one mRNA).

In some embodiments, the methods for developing the mRNA encoding the HIV antibodies of the invention involves selecting broadly neutralizing antibodies. Key antigen binding components of such antibodies may be identified and used to build variants. For instance, the composition in some embodiments comprises a combination of mRNA encoding at least one antibody that binds CD4 binding site of gp120, at least one antibody that blocks the V1/V2 site of gp120, and/or at least one antibody that blocks the V3 site of gp120. The antibodies may be any combination of intact antibodies and scFv. A composition of antibodies that bind to and block these three regions of the HIV virus have been shown to be highly effective at preventing HIV transmission and are thus useful therapeutically in subjects at risk of infection with HIV as well as subjects infected with HIV, in preventing further infection.

Thus, compositions of three or more ribonucleic acid (RNA) polynucleotides each having an open reading frame (ORF) encoding an HIV antibody or scFv are encompassed within the invention. These compositions include therapeutically effective antibody combinations. Such combinations may include for instance, a combination of an IgG and two or more scFv.

In one embodiment, the intact IgG antibody may be one that blocks the CD4 binding site of gp120, e.g., a 3BNC117, NIH45-46, or an N6 IgG. The scFv may be an scFv antibody that blocks the V1/V2 site of gp120, e.g., PDGM_1400 (such as scFV-FC_var7) and/or an scFv that blocks the V3 site of gp120, e.g., PGT_121 or 10-1074. In one embodiment, a composition of the invention comprises a nucleotide sequence encoding an the intact IgG antibody comprising the CDRs of N6 IgG, an scFv antibody comprising the CDRs of PDGM1400 and an scFv comprising the CDRs of PGT-121.

The antibodies described herein may include modified or variant variable domains from the sequences disclosed herein. The variant or modification may be, for instance, amino acid substitutions, compared to the sequences disclosed herein . Modifications can also include amino acid deletions. For example, one or two amino acids may be deleted from a variant VH and/or VL domain. The deleted amino acids typically may be from the carboxyl or amino terminal ends of the VH and/or VL domains.

A variable domain of the antibodies described herein comprises three

complementarity determining regions (CDRs), each of which is flanked by a framework region (FW). For example, a VH domain may comprise a set of three heavy chain CDRs, HCDR1, HCDR2, and HCDR3. A VL domain may comprise a set of three light chain CDRs, LCDR1, LCDR2, and LCDR3. A set of HCDRs disclosed herein can be provided in a VH domain that is used in combination with a VL domain. A VH domain may be provided with a set of HCDRs as disclosed herein, and if such a VH domain is paired with a VL domain, then the VL domain may be provided with a set of LCDRs disclosed herein. Exemplary CDRs useful in the antibodies (intact antibody, antigen binding fragment thereof or scFv) are included herein as SEQ ID NOs.200-217). Exemplary PGT-121 CDRs and PGDM-1400 CDRs identified based on two different labeling scheme, Kabat and Chothia are presented below.

217)

In some embodiments the HIV antibody is a single chain Fv (scFv). As used herein, the term“single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. An scFv has a variable domain of light chain (VL) connected from its C- terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately the scFv comprises of polypeptide chain where in the C-terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain. In some embodiments the scFv constructs may be oriented in a variety of ways. For instance the order to VH and VL in the construct may vary and alter the expression and/or activity of the scFv. In some embodiments the scFv constructs are oriented, from N to C terminus, VL-linker-VH-linker- CH2-CH3. It was discovered that this orientation may produce optimal results.

The scFv fragments, which may be attached to one another and to Fc domains by flexible linkers. In some embodiments, the variable heavy and variable light chains are covalently attached using flexible linkers. Traditionally, flexible linkers such as those containing glycine and serine are used. The scFV-FC synthesis from a typical antibody format involved the addition of linkers. Typically an ideal linker is considered to be (G₄S)₃. However, it was discovered that longer linkers were more effective in producing highly neutralizing scFv. For instance, it was found that increasing the VL-VH linker length could reduce strain and oligomerization. The (G4S)₃ linker is a shorter linker, which tends to produce more strain. The (G4S)₄ linker (also referred to as Linker20) is a longer linker with less strain. Increasing the length of the linker between the VL and VH domains reduced the strain, causing opening of the VL-VH interface and domain swapping, in particular in the PGT-121. It is desirable for the linker to have greater than 15 amino acids in length. In some embodiments it is desirable to have a linker of 16-30 amino acids. In other embodiments the linker has 16—25, 16-20, 18-30, 18-25, 18-20, 19-30, 19-25, 19-20, 20-30, 20-25, or 20-22 amino acids. In some embodiments the linker is (G₄S)₄.

During scFv initially, constant domains are removed. Two linkers are added to connect the FV region which binds the antigen, and the FC region. In some embodiments the FC region is a wild type of FC region. In other embodiments it is a variant of wild type. In some embodiments a wild t pe constant re ion is a wild t pe I G1 constant re ion (e. .,

SFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO. 220)).

In some embodiments, the antibody constructs may be enhanced by modifying glycan stability to enhance the effectivity of the antibody. For instance, as shown in the Examples presented herein adding glycan stability to antibodies such as scFv i.e., PGT-121 variants, improved the activity of the antibody.

The compositions and combinations of compositions described herein are useful for treating HIV infection, typically by blocking entry of HIV into host cells, and thus blocking infection. Latent reservoirs of HIV-1 infected cells are difficult to treat with traditional HIV medicine. The ability to block entry of HIV provides a significant advance in the treatment and prevention of HIV-1 infection. In humans, a latent reservoir is established within days of initial infection and persists for the lifetime of the individual. The combinations of antibodies disclosed herein can be used in preventing the establishment of the reservoir.

In some embodiments the antibodies are administered as a bolus IV injection or bolus. This form of delivery can produce high levels of expressed antibody.

Staphylococcus is a genus of Gram-positive bacteria. Staphylococci, gram positive bacteria including coagulase-negative staphylococci (CONS) and Staphylococcus aureus, are responsible for between 56 and >75% of hospital-acquired, late-onset neonatal sepsis.

In one embodiment, the polynucleotides of the invention may be used in the treatment or prevention Staphylococcus infection. As used herein“Staphylococcus” means a bacteria that can cause sepsis. In one embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for the treatment or prevention of sepsis caused by Staphylococcus.

Anthrax is a serious infectious disease caused by gram-positive bacteria known as Bacillus anthracis (B. anthracis). Although rare, human can get infected with anthrax if they come in contact with infected animals or contaminated animal products. Bacillus anthracis has also long been considered a potential biological warfare agent.

Anthrax toxin is composed of a cell-binding protein, known as protective antigen (PA), and two enzyme components, called edema factor (EF) and lethal factor (LF). These three protein components act together to impart their physiological effects. PA is a protein component of the toxins produced by the bacterium. It initiates the activity of the toxins by attaching to cells in the infected person, and then facilitates the entry of additional destructive factors– LF and EF into the cells. PA comprises a protein having a weight of about 83 kD (PA83) that is cleaved into a protein having a weight of about 63 kD (PA63). Three forms of human anthrax disease exist based on their portal of entry: cutaneous (most common), causes a localized inflammatory necrotic lesion; pulmonary (highly fatal), causes sudden, massive chest edema succeeded by cardiovascular shock; gastrointestinal, a (rare but also fatal) from ingestion of spores.

In one embodiment, the polynucleotides of the invention may be used in the treatment or prevention of Bacillus anthracis (B. anthracis) infection and anthrax. As used herein “Bacillus anthracis” or“B. anthracis” is the bacterium that causes anthrax. In one

embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for the treatment or prevention of anthrax.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic-uremic syndrome (HUS). Diarrhea-associated HUS is a common cause of acute renal failure and up to 50% of patients with HUS develop some degree of renal impairment. The Shiga toxins produced by E. coli are majorly Stx1 and Stx2. (Thorpe, Clinical Infectious Disease, vol.38(9), 1298-1303 (2004), the contents of which are incorporated herein by reference in their entirety).

In one embodiment, the polynucleotides of the invention may encode any antibody that targets a Shiga toxin, including, but not limited to the shigamabs antibody, for the prevention or treatment of STEC and HUS. The polynucleotides may be used in combination with antibiotic therapies in the prevention and/or treatment of STEC and HUS.

Clostridium difficile (C. difficile) is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. C. difficile most commonly affects older adults in hospitals or in long-term care facilities and often occurs after the use of antibiotics. The most well-understood toxins produced by pathogenic C. difficile strains are enterotoxin (Clostridium difficile toxin A) and cytotoxin (Clostridium difficile toxin B), both of which can produce diarrhea and inflammation in infected patients.

In one embodiment, the polynucleotides of the invention may be used in the treatment or prevention of Clostridium difficile (C. difficile) infection. In one embodiment, the polynucleotides of the invention may encode at least one antibody, fragment or variant thereof which may be used for the treatment or prevention of C. difficile infection.

In one embodiment, the polynucleotides described herein encode a monoclonal antibody that is directed to toxin A and/or toxin B for C. difficile, which may be used for the treatment or prevention of C. difficile infection. Besides Pseudomonas aeruginosa, there are other genera of Gram-negative bacteria, such as the Acinetobacter species, that often produce multidrug-resistant and even pan- resistant strains.

Acinetobacter baumannii is a Gram-negative bacterium that has been isolated form water and soil samples. A. baumannii affects people with compromised immune systems, and is becoming increasingly more frequent as a hospital-derived infection. Due to its ability to form a biofilm, it can persist on artificial surfaces and infect new patients. It is thought that the ability of A. baumannii to form biofilms is correlated with multi drug resistance (MDR). A. baumannii also forms protective capsules composed of polysaccharides around each individual cell, further providing additional protection from antibiotics and antibacterial agents. In a 2009 study, A. baumannii was found to be responsible for 19.1% of ventilator- associated pneumonia (VAP) cases in European intensive care units.

Although the passive immunization approach provides only temporary immunity, it may be sufficient to clear an acute A. baumannii infection, alone or in combination with other antimicrobials. Passive immunization may therefore become an important therapeutic approach, in particular given the high incidence of multi-drug resistant strains.

In one embodiment, the antibodies encoded by the polynucleotides of the present invention bind or target one or more proteins or peptides of Acinetobacter baumannii.

Hepatitis B virus (HBV) causes an infectious illness of the liver and has caused epidemics in parts of Asia and Africa, and is still endemic in China. The virus is transmitted by exposure to infectious blood or body fluids such as semen and vaginal fluids. Perinatal infection is a major route of infection in developing countries. The acute illness causes liver inflammation, vomiting, jaundice. HBV results in one million deaths annually, primarily due to cirrhosis and liver cancer.

The hepatitis B virus is a partially double stranded DNA virus composed of a 27 nm nucleocapsid core (HBcAg), surrounded by an outer lipoprotein coat (envelope) containing the surface antigen (HBsAg). The nucleocapsid has been found to be very immunogenic and a number of antibodies with nucleocapsid epitopes have been described. The nucleocapsid is dimorphic and is comprised of either 90 or 120 dimers arranged such that the four-helix bundles project from the surface as 25 Å-long spike. Together these two capsid forms are known as core antigen (HBcAg). The principal antigenic determinant of HBcAg, the immunodominant loop, is located at the apices of the capsid spikes (Watts et al, Non- Canonical Binding Of An Antibody Resembling A Naïve B Cell Receptor Immunoglobin To Hepatitis B Virus Capsids J Mol Biol. Jun 20, 2008; 379(5): 1119–1129, the contents of which are herein incorporated by reference in its entirety).

In some embodiments, the polynucleotides of the invention may encode at least one antibody that binds to the nucleocapsid of HBV.

In some embodiments, the polynucleotides of the invention may encode at least one antibody that binds to the nucleocapsid of HBV for the prevention, management, or treatment of HBV infections.

In some embodiments, the polynucleotides of the invention may encode at least one antibody that binds to the nucleocapsid of HBV that can be used in combination with other HBV treatments, including existing HBV vaccines.

Cancer is one of the leading causes of death in the United States. Conventional methods of cancer treatment like chemotherapy, surgery or radiation therapy, can be limited in their efficacy since they are often nonspecific to the cancer. In many cases tumors, however, can specifically express genes whose products are required for inducing or maintaining the malignant state. These proteins may serve as antigen markers for the development and establishment of efficient anti-cancer treatments. The polynucleotides of the invention may encode anti-cancer antibodies. Such antibodies may be used to target cancer cells by binding cancer antigens.

Cancer antigens can elicit an immune response. These antigens can be either proteins, polysaccharides, lipids, or glycolipids, which can be recognized as foreign by immune cells, such as T cells and B cells. Exposure of immune cells to one or more of these antigens can elicit a rapid cell division and differentiation response resulting in the formation of clones of the exposed T cells and B cells. B cells can differentiate into plasma cells which in turn can produce antibodies which selectively bind to the antigens.

In cancer, there are four general groups of tumor antigens: (i) viral tumor antigens which can be identical for any viral tumor of this type, (ii) carcinogenic tumor antigens which can be specific for patients and for the tumors, (iii) isoantigens of the transplantation type or tumor-specific transplantation antigens which can be different in all individual types of tumor but can be the same in different tumors caused by the same virus; and (iv) embryonic antigens. The polynucleotides of the ivention encode any tyoe of cancer antigen including any of these 4 classes of antigens.

In addition to the development of antibodies against tumor antigens for cancer treatment, antibodies that target immune cells to boost the immune response have also been developed. For example, an anti-CD40 antibody that is a CD40 agonist can be used to activate dendritic cells to enhance the immune response.

Additionally antibodies may function as immune checkpoint modulators. In some preferred embodiments of the invention the polynucleotides encode an antibody that is a T cell activator such as an immune checkpoint modulator. Immune checkpoint modulators include both stimulatory checkpoint molecules and inhibitory checkpoint molecules i.e., an anti-CTLA4 and anti-PD1 antibody.

Stimulatory checkpoint inhibitors function by promoting the checkpoint process.

Several stimulatory checkpoint molecules are members of the tumor necrosis factor (TNF) receptor superfamily - CD27, CD40, OX40, GITR and CD137, while others belong to the B7-CD28 superfamily - CD28 and ICOS. OX40 (CD134), is involved in the expansion of effector and memory T cells. Anti-OX40 monoclonal antibodies have been shown to be effective in treating advanced cancer. MEDI0562 is a humanized OX40 agonist. GITR, Glucocorticoid-Induced TNFR family Related gene, is involved in T cell expansion Several antibodies to GITR have been shown to promote an anti-tumor responses. ICOS, Inducible T- cell costimulator, is important in T cell effector function. CD27 supports antigen-specific expansion of naïve T cells and is involved in the generation of T and B cell memory. Several agonistic anti-CD27 antibodies are in development. CD122 is the Interleukin-2 receptor beta sub-unit. NKTR-214 is a CD122-biased immune-stimulatory cytokine.

Inhibitory checkpoint molecules include but are not limited to PD-1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3. CTLA-4, PD-1 and its ligands are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T-cell function and other cell functions. CTLA-4, Cytotoxic T- Lymphocyte-Associated protein 4 (CD152) , is involved in controlling T cell proliferation.

The PD-1 receptor is expressed on the surface of activated T cells (and B cells) and, under normal circumstances, binds to its ligands (PD-L1 and PD-L2) that are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages. This interaction sends a signal into the T cell and inhibits it. Cancer cells take advantage of this system by driving high levels of expression of PD-L1 on their surface. This allows them to gain control of the PD-1 pathway and switch off T cells expressing PD-1 that may enter the tumor microenvironment, thus suppressing the anticancer immune response. Pembrolizumab (formerly MK-3475 and lambrolizumab, trade name Keytruda) is a human antibody used in cancer immunotherapy. It targets the PD-1 receptor. The checkpoint inhibitor is a molecule such as a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein or a combination thereof or a small molecule. For instance, the checkpoint inhibitor inhibits a checkpoint protein which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or a

combination thereof. Ligands of checkpoint proteins include but are not limited to CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and B-7 family ligands. In some embodiments the anti-PD-1 antibody is BMS-936558 (nivolumab). In other embodiments the anti-CTLA-4 antibody is ipilimumab (trade name Yervoy, formerly known as MDX-010 and MDX-101).

The RNA polynucleotide of the ivnetnion may encode an antibody against any cancer antigen. As used herein, the terms“cancer antigen” and“tumor antigen” are used interchangeably to refer to antigens which are differentially expressed by cancer cells and can thereby be exploited in order to target cancer cells. Cancer antigens are antigens which can potentially stimulate apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses.

An antibody specific for a cell surface antigen of, for example, a cancer cell, may promote an immune response resulting in antibody dependent cellular cytotoxicity (ADCC). In one embodiment, the antibody may be selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE- 1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000,

LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX- 260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab and ImmuRAIT-CEA. Intrabody Constructs

According to the present invention, an intrabody construct is a polynucleotide which has been modified for expression inside a target cell and where the expression product binds an intracellular protein. Such constructs may have sub picomolar binding affinities and may be formulated for targeting to particular sites or tissues. For example, intrabody constructs may be formulated in any of the lipid nanoparticle formulations disclosed herein.

Bicistronic and/or Pseudo-bicistronic Constructs

According to the present invention, a bicistronic construct is a polynucleotide encoding a two-protein chain antibody on a single polynucleotide strand. (Fig.2B) A pseudo- bicistronic construct is a polynucleotide encoding a single chain antibody discontinuously on a single polynucleotide strand. For bicistronic constructs, the encoded two strands or two portions/regions and/or domains (as is the case with pseudo-bicistronic) are separated by at least one nucleotide not encoding the strands or domains. More often the separation comprises a cleavage signal or site or a non-coding region of nucleotides.Such cleavage sites include, for example, furin cleavage sites encoded as an“RKR” site in the resultant polypeptide.

Single Domain Constructs

According to the present invention, a single domain construct comprises one or two polynucleotides ecoding a single monomeric variable antibody domain. See Figs 3B and 4B for examples. Typically single domain antibodies comprise one variable domain (VH) of a heavy-chain antibody.

Single chain Fv Constructs

According to the present invention, a single chain Fv constructs is a polynucleotide encoding at least two coding regions and a linker region. The scFv construct may encode a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of

immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. See Fig.3A for an example. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C- terminus of the VL, or vice versa. Other linkers include those known in the art and disclosed herein.

Single chain antibodies may be camelid antibodies. They may also be human heavy chain only antibodies such as those made by Crescendo Biologics. Bispecific Constructs

According to the present invention, a bispecific construct is a polynucleotide encoding portions or regions of two different antibodies. Bispecific constructs encode polypeptides which may bind two different antigens. See Fig.4A for an example. Polynucleotides of the present invention may also encode trispecific antibodies having an affinity for three antigens.

Modular IgA Antibodies

According to the present invention, the polynucleotides can be designed as modular IgA antibodies. These antibodies may be monomers or dimers or multimers.

Modular IgA constructs comprise a VHH antigen binding domain and an IgA2 backbone which can provide protection from bacterial IgA1 protease.

Multimeric IgA constructs can be designed using an IgJ chain for polymerization and may be encoded on a polycistronic transcript using a 2A peptide or PACE cleavage. Further modification can include a secretory component of for example polyimmunoglobulin receptor (PIGR) for production of secreted IgA.

Linkers

Examples of linkers which may be used in the polynucleotides of the present invention include those in Table 4.

Table 4. Linkers

Table references: Merutka G, Shalongo W, Stellwagen E. (1991) A model peptide with enhanced helicity. Biochem.30: 4245-4248 and Sommese RF, Sivaramakrishnan S, Baldwin RL, Spudich JA. (2010) Helicity of short E-R/K peptides. Protein Sci.19: 2001- 2005.

In one embodiment, the length of a region encoding at least one peptide polypeptide of interest of the polynucleotides present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). As used herein, such a region may be referred to as a “coding region” or“region encoding.”

In one embodiment, the polynucleotides of the present invention is or functions as a messenger RNA (mRNA). As used herein, the term“messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo.

Influenza virus treatments, as provided herein, comprise at least one (e.g., one or more) RNA (e.g., mRNA) polynucleotide having an open reading frame encoding at least one antibody, antibody domain, antibody portion, and/or antibody fragment thereof that binds to an influenza virus HA protein. The terms“polynucleotide” and“nucleic acid,” in their broadest sense, include any compound and/or substance that comprises a polymer of nucleotides. Polynucleotides (also referred to as nucleic acids) may be or may include, for example, RNAs, deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β- D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- α- LNA having a 2′-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA).“Messenger RNA” (mRNA) refers to any polynucleotide that encodes at least one polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo.

The basic components of an mRNA molecule typically include at least one coding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap, and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

In some embodiments, an RNA polynucleotide encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5- 6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antibodies and antigen binding fragment polypeptides. In some embodiments, an RNA polynucleotide encodes at least 10, 20, 30, 40, 50 , 60, 70, 80, 90 or 100 antibodies and antigen binding fragment polypeptides. In some embodiments, an RNA polynucleotide encodes at least 100 or at least 200 antibodies and antigen binding fragment polypeptides. In some embodiments, an RNA polynucleotide encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or 2- 100 antibodies and antigen binding fragment polypeptides. Signal Sequences

The polynucleotides (e.g., a RNA, e.g., an mRNA) of the present disclosure can also comprise nucleotide sequences that encode additional features that facilitate trafficking of the encoded polypeptides to therapeutically relevant sites. One such feature that aids in protein trafficking is the signal sequence, or targeting sequence. The peptides encoded by these signal sequences are known by a variety of names, including targeting peptides, transit peptides, and signal peptides. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotide sequence (e.g., an ORF) that encodes a signal peptide operably linked a nucleotide sequence that encodes an antibody described herein.

In some embodiments, the "signal sequence" or "signal peptide" is a polynucleotide or polypeptide, respectively, which is from about 30-210, e.g., about 45-80 or 15-60nucleotides (3-70 amino acids) in length that, optionally, is incorporated at the 5′ (or N-terminus) of the coding region or the polypeptide, respectively. Addition of these sequences results in trafficking the encoded polypeptide to a desired site, such as the endoplasmic reticulum or the mitochondria through one or more targeting pathways. Some signal peptides are cleaved from the protein, for example by a signal peptidase after the proteins are transported to the desired site.

In some embodiments, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an antibody, wherein the nucleotide sequence further comprises a 5' nucleic acid sequence encoding a native signal peptide. In another

embodiment, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an antibody, wherein the nucleotide sequence lacks the nucleic acid sequence encoding a native signal peptide.

In some embodiments, the polynucleotide of the present disclosure comprises a nucleotide sequence encoding an antibody, wherein the nucleotide sequence further comprises a 5' nucleic acid sequence encoding a heterologous signal peptide. Fusion Proteins

In some embodiments, the polynucleotide of the present disclosure (e.g., a RNA, e.g., an mRNA) can comprise more than one nucleic acid sequence (e.g., an ORF) encoding a polypeptide of interest. In some embodiments, polynucleotides of the present disclosure comprise a single ORF encoding an antibody, a functional fragment, or a variant thereof. However, in some embodiments, the polynucleotide of the present disclosure can comprise more than one ORF, for example, a first ORF encoding an antibody (a first polypeptide of interest), a functional fragment, or a variant thereof, and a second ORF expressing a second polypeptide of interest. In some embodiments, two or morepolypeptides of interest can be genetically fused, i.e., two or more polypeptides can be encoded by the same ORF. In some embodiments, the polynucleotide can comprise a nucleic acid sequence encoding a linker (e.g., a (G₄S)_n peptide linker (wherein n is 2, 3, 4, 5, 6, 7, 8, 9 or 10) or another linker known in the art) between two or more polypeptides of interest. In some embodiments, a polynucleotide of the present disclosure (e.g., a RNA, e.g., an mRNA) can comprise two, three, four, or more ORFs, each expressing a polypeptide of interest.

In some embodiments, the polynucleotide of the present disclosure (e.g., a RNA, e.g., an mRNA) can comprise a first nucleic acid sequence (e.g., a first ORF) encoding an antibody and a second nucleic acid sequence (e.g., a second ORF) encoding a second polypeptide of interest such as an antibody Fc domain. Sequence Optimization of Nucleotide Sequence Encoding an antibody

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure is sequence optimized. In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a nucleotide sequence (e.g., an ORF) encoding an antibody, optionally, a nucleotide sequence (e.g, an ORF) encoding another polypeptide of interest, a 5'-UTR, a 3'-UTR, the 5’ UTR or 3’ UTR optionally comprising at least one microRNA binding site, optionally a nucleotide sequence encoding a linker, a polyA tail, or any combination thereof), in which the ORF(s) that are sequence optimized.

A sequence-optimized nucleotide sequence, e.g., an codon-optimized mRNA sequence encoding an antibody, is a sequence comprising at least one synonymous nucleobase substitution with respect to a reference sequence (e.g., a wild type nucleotide sequence encoding an antibody).

A sequence-optimized nucleotide sequence can be partially or completely different in sequence from the reference sequence. For example, a reference sequence encoding polyserine uniformly encoded by TCT codons can be sequence-optimized by having 100% of its nucleobases substituted (for each codon, T in position 1 replaced by A, C in position 2 replaced by G, and T in position 3 replaced by C) to yield a sequence encoding polyserine which would be uniformly encoded by AGC codons. The percentage of sequence identity obtained from a global pairwise alignment between the reference polyserine nucleic acid sequence and the sequence-optimized polyserine nucleic acid sequence would be 0%.

However, the protein products from both sequences would be 100% identical.

Some sequence optimization (also sometimes referred to codon optimization) methods are known in the art (and discussed in more detail below) and can be useful to achieve one or more desired results. These results can include, e.g., matching codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability or reduce secondary structures; minimizing tandem repeat codons or base runs that can impair gene construction or expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; removing/adding post translation modification sites in an encoded protein (e.g., glycosylation sites); adding, removing or shuffling protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting translational rates to allow the various domains of the protein to fold properly; and/or reducing or eliminating problem secondary structures within the polynucleotide. Sequence optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.

Codon options for each amino acid are given in the following TABLE.

In some embodiments, a polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding an antibody, a functional fragment, or a variant thereof, wherein the antibody, functional fragment, or a variant thereof encoded by the sequence-optimized nucleotide sequence has improved properties (e.g., compared to an antibody, functional fragment, or a variant thereof encoded by a reference nucleotide sequence that is not sequence optimized), e.g., improved properties related to expression efficacy after administration in vivo. Such properties include, but are not limited to, improving nucleic acid stability (e.g., mRNA stability), increasing translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding or prevent misfolding of the expressed proteins, reducing toxicity of the expressed products, reducing cell death caused by the expressed products, increasing and/or decreasing protein aggregation.

In some embodiments, the sequence-optimized nucleotide sequence (e.g., an ORF) is codon optimized for expression in human subjects, having structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity; overcoming a threshold of expression; improving expression rates; half-life and/or protein concentrations; optimizing protein localization; and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.

In some embodiments, the polynucleotides of the present disclosure comprise a nucleotide sequence (e.g., a nucleotide sequence (e.g, an ORF) encoding an antibody, a 5'- UTR, a 3'-UTR, a microRNA, a nucleic acid sequence encoding a linker, or any combination thereof) that is sequence-optimized according to a method comprising:

(i) substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding an antibody) with an alternative codon to increase or decrease uridine content to generate a uridine-modified sequence;

(ii) substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding an antibody) with an alternative codon having a higher codon frequency in the synonymous codon set;

(iii) substituting at least one codon in a reference nucleotide sequence (e.g., an ORF encoding an antibody) with an alternative codon to increase G/C content; or

(iv) a combination thereof.

In some embodiments, the sequence-optimized nucleotide sequence (e.g., an ORF encoding an antibody) has at least one improved property with respect to the reference nucleotide sequence. In some embodiments, the sequence optimization method is multiparametric and comprises one, two, three, four, or more methods disclosed herein and/or other optimization methods known in the art.

Features, which can be considered beneficial in some embodiments of the present disclosure, can be encoded by or within regions of the polynucleotide and such regions can be upstream (5') to, downstream (3') to, or within the region that encodes the antibody. These regions can be incorporated into the polynucleotide before and/or after sequence-optimization of the protein encoding region or open reading frame (ORF). Examples of such features include, but are not limited to, untranslated regions (UTRs), microRNA sequences, Kozak sequences, oligo(dT) sequences, poly-A tail, and detectable tags and can include multiple cloning sites that can have XbaI recognition.

In some embodiments, the polynucleotide of the present disclosure comprises a 5′ UTR. a 3′ UTR and/or a miRNA binding site. In some embodiments, the polynucleotide comprises two or more 5′ UTRs and/or 3′ UTRs, which can be the same or different sequences. In some embodiments, the polynucleotide comprises two or more miRNA binding site, which can be the same or different sequences. Any portion of the 5’ UTR, 3’ UTR, and/or miRNA binding site, including none, can be sequence-optimized and can

independently contain one or more different structural or chemical modifications, before and/or after sequence optimization.

In some embodiments, after optimization, the polynucleotide is reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, the optimized polynucleotide can be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.

Exemplary amino acid sequences and nucleotide sequences encoding human antibody are shown in tables included herein and in the Sequence Appendix. Sequence-Optimized Nucleotide Sequences Encoding Antibodies

In some embodiments, the polynucleotide of the present disclosure comprises a sequence-optimized nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the polynucleotide of the present disclosure comprises an open reading frame (ORF) encoding an antibody, wherein the ORF has been sequence optimized.

The sequence-optimized nucleotide sequences disclosed herein are distinct from the corresponding wild type nucleotide acid sequences and from other known sequence- optimized nucleotide sequences, e.g., these sequence-optimized nucleic acids have unique compositional characteristics.

In some embodiments, the percentage of uracil or thymine nucleobases in a sequence- optimized nucleotide sequence (e.g., encoding an antibody, a functional fragment, or a variant thereof) is modified (e.g,. reduced) with respect to the percentage of uracil or thymine nucleobases in the reference wild-type nucleotide sequence. Such a sequence is referred to as a uracil-modified or thymine-modified sequence. The percentage of uracil or thymine content in a nucleotide sequence can be determined by dividing the number of uracils or thymines in a sequence by the total number of nucleotides and multiplying by 100. In some embodiments, the sequence-optimized nucleotide sequence has a lower uracil or thymine content than the uracil or thymine content in the reference wild-type sequence. In some embodiments, the uracil or thymine content in a sequence-optimized nucleotide sequence of the present disclosure is greater than the uracil or thymine content in the reference wild-type sequence and still maintain beneficial effects, e.g., increased expression and/or reduced Toll-Like Receptor (TLR) response when compared to the reference wild-type sequence. Methods for optimizing codon usage are known in the art. Characterization of Sequence Optimized Nucleic Acids

In some embodiments of the present disclosure, the polynucleotide (e.g., a RNA, e.g., an mRNA) comprising a sequence optimized nucleic acid disclosed herein encoding an antibody can be can be tested to determine whether at least one nucleic acid sequence property (e.g., stability when exposed to nucleases) or expression property has been improved with respect to the non-sequence optimized nucleic acid.

As used herein, "expression property" refers to a property of a nucleic acid sequence either in vivo (e.g., translation efficacy of a synthetic mRNA after administration to a subject in need thereof) or in vitro (e.g., translation efficacy of a synthetic mRNA tested in an in vitro model system). Expression properties include but are not limited to the amount of protein produced by an mRNA encoding an antibody after administration, and the amount of soluble or otherwise functional protein produced. In some embodiments, sequence optimized nucleic acids disclosed herein can be evaluated according to the viability of the cells expressing a protein encoded by a sequence optimized nucleic acid sequence (e.g., a RNA, e.g., an mRNA) encoding an antibody disclosed herein.

In a particular embodiment, a plurality of sequence optimized nucleic acids disclosed herein (e.g., a RNA, e.g., an mRNA) containing codon substitutions with respect to the non- optimized reference nucleic acid sequence can be characterized functionally to measure a property of interest, for example an expression property in an in vitro model system, or in vivo in a target tissue or cell. a. Optimization of Nucleic Acid Sequence Intrinsic Properties

In some embodiments of the present disclosure, the desired property of the polynucleotide is an intrinsic property of the nucleic acid sequence. For example, the nucleotide sequence (e.g., a RNA, e.g., an mRNA) can be sequence optimized for in vivo or in vitro stability. In some embodiments, the nucleotide sequence can be sequence optimized for expression in a particular target tissue or cell. In some embodiments, the nucleic acid sequence is sequence optimized to increase its plasma half by preventing its degradation by endo and exonucleases.

In other embodiments, the nucleic acid sequence is sequence optimized to increase its resistance to hydrolysis in solution, for example, to lengthen the time that the sequence optimized nucleic acid or a pharmaceutical composition comprising the sequence optimized nucleic acid can be stored under aqueous conditions with minimal degradation.

In other embodiments, the sequence optimized nucleic acid can be optimized to increase its resistance to hydrolysis in dry storage conditions, for example, to lengthen the time that the sequence optimized nucleic acid can be stored after lyophilization with minimal degradation. b. Nucleic Acids Sequence Optimized for Protein Expression

In some embodiments of the present disclosure, the desired property of the polynucleotide is the level of expression of an antibody encoded by a sequence optimized sequence disclosed herein. Protein expression levels can be measured using one or more expression systems. In some embodiments, expression can be measured in cell culture systems, e.g., CHO cells or HEK293 cells. In some embodiments, expression can be measured using in vitro expression systems prepared from extracts of living cells, e.g., rabbit reticulocyte lysates, or in vitro expression systems prepared by assembly of purified individual components. In other embodiments, the protein expression is measured in an in vivo system, e.g., mouse, rabbit, monkey, etc.

In some embodiments, protein expression in solution form can be desirable.

Accordingly, in some embodiments, a reference sequence can be sequence optimized to yield a sequence optimized nucleic acid sequence having optimized levels of expressed proteins in soluble form. Levels of protein expression and other properties such as solubility, levels of aggregation, and the presence of truncation products (i.e., fragments due to proteolysis, hydrolysis, or defective translation) can be measured according to methods known in the art, for example, using electrophoresis (e.g., native or SDS-PAGE) or chromatographic methods (e.g., HPLC, size exclusion chromatography, etc.). c. Optimization of Target Tissue or Target Cell Viability

In some embodiments, the expression of heterologous therapeutic proteins encoded by a nucleic acid sequence can have deleterious effects in the target tissue or cell, reducing protein yield, or reducing the quality of the expressed product (e.g., due to the presence of protein fragments or precipitation of the expressed protein in inclusion bodies), or causing toxicity.

Accordingly, in some embodiments of the present disclosure, the sequence optimization of a nucleic acid sequence disclosed herein, e.g., a nucleic acid sequence encoding an antibody, can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid.

Heterologous protein expression can also be deleterious to cells transfected with a nucleic acid sequence for autologous or heterologous transplantation. Accordingly, in some embodiments of the present disclosure the sequence optimization of a nucleic acid sequence disclosed herein can be used to increase the viability of target cells expressing the protein encoded by the sequence optimized nucleic acid sequence. Changes in cell or tissue viability, toxicity, and other physiological reaction can be measured according to methods known in the art. d. Reduction of Immune and/or Inflammatory Response

In some cases, the administration of a sequence optimized nucleic acid encoding antibody or a functional fragment thereof can trigger an immune response, which could be caused by (i) the therapeutic agent (e.g., an mRNA encoding an antibody), or (ii) the expression product of such therapeutic agent (e.g., the antibody encoded by the mRNA), or (iv) a combination thereof. Accordingly, in some embodiments of the present disclosure the sequence optimization of nucleic acid sequence (e.g., an mRNA) disclosed herein can be used to decrease an immune or inflammatory response (other than coagulation pathway activation) triggered by the administration of a nucleic acid encoding an antibody or by the expression product of antibody encoded by such nucleic acid. In some aspects, an inflammatory response can be measured by detecting increased levels of one or more inflammatory cytokines using methods known in the art, e.g., ELISA. The term "inflammatory cytokine" refers to cytokines that are elevated in an inflammatory response. Examples of inflammatory cytokines include interleukin-6 (IL-6), CXCL1

(chemokine (C-X-C motif) ligand 1; also known as GROα, interferon-γ (IFNγ), tumor necrosis factor α (TNFα), interferon γ-induced protein 10 (IP-10), or granulocyte-colony stimulating factor (G-CSF). The term inflammatory cytokines includes also other cytokines associated with inflammatory responses known in the art, e.g., interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-13 (Il-13), interferon α (IFN-α), etc. Modified Nucleotide Sequences Encoding Antibodies

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosure comprises a chemically modified nucleobase, , for example, a chemically modified uracil, e.g., pseudouracil, 1-methylpseuodouracil, 5-methoxyuracil, or the like. In some embodiments, the mRNA is a uracil-modified sequence comprising an ORF encoding an antibody, wherein the mRNA comprises a chemically modified nucleobase, for example, a chemically modified uracil, e.g., pseudouracil, 1-methylpseuodouracil, or 5-methoxyuracil.

In certain aspects of the present disclosure, when the modified uracil base is connected to a ribose sugar, as it is in polynucleotides, the resulting modified nucleoside or nucleotide is refered to as modified uradine. In some embodiments, uracil in the

polynucleotide is at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least 90%, at least 95%, at least 99%, or about 100% modified uracil. In one embodiment, uracil in the polynucleotide is at least 95% modified uracil. In another embodiment, uracil in the polynucleotide is 100% modified uracil.

In some embodiments, a binding peptide, e.g., antibody, such as an influenza virus binding polypeptide, is longer than 4 amino acids and shorter than 30 amino acids. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. Polypeptides may also comprise single chain or multichain polypeptides such as antibodies and may be associated or linked. Most commonly, disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which at least one amino acid residue is an artificial chemical analogue of a corresponding naturally-occurring amino acid.

The term“polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a native or reference sequence.

In some embodiments“variant mimics” are provided. As used herein, the term “variant mimic” is one which contains at least one amino acid that would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, for example, phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.

“Orthologs” refers to genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Identification of orthologs is critical for reliable prediction of gene function in newly sequenced genomes.

“Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations, for example, substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

The present disclosure provides several types of compositions that are polynucleotide or polypeptide based, including variants and derivatives. These include, for example, substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.

As such, polynucleotides encoding peptides or polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends).

Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

As used herein, the term“conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue. “Features” when referring to polypeptide or polynucleotide are defined as distinct amino acid sequence-based or nucleotide-based components of a molecule respectively. Features of the polypeptides encoded by the polynucleotides include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

As used herein, when referring to polypeptides, the term“domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

As used herein, when referring to polypeptides, the terms“site” as it pertains to amino acid based embodiments is used synonymously with“amino acid residue” and“amino acid side chain.” As used herein, when referring to polynucleotides, the terms“site” as it pertains to nucleotide based embodiments is used synonymously with“nucleotide.” A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide or polynucleotide based molecules.

As used herein, the terms“termini” or“terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide, respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH₂)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (e.g., multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non- polypeptide based moiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% or 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with the reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, with art-described molecules (e.g., engineered or designed molecules or wild-type molecules). The term“identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g.,“algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on the Smith- Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamic programming is the Needleman–Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970)“A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453.). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman–Wunsch algorithm. Other tools are described herein, specifically in the definition of“identity” below.

As used herein, the term“homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Polymeric molecules (e.g., nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules) that share a threshold level of similarity or identity determined by alignment of matching residues are termed homologous. Homology is a qualitative term that describes a relationship between molecules and can be based upon the quantitative similarity or identity. Similarity or identity is a quantitative term that defines the degree of sequence match between two compared sequences. In some embodiments, polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term“homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). Two polynucleotide sequences are considered homologous if the polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least 20 amino acids. In some embodiments, homologous

polynucleotide sequences are characterized by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. Two protein sequences are considered homologous if the proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least 20 amino acids.

Homology implies that the compared sequences diverged in evolution from a common origin. The term“homolog” refers to a first amino acid sequence or nucleic acid sequence (e.g., gene (DNA or RNA) or protein sequence) that is related to a second amino acid sequence or nucleic acid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteins separated by the event of speciation or to the relationship between genes and/or proteins separated by the event of genetic duplication.“Orthologs” are genes (or proteins) in different species that evolved from a common ancestral gene (or protein) by speciation. Typically, orthologs retain the same function in the course of evolution.“Paralogs” are genes (or proteins) related by duplication within a genome. Orthologs retain the same function in the course of evolution, whereas paralogs evolve new functions, even if these are related to the original one.

The term“identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988;

Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J.

Molec. Biol., 215, 403 (1990)). RNA (e.g., mRNA) treatments of the present disclosure comprise at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one binding polypeptide that comprises at least one chemical modification.

Therapeutic compositions of the present disclosure comprise, in some embodiments, at least one nucleic acid (e.g., RNA) having an open reading frame encoding at least one antibody, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177;

PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; or PCT/IB2017/051367 all of which are incorporated by reference herein.

In some embodiments, at least one RNA (e.g., mRNA) of the present disclosure is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof. Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A“nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). A“nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standard adenosine- thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (m1ψ), 1-ethyl- pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (ψ). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

In some embodiments, a RNA nucleic acid of the disclosure comprises 1-methyl- pseudouridine

substitutions at one or more or all uridine positions of the nucleic acid.

substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid..

In some embodiments, a RNA nucleic acid of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1- methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

The nucleic acids may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

Antibodies and antigen binding fragments thereof of the present disclosure comprise at least one RNA polynucleotide, such as an mRNA (e.g., modified mRNA). mRNA, for example, is transcribed in vitro from template DNA, referred to as an“in vitro transcription template.” In some embodiments, an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a polyA tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template. Untranslated Regions (UTRs)

Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and regulated by a variety of mechanisms that are provided by various cis-acting nucleic acid structures. For example, naturally-occurring, cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element influences or modulates the initiation of polynucleotide translation, particularly when the RNA element is positioned in the 5′ UTR close to the 5’-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):515-526; Kozak (1986) Proc Natl Acad Sci 83:2850-2854).

Untranslated regions (UTRs) are nucleic acid sections of a polynucleotide before a start codon (5' UTR) and after a stop codon (3' UTR) that are not translated. In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the invention comprising an open reading frame (ORF) encoding an antibody further comprises UTR (e.g., a 5′UTR or functional fragment thereof, a 3′UTR or functional fragment thereof, or a combination thereof).

Cis-acting RNA elements can also affect translation elongation, being involved in numerous frameshifting events (Namy et al., (2004) Mol Cell 13(2):157-168). Internal ribosome entry sequences (IRES) represent another type of cis-acting RNA element that are typically located in 5′ UTRs, but have also been reported to be found within the coding region of naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet 16(10):469-473). In cellular mRNAs, IRES often coexist with the 5′-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol 4(7):a012245). Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs). Naturally-occurring uORFs occur singularly or multiply within the 5′ UTRs of numerous mRNAs and influence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under conditions of increased eIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)). Additional exemplary translational regulatory activities provided by components, structures, elements, motifs, and/or specific sequences comprising

polynucleotides (e.g., mRNA) include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol 16(3):293-299), translational activation (Villalba et al., (2011) Curr Opin Genet Dev 21(4):452-457), and translational repression (Blumer et al., (2002) Mech Dev 110(1-2):97-112). Studies have shown that naturally-occurring, cis-acting RNA elements can confer their respective functions when used to modify, by incorporation into, heterologous polynucleotides (Goldberg-Cohen et al., (2002) J Biol Chem 277(16):13635-13640).

A UTR can be homologous or heterologous to the coding region in a polynucleotide. In some embodiments, the UTR is homologous to the ORF encoding the antibody. In some embodiments, the UTR is heterologous to the ORF encoding the antibody. In some embodiments, the polynucleotide comprises two or more 5′UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences. In some

embodiments, the polynucleotide comprises two or more 3′UTRs or functional fragments thereof, each of which have the same or different nucleotide sequences.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof is sequence optimized.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.

UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency. A polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods. In some embodiments, a functional fragment of a 5'UTR or 3'UTR comprises one or more regulatory features of a full length 5' or 3' UTR, respectively. Natural 5′UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5′UTRs also have been known to form secondary structures that are involved in elongation factor binding.

By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of a polynucleotide. For example, introduction of 5′UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or antibody, can enhance expression of polynucleotides in hepatic cell lines or liver. Likewise, use of 5′UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i- NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).

In some embodiments, UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.

In some embodiments, the 5’UTR and the 3’UTR can be heterologous. In some embodiments, the 5'UTR can be derived from a different species than the 3'UTR. In some embodiments, the 3'UTR can be derived from a different species than the 5'UTR.

Co-owned International Patent Application No. PCT/US2014/021522 (Publ. No. WO/2014/164253, incorporated herein by reference in its entirety) provides a listing of exemplary UTRs that can be utilized in the polynucleotide of the present disclosure as flanking regions to an ORF.

Exemplary UTRs of the application include, but are not limited to, one or more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: a globin, such as an α- or β- globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 α polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-β) dehydrogenase); a virus (e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitis virus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), a sindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein (e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucose transporter (e.g., hGLUT1 (human glucose transporter 1)); an actin (e.g., human α or β actin); a GAPDH; a tubulin; a histone; a citric acid cycle enzyme; a topoisomerase (e.g., a 5′UTR of a TOP gene lacking the 5′ TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32 (L32); a ribosomal protein (e.g., human or mouse ribosomal protein, such as, for example, rps9); an ATP synthase (e.g., ATP5A1 or the β subunit of mitochondrial H⁺-ATP synthase); a growth hormone e (e.g., bovine (bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1 α1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyte enhancer factor 2A (MEF2A); a β-F1-ATPase, a creatine kinase, a myoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen (e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1 (Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1

(Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low density lipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); a procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); and a nucleobindin (e.g., Nucb1).

In some embodiments, the 5'UTR is selected from the group consisting of a β-globin 5’UTR; a 5′UTR containing a strong Kozak translational initiation signal; a cytochrome b- 245 α polypeptide (CYBA) 5'UTR; a hydroxysteroid (17-β) dehydrogenase (HSD17B4) 5'UTR; a Tobacco etch virus (TEV) 5'UTR; a Venezuelen equine encephalitis virus (TEEV) 5'UTR; a 5' proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5'UTR; a heat shock protein 70 (Hsp70) 5'UTR; a eIF4G 5'UTR; a GLUT15'UTR; functional fragments thereof and any combination thereof.

In some embodiments, the 3'UTR is selected from the group consisting of a β-globin 3’UTR; a CYBA 3'UTR; an albumin 3'UTR; a growth hormone (GH) 3'UTR; a VEEV 3'UTR; a hepatitis B virus (HBV) 3'UTR; α-globin 3′UTR; a DEN 3'UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3'UTR; an elongation factor 1 α1 (EEF1A1) 3'UTR; a manganese superoxide dismutase (MnSOD) 3'UTR; a β subunit of mitochondrial H(+)-ATP synthase (β-mRNA) 3'UTR; a GLUT13'UTR; a MEF2A 3'UTR; a β-F1-ATPase 3'UTR; functional fragments thereof and combinations thereof. Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the present disclosure. In some embodiments, a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. In some embodiments, variants of 5′ or 3′ UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.

Additionally, one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs.

UTRs or portions thereof can be placed in the same orientation as in the transcript from which they were selected or can be altered in orientation or location. Hence, a 5′ and/or 3′ UTR can be inverted, shortened, lengthened, or combined with one or more other 5′ UTRs or 3′ UTRs.

In some embodiments, the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5’UTR or 3’UTR. For example, a double UTR comprises two copies of the same UTR either in series or substantially in series. For example, a double beta-globin 3′UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).

In certain embodiments, the polynucleotides of the present disclosure comprise a 5'UTR and/or a 3'UTR selected from any of the UTRs disclosed herein. In some

embodiments the 5'UTR com rises:

In certain embodiments, the 5'UTR and/or 3'UTR sequence of the present disclosure comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5'UTR sequences comprising any of SEQ ID NOs: 71-88, 160, and 162 and/or 3'UTR sequences comprises any of SEQ ID NOs: 89-99, 161 and 163, and any combination thereof. The polynucleotides of the present disclosure can comprise combinations of features. For example, the ORF can be flanked by a 5′UTR that comprises a strong Kozak translational initiation signal and/or a 3′UTR comprising an oligo(dT) sequence for templated addition of a poly-A tail. A 5′UTR can comprise a first polynucleotide fragment and a second

polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).

Other non-UTR sequences can be used as regions or subregions within the

polynucleotides of the present disclosure. For example, introns or portions of intron sequences can be incorporated into the polynucleotides of the present disclosure.

Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels. In some embodiments, the polynucleotide of the present disclosure comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun.2010394(1):189-193, the contents of which are incorporated herein by reference in their entirety). In some embodiments, the polynucleotide comprises an IRES instead of a 5’UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5'UTR in combination with a non-synthetic 3'UTR.

In some embodiments, the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements

(collectively, "TEE," which refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide. As a non-limiting example, the TEE can be located between the transcription promoter and the start codon. In some

embodiments, the 5'UTR comprises a TEE.

In some embodiments, a 5′UTR and/or 3'UTR comprising at least one TEE described herein can be incorporated in a monocistronic sequence such as, but not limited to, a vector system or a nucleic acid vector.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of the present disclosure comprises a TEE or portion thereof described herein. In some embodiments, the TEEs in the 3′UTR can be the same and/or different from the TEE located in the 5′UTR.

In some embodiments, a 5'UTR and/or 3'UTR of a polynucleotide of the present disclosure can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In one embodiment, the 5′UTR of a polynucleotide of the present disclosure can include 1-60, 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1- 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 TEE sequences. The TEE sequences in the 5′UTR of the polynucleotide of the present disclosure can be the same or different TEE sequences. A combination of different TEE sequences in the 5′UTR of the polynucleotide of the present disclosure can include combinations in which more than one copy of any of the different TEE sequences are incorporated.

In some embodiments, the 5′UTR and/or 3'UTR comprises a spacer to separate two TEE sequences. As a non-limiting example, the spacer can be a 15 nucleotide spacer and/or other spacers known in the art. As another non-limiting example, the 5′UTR and/or 3'UTR comprises a TEE sequence-spacer module repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more than 10 times in the 5′UTR and/or 3'UTR, respectively. In some embodiments, the 5′UTR and/or 3'UTR comprises a TEE sequence- spacer module repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the spacer separating two TEE sequences can include other sequences known in the art that can regulate the translation of the polynucleotide of the present disclosure, e.g., miR binding site sequences described herein (e.g., miR binding sites and miR seeds). As a non-limiting example, each spacer used to separate two TEE sequences can include a different miR binding site sequence or component of a miR sequence (e.g., miR seed sequence).

The present disclosure provides synthetic polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity. In some embodiments, the disclosure provides a polynucleotide comprising a 5’ untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3’ UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and/or enhances the translational fidelity of mRNA translation. In some embodiments, the desired translational regulatory activity is a cis-acting regulatory activity. In some

embodiments, the desired translational regulatory activity is an increase in the residence time of the 43S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some embodiments, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some embodiments, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the production of aberrant translation products. In some embodiments, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.

Accordingly, the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein. In some aspects, the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that promotes and/or enhances the translational fidelity of mRNA translation. In some aspects, the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and/or reducing leaky scanning. In some aspects, the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that inhibits and/or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.

In some embodiments, the RNA element comprises natural and/or modified nucleotides. In some embodiments, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof, that provides a desired translational regulatory activity as described herein. In some embodiments, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof, that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein. RNA elements can be identified and/or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g. stem-loop), by the location of the element within the RNA molecule (e.g., located within the 5’ UTR of an mRNA), by the biological function and/or activity of the element (e.g.,“translational enhancer element”), and any combination thereof.

In some aspects, the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and/or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNA element. In some aspects, the disclosure provides a modified mRNA comprising at least one

modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15- 25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%- 60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 3- 30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.

In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine. In any of the foregoing or related aspects, the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine. In some embodiments, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine. In some embodiments, the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.

In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about 12, about 10, about 6 or about 3 nucleotides, or derivatives or analogues thereof, wherein the sequence comprises a repeating GC-motif, wherein the repeating GC-motif is [CCG]n, wherein n = 1 to 10, n= 2 to 8, n= 3 to 6, or n= 4 to 5. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, 3, 4 or 5. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1, 2, or 3. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 1. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 2. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 3. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 4. In some embodiments, the sequence comprises a repeating GC-motif [CCG]n, wherein n = 5.

In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5’ UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the sequences set forth in Table 5. In one embodiment, the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V1 [CCCCGGCGCC] (SEQ ID NO: 100) as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V2 [CCCCGGC] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence V2 as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence V2 as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence V2 as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence EK [GCCGCC] as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC- rich element comprises the sequence EK as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In some embodiments, the GC-rich element comprises the sequence EK as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence EK as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA.

In yet other aspects, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V1 [CCCCGGCGCC] (SEQ ID NO: 100) as set forth in Table 5, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 5:

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 101).

In some embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5’ UTR sequence shown in Table 5. In some embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 5:

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 101).

In other embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 5 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the following sequence shown in Table 5:

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGA (SEQ ID NO: 101).

In some embodiments, the 5’ UTR comprises the following sequence set forth in Table 5:

GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGC CACC (SEQ ID NO: 87)

Table 5

In another aspect, the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a stable RNA secondary structure comprising a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop. In one embodiment, the stable RNA secondary structure is upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal/mol, about -20 to -30 kcal/mol, about -20 kcal/mol, about -10 to -20 kcal/mol, about -10 kcal/mol, about -5 to -10 kcal/mol.

In another embodiment, the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous. In another embodiment, the sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.

RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling . Ribosome profiling is a technique that allows the determination of the positions of PICs and/or ribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion. Protection results in the generation of a 30-bp fragment of RNA termed a‘footprint’. The sequence and frequency of RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq). The footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and/or ribosome exhibits decreased processivity and fewer footprints where the PIC and/or ribosome exhibits increased processivity (Gardin et al., (2014) eLife 3:e03735). In some embodiments, residence time or the time of occupancy of a the PIC or ribosome at a discrete position or location along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.

In some embodiments, a polynucleotide of the present disclosure comprises a miR and/or TEE sequence. In some embodiments, the incorporation of a miR sequence and/or a TEE sequence into a polynucleotide of the present disclosure can change the shape of the stem loop region, which can increase and/or decrease translation. See e.g., Kedde et al., Nature Cell Biology 201012(10):1014-20, herein incorporated by reference in its entirety). MicroRNA (miRNA) Binding Sites

Polynucleotides of the present disclosure can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof. In some embodiments, polynucleotides including such regulatory elements are referred to as including “sensor sequences”.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of the present disclosure comprises an open reading frame (ORF) encoding a polypeptide of interest and further comprises one or more miRNA binding site(s). Inclusion or incorporation of miRNA binding site(s) provides for regulation of polynucleotides of the present disclosure, and in turn, of the polypeptides encoded therefrom, based on tissue-specific and/or cell-type specific expression of naturally-occurring miRNAs.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 nucleotide long noncoding RNA that binds to a polynucleotide and down-regulates gene expression either by reducing stability or by inhibiting translation of the polynucleotide. A miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA. A miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA.

As used herein, the term“microRNA (miRNA or miR) binding site” refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA. In some embodiments, a polynucleotide of the present disclosure comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s). In exemplary

embodiments, a 5'UTR and/or 3'UTR of the polynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) comprises the one or more miRNA binding site(s).

A miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide. In exemplary aspects of the present disclosure, a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA- induced silencing complex (RISC)-mediated cleavage of mRNA. The miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence. A miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence. Full or complete complementarity (e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA) is preferred when the desired regulation is mRNA degradation.

In some embodiments, a miRNA binding site includes a sequence that has

complementarity (e.g., partial or complete complementarity) with an miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some

embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.

In some embodiments, the miRNA binding site is the same length as the

corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5' terminus, the 3' terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5' terminus, the 3' terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA. By engineering one or more miRNA binding sites into a polynucleotide of the present disclosure, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the present disclosure is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5′UTR and/or 3′UTR of the polynucleotide.

Conversely, miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.

In one embodiment, a polynucleotide of the present disclosure can include at least one miRNA-binding site in the 5'UTR and/or 3′UTR in order to regulate cytotoxic or

cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells. In another embodiment, a polynucleotide of the present disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5'-UTR and/or 3′-UTR in order to regulate cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.

Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites. The decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings in tissues and/or cells in development and/or disease. Identification of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 201118:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007129:1401-1414; Gentner and Naldini, Tissue

Antigens.201280:393-403 and all references therein; each of which is incorporated herein by reference in its entirety).

Examples of tissues where miRNA are known to regulate mRNA, and thereby protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR- 208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR- 16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR- 149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR- 126).

Specifically, miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc. Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune-response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cells specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells). For example, miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR- 142 binding sites to the 3′-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med.2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.

Introducing a miR-142 binding site into the 5'UTR and/or 3′UTR of a polynucleotide of the present disclosure can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polynucleotide. The polynucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.

In one embodiment, binding sites for miRNAs that are known to be expressed in immune cells, in particular, antigen presenting cells, can be engineered into a polynucleotide of the present disclosure to suppress the expression of the polynucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen-mediated immune response. Expression of the polynucleotide is maintained in non-immune cells where the immune cell specific miRNAs are not expressed. For example, in some embodiments, to prevent an immunogenic reaction against a liver specific protein, any miR- 122 binding site can be removed and a miR-142 (and/or mirR-146) binding site can be engineered into the 5'UTR and/or 3'UTR of a polynucleotide of the present disclosure.

To further drive the selective degradation and suppression in APCs and macrophage, a polynucleotide of the present disclosure can include a further negative regulatory element in the 5'UTR and/or 3'UTR, either alone or in combination with miR-142 and/or miR-146 binding sites. As a non-limiting example, the further negative regulatory element is a Constitutive Decay Element (CDE).

Immune cell specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let- 7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa- let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1--3p, hsa-let-7f-2-- 5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-143-3p, miR-143- 5p, miR-146a-3p, miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR- 148a-5p, miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p, miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p, miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p, miR-181a-2-3p, miR-182-3p, miR-182-5p, miR- 197-3p, miR-197-5p, miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR- 223-5p, miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p, miR-26b-3p, miR-26b-5p, miR-27a- 3p, miR-27a-5p, miR-27b-3p,miR-27b-5p, miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p, miR-29b-2-5p, miR-29c-3p, miR-29c-5p,, miR-30e-3p, miR- 30e-5p, miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346, miR- 34a-3p, miR-34a-5p, , miR-363-3p, miR-363-5p, miR-372, miR-377-3p, miR-377-5p, miR- 493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p, miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935, miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR- 99b-5p. Furthermore, novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety.)

miRNAs that are known to be expressed in the liver include, but are not limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p. MiRNA binding sites from any liver specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the liver. Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a

polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the lung include, but are not limited to, let- 7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR- 130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR- 18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381- 5p. miRNA binding sites from any lung specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the lung. Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR- 208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR- 499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b- 5p. mMiRNA binding sites from any heart specific microRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the heart. Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a

polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR- 125b-5p,miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR- 212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR- 30d-5p, miR-329, miR-342-3p, miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410, miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR- 516a-3p, miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p, miR-7-5p, miR- 802, miR-922, miR-9-3p, and miR-9-5p. miRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR- 212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR- 3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the nervous system. Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a

polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a- 5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR- 33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.

MiRNA binding sites from any pancreas specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the pancreas. Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g. APC) miRNA binding sites in a polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c- 2-3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562. miRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the kidney. Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a

polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR- 208b, miR-25-3p, and miR-25-5p. MiRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the muscle. Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the present disclosure.

miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.

miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR- 126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2- 5p, miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR- 221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p, miR- 361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p, miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered in endothelial cells from deep-sequencing analysis (e.g., Voellenkle C et al., RNA, 2012, 18, 472-484, herein incorporated by reference in its entirety). miRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the endothelial cells.

miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR- 802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. miRNA binding sites from any epithelial cell specific miRNA can be introduced to or removed from a polynucleotide of the present disclosure to regulate expression of the polynucleotide in the epithelial cells.

In addition, a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy KT et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal JA and Ventura A, Semin Cancer Biol.2012, 22(5-6), 428-436; Goff LA et al., PLoS One, 2009, 4:e7192; Morin RD et al., Genome Res,2008,18, 610-621; Yoo JK et al., Stem Cells Dev.2012, 21(11), 2049-2057, each of which is herein incorporated by reference in its entirety). MiRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2- 3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154- 3p, miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR- 302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d- 3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p, miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p, miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f, miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-548l, miR- 548m, miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p, miR- 664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p, miR-885-5p,miR-93-3p, miR-93-5p, miR-941,miR-96-3p, miR-96-5p, miR-99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered by deep sequencing in human embryonic stem cells (e.g., Morin RD et al., Genome Res,2008,18, 610-621; Goff LA et al., PLoS One, 2009, 4:e7192; Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each of which is incorporated herein by reference in its entirety).

In one embodiment, the binding sites of embryonic stem cell specific miRNAs can be included in or removed from the 3'UTR of a polynucleotide of the present disclosure to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g. degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g. cancer stem cells).

As a non-limiting example, miRNA binding sites for miRNAs that are over-expressed in certain cancer and/or tumor cells can be removed from the 3'UTR of a polynucleotide of the present disclosure, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death.

Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected.

miRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the polynucleotides of the present disclosure, miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological processes. In this context, the polynucleotides of the present disclosure are defined as auxotrophic polynucleotides.

In some embodiments, a polynucleotide of the present disclosure comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from Table 6, including one or more copies of any one or more of the miRNA binding site sequences. In some embodiments, a polynucleotide of the present disclosure further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from Table 6, including any combination thereof. In some embodiments, the miRNA binding site binds to miR-142 or is

complementary to miR-142. In some embodiments, the miR-142 comprises SEQ ID NO:113. In some embodiments, the miRNA binding site binds to miR-142-3p or miR-142-5p. In some embodiments, the miR-142-3p binding site comprises SEQ ID NO:106. In some

embodiments, the miR-142-5p binding site comprises SEQ ID NO:108. In some

embodiments, the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:106 or SEQ ID NO:108.

Table 6. miR-142 and miR-142 binding sites

In some embodiments, a miRNA binding site is inserted in the polynucleotide of the present disclosure in any position of the polynucleotide (e.g., the 5'UTR and/or 3'UTR). In some embodiments, the 5'UTR comprises a miRNA binding site. In some embodiments, the 3'UTR comprises a miRNA binding site. In some embodiments, the 5'UTR and the 3'UTR comprise a miRNA binding site. The insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.

In some embodiments, a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the present disclosure comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the present disclosure. In some embodiments, a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the present disclosure.

miRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence. The miRNA can be influenced by the 5′UTR and/or 3′UTR. As a non-limiting example, a non- human 3′UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elements of the 5′UTR can influence miRNA mediated gene regulation. One example of a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5′UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A2 binding to this secondarily structured element in the 5′-UTR is necessary for miRNA mediated gene expression (Meijer HA et al., Science, 2013, 340, 82- 85, herein incorporated by reference in its entirety). The polynucleotides of the present disclosure can further include this structured 5′UTR in order to enhance microRNA mediated gene regulation. At least one miRNA binding site can be engineered into the 3′UTR of a polynucleotide of the present disclosure. In this context, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3′UTR of a polynucleotide of the present disclosure. For example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of a polynucleotide of the present disclosure. In one embodiment, miRNA binding sites incorporated into a polynucleotide of the present disclosure can be the same or can be different miRNA sites. A combination of different miRNA binding sites incorporated into a polynucleotide of the present disclosure can include combinations in which more than one copy of any of the different miRNA sites are incorporated. In another embodiment, miRNA binding sites incorporated into a polynucleotide of the present disclosure can target the same or different tissues in the body. As a non-limiting example, through the introduction of tissue-, cell-type-, or disease-specific miRNA binding sites in the 3′-UTR of a polynucleotide of the present disclosure, the degree of expression in specific cell types (e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR, about halfway between the 5′ terminus and 3′ terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in a polynucleotide of the present disclosure. As a non-limiting example, a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR. As another non- limiting example, a miRNA binding site can be engineered near the 3′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR. As yet another non-limiting example, a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites. The miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.

In one embodiment, a polynucleotide of the present disclosure can be engineered to include more than one miRNA site expressed in different tissues or different cell types of a subject. As a non-limiting example, a polynucleotide of the present disclosure can be engineered to include miR-192 and miR-122 to regulate expression of the polynucleotide in the liver and kidneys of a subject. In another embodiment, a polynucleotide of the present disclosure can be engineered to include more than one miRNA site for the same tissue. In some embodiments, the expression of a polynucleotide of the present disclosure can be controlled by incorporating at least one miR binding site in the polynucleotide and formulating the polynucleotide for administration. As a non-limiting example, a

polynucleotide of the present disclosure can be targeted to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising an ionizable e.g., an ionizable amino lipid, sometimes referred to in the prior art as an“ionizable cationic lipid”, including any of the lipids described herein.

A polynucleotide of the present disclosure can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions. Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the present disclosure can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.

In some embodiments, a polynucleotide of the present disclosure can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences. In some embodiments, a polynucleotide of the present disclosure can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences. The miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced downmodulation of the polynucleotide. In essence, the degree of match or mis-match between the miRNA binding site and the miRNA seed can act as a rheostat to more finely tune the ability of the miRNA to modulate protein expression. In addition, mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop of a stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5′ or 3′ stem of the stem loop.

In one embodiment the miRNA sequence in the 5′UTR can be used to stabilize a polynucleotide of the present disclosure described herein.

In another embodiment, a miRNA sequence in the 5′UTR of a polynucleotide of the present disclosure can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon. See, e.g., Matsuda et al., PLoS One.2010 11(5):e15057; incorporated herein by reference in its entirety, which used antisense locked nucleic acid (LNA) oligonucleotides and exon-junction complexes (EJCs) around a start codon (-4 to +37 where the A of the AUG codons is +1) in order to decrease the accessibility to the first start codon (AUG). Matsuda showed that altering the sequence around the start codon with an LNA or EJC affected the efficiency, length and structural stability of a polynucleotide. A polynucleotide of the present disclosure can comprise a miRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation. The site of translation initiation can be prior to, after or within the miRNA sequence. As a non-limiting example, the site of translation initiation can be located within a miRNA sequence such as a seed sequence or binding site. As another non-limiting example, the site of translation initiation can be located within a miR-122 sequence such as the seed sequence or the mir-122 binding site.

In some embodiments, a polynucleotide of the present disclosure can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells. The miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof. As a non-limiting example, a miRNA incorporated into a polynucleotide of the present disclosure can be specific to the

hematopoietic system. As another non-limiting example, a miRNA incorporated into a polynucleotide of the present disclosure to dampen antigen presentation is miR-142-3p.

In some embodiments, a polynucleotide of the present disclosure can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest. As a non-limiting example, a polynucleotide of the present disclosure can include at least one miR-122 binding site in order to dampen expression of an encoded polypeptide of interest in the liver. As another non-limiting example a polynucleotide of the present disclosure can include at least one miR-142-3p binding site, miR-142-3p seed sequence, miR- 142-3p binding site without the seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.

In some embodiments, a polynucleotide of the present disclosure can comprise at least one miRNA binding site in the 3′UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery. As a non-limiting example, the miRNA binding site can make a polynucleotide of the present disclosure more unstable in antigen presenting cells. Non-limiting examples of these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p, and mir-146-3p.

In one embodiment, a polynucleotide of the present disclosure comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.

In some embodiments, the polynucleotide of the present disclosure (e.g., a RNA, e.g., an mRNA) comprising (i) a sequence-optimized nucleotide sequence (e.g., an ORF) encoding an antibody (e.g., the wild-type sequence, functional fragment, or variant thereof) and (ii) a miRNA binding site (e.g., a miRNA binding site that binds to miR-142).

In some embodiments, the polynucleotide of the present disclosure (e.g., a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g., an ORF) encoding an antibody (e.g., full length antibody, scFv, functional fragment, or variant thereof), wherein the polynucleotide comprises 1-methylpseudouridines. In some embodiments, the polynucleotide further comprises a 5’ UTR having SEQ ID NO.160 or 162 and a 3’UTR having SEQ ID NO.161 and 163. In some embodiments, the polynucleotide disclosed herein is formulated with a delivery agent, e.g., a lipid nanoparticle comprised of an ionizable lipid of compound 18 or 25, a neutral lipid, a structural lipid and a PEG lipid. In some

embodiments the delivery agent is an LNP comprised of:

an ionizable cationic lipid of

,

and a PEG lipid comprising Formula VI, or an ionizable cationic lipid of

,

and an alternative lipid comprising oleic acid, or

an ionizable cationic lipid of

an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI. 3′ UTRs

In certain embodiments, a polynucleotide of the present disclosure (e.g., a

polynucleotide comprising a nucleotide sequence encoding an antibody of the present disclosure) further comprises a 3' UTR.

3'-UTR is the section of mRNA that immediately follows the translation termination codon and often contains regulatory regions that post-transcriptionally influence gene expression. Regulatory regions within the 3'-UTR can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. In one embodiment, the 3'-UTR useful for the present disclosure comprises a binding site for regulatory proteins or microRNAs. Regions having a 5′ Cap

The present disclosure also includes a polynucleotide that comprises both a 5′ Cap and a polynucleotide of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody ).

The 5′ cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5′ proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5′-end capped generating a 5′-ppp-5′- triphosphate linkage between a terminal guanosine cap residue and the 5′-terminal transcribed sense nucleotide of the mRNA molecule. This 5′-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5′ end of the mRNA can optionally also be 2′-O- methylated.5′-decapping through hydrolysis and cleavage of the guanylate cap structure can target a nucleic acid molecule, such as an mRNA molecule, for degradation. In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody ) incorporate a cap moiety.

In some embodiments, polynucleotides of the present disclosure (e.g., a

polynucleotide comprising a nucleotide sequence encoding an antibody) comprise a non- hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life.

Because cap structure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiester linkages, modified nucleotides can be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio- guanosine nucleotides according to the manufacturer's instructions to create a

phosphorothioate linkage in the 5′-ppp-5′ cap. Additional modified guanosine nucleotides can be used such as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as a

polynucleotide that functions as an mRNA molecule. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type or physiological) 5′- caps in their chemical structure, while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the present disclosure.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5′-5′-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3′-O-methyl group (i.e., N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G- 3′mppp-G; which can equivalently be designated 3′ O-Me-m7G(5')ppp(5')G). The 3′-O atom of the other, unmodified, guanine becomes linked to the 5′-terminal nucleotide of the capped polynucleotide. The N7- and 3′-O-methlyated guanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a 2′-O-methyl group on guanosine (i.e., N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm- ppp-G).

In some embodiments, the cap is a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog can be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is a N7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analog known in the art and/or described herein. Non- limiting examples of a N7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and a N7-(4- chlorophenoxyethyl)-m^3'-OG(5')ppp(5')G cap analog (See, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by reference in its entirety). In another embodiment, a cap analog of the present disclosure is a 4- chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotide or a region thereof, in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5′-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, can lead to reduced translational competency and reduced cellular stability.

Polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) can also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes, in order to generate more authentic 5′-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5′cap structures of the present disclosure are those that, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5′ endonucleases and/or reduced 5′decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural or physiological 5′cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphate linkage between the 5′-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5′- terminal nucleotide of the mRNA contains a 2′-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5′cap analog structures known in the art. Cap structures include, but are not limited to,

7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')- ppp(5')NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotides post-manufacture can be more efficient as nearly 100% of the chimeric polynucleotides can be capped. This is in contrast to ~80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.

According to the present disclosure, 5′ terminal caps can include endogenous caps or cap analogs. According to the present disclosure, a 5′ terminal cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA- guanosine, and 2-azido-guanosine. Poly-A Tails

In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) further comprise a poly-A tail. In further embodiments, terminal groups on the poly-A tail can be incorporated for stabilization. In other embodiments, a poly-A tail comprises des-3' hydroxyl tails.

During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide such as an mRNA molecule in order to increase stability.

Immediately after transcription, the 3' end of the transcript can be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.

PolyA tails can also be added after the construct is exported from the nucleus.

According to the present disclosure, terminal groups on the poly A tail can be incorporated for stabilization. Polynucleotides of the present disclosure can include des-3' hydroxyl tails. They can also include structural moieties or 2'-Omethyl modifications as taught by Junjie Li, et al. (Current Biology, Vol.15, 1501–1507, August 23, 2005, the contents of which are incorporated herein by reference in its entirety). The polynucleotides of the present disclosure can be designed to encode transcripts with alternative polyA tail structures including histone mRNA. According to Norbury, "Terminal uridylation has also been detected on human replication-dependent histone mRNAs. The turnover of these mRNAs is thought to be important for the prevention of potentially toxic histone accumulation following the completion or inhibition of

chromosomal DNA replication. These mRNAs are distinguished by their lack of a 3ʹ poly(A) tail, the function of which is instead assumed by a stable stem–loop structure and its cognate stem–loop binding protein (SLBP); the latter carries out the same functions as those of PABP on polyadenylated mRNAs" (Norbury, "Cytoplasmic RNA: a case of the tail wagging the dog," Nature Reviews Molecular Cell Biology; AOP, published online 29 August 2013; doi:10.1038/nrm3645) the contents of which are incorporated herein by reference in its entirety.

Unique poly-A tail lengths provide certain advantages to the polynucleotides of the present disclosure. Generally, the length of a poly-A tail, when present, is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).

In some embodiments, the polynucleotide or region thereof includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design can be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail can also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein can enhance expression.

Additionally, multiple distinct polynucleotides can be linked together via the PABP (Poly-A binding protein) through the 3′-end using modified nucleotides at the 3′-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72hr and day 7 post- transfection.

In some embodiments, the polynucleotides of the present disclosure are designed to include a polyA-G Quartet region. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone. Start codon region

The present disclosure also includes a polynucleotide that comprises both a start codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody). In some embodiments, the polynucleotides of the present disclosure can have regions that are analogous to or function like a start codon region.

In some embodiments, the translation of a polynucleotide can initiate on a codon that is not the start codon AUG. Translation of the polynucleotide can initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG,

ATA/AUA, ATT/AUU, TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 20105:11; the contents of each of which are herein incorporated by reference in its entirety).

As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG. Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 20105:11; the contents of which are herein incorporated by reference in its entirety).

Masking any of the nucleotides flanking a codon that initiates translation can be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.

In some embodiments, a masking agent can be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) polynucleotides and exon- junction complexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agents LNA polynucleotides and EJCs (PLoS ONE, 20105:11); the contents of which are herein incorporated by reference in its entirety).

In another embodiment, a masking agent can be used to mask a start codon of a polynucleotide in order to increase the likelihood that translation will initiate on an alternative start codon. In some embodiments, a masking agent can be used to mask a first start codon or alternative start codon in order to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

In some embodiments, a start codon or alternative start codon can be located within a perfect complement for a miR binding site. The perfect complement of a miR binding site can help control the translation, length and/or structure of the polynucleotide similar to a masking agent. As a non-limiting example, the start codon or alternative start codon can be located in the middle of a perfect complement for a miRNA binding site. The start codon or alternative start codon can be located after the first nucleotide, second nucleotide, third nucleotide, fourth nucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide, eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventh nucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenth nucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenth nucleotide, eighteenth nucleotide, nineteenth nucleotide, twentieth nucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide can be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon that is not the start codon. Translation of the polynucleotide can begin on the codon following the removed start codon or on a downstream start codon or an alternative start codon. In a non-limiting example, the start codon ATG or AUG is removed as the first 3 nucleotides of the polynucleotide sequence in order to have translation initiate on a downstream start codon or alternative start codon. The polynucleotide sequence where the start codon was removed can further comprise at least one masking agent for the downstream start codon and/or alternative start codons in order to control or attempt to control the initiation of translation, the length of the polynucleotide and/or the structure of the

polynucleotide. Stop Codon Region

The present disclosure also includes a polynucleotide that comprises both a stop codon region and the polynucleotide described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody). In some embodiments, the polynucleotides of the present disclosure can include at least two stop codons before the 3' untranslated region (UTR). The stop codon can be selected from TGA, TAA and TAG in the case of DNA, or from UGA, UAA and UAG in the case of RNA. In some embodiments, the polynucleotides of the present disclosure include the stop codon TGA in the case or DNA, or the stop codon UGA in the case of RNA, and one additional stop codon. In a further embodiment the addition stop codon can be TAA or UAA. In another embodiment, the polynucleotides of the present disclosure include three consecutive stop codons, four stop codons, or more.

In certain embodiments, the mRNAs of the disclosure encode more than one peptide, referred to herein as multimer constructs. In certain embodiments of the multimer constructs, the mRNA further encodes a linker located between each domain. The linker can be, for example, a cleavable linker or protease-sensitive linker. In certain embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE6:e18556). In certain embodiments, the linker is an F2A linker. In certain embodiments, the linker is a GGGS linker. In certain embodiments, the multimer construct contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain. The skilled artisan will likewise appreciate that other multicistronic constructs may be suitable for use in the invention. In exemplary embodiments, the construct design yields approximately equimolar amounts of intrabody and/or domain thereof encoded by the constructs of the invention. In one embodiment, the self-cleaving peptide may be, but is not limited to, a 2A peptide. A variety of 2A peptides are known and available in the art and may be used, including e.g., the foot and mouth disease virus (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and the porcine teschovirus-12A peptide. 2A peptides are used by several viruses to generate two proteins from one transcript by ribosome-skipping, such that a normal peptide bond is impaired at the 2A peptide sequence, resulting in two discontinuous proteins being produced from one translation event. As a non-limiting example, the 2A peptide may have the protein sequence:

GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 109), fragments or variants thereof. In one embodiment, the 2A peptide cleaves between the last glycine and last proline. The polynucleotide sequence of the 2A peptide may be modified or codon optimized by the methods described herein and/or are known in the art. Polynucleotide Comprising an mRNA Encoding an Antibody

In certain embodiments, a polynucleotide of the present disclosure, for example a polynucleotide comprising an mRNA nucleotide sequence encoding an antibody, comprises from 5’ to 3’ end:

(i) a 5' cap provided above;

(ii) a 5' UTR, such as the sequences provided above;

(iii) an open reading frame encoding an antibody, e.g., a sequence optimized nucleic acid sequence encoding an antibody disclosed herein;

(iv) at least one stop codon;

(v) a 3' UTR, such as the sequences provided above; and

(vi) a poly-A tail provided above.

In some embodiments, the polynucleotide further comprises a miRNA binding site, e.g, a miRNA binding site that binds to miRNA-142. In some embodiments, the 5’UTR comprises the miRNA binding site.

In some embodiments, a polynucleotide of the present disclosure comprises a nucleotide sequence encoding a polypeptide sequence at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100% identical to the protein sequence of an antibody described herein. Methods of Making Polynucleotides

The present disclosure also provides methods for making a polynucleotide of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) or a complement thereof.

In some aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding an antibody, can be constructed using in vitro transcription. In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding an antibody, can be constructed by chemical synthesis using an oligonucleotide synthesizer.

In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding an antibody is made by using a host cell. In certain aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, and encoding an antibody is made by one or more combination of the IVT, chemical synthesis, host cell expression, or any other methods known in the art.

Naturally occurring nucleosides, non-naturally occurring nucleosides, or

combinations thereof, can totally or partially naturally replace occurring nucleosides present in the candidate nucleotide sequence and can be incorporated into a sequence-optimized nucleotide sequence (e.g., a RNA, e.g., an mRNA) encoding an antibody. The resultant polynucleotides, e.g., mRNAs, can then be examined for their ability to produce protein and/or produce a therapeutic outcome. a. In Vitro Transcription / Enzymatic Synthesis

The polynucleotides of the present disclosure disclosed herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) can be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs can be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase can be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides disclosed herein. See U.S. Publ. No. US20130259923, which is herein incorporated by reference in its entirety.

Any number of RNA polymerases or variants can be used in the synthesis of the polynucleotides of the present disclosure. RNA polymerases can be modified by inserting or deleting amino acids of the RNA polymerase sequence. As a non-limiting example, the RNA polymerase can be modified to exhibit an increased ability to incorporate a 2´-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Patent 8,101,385; herein incorporated by reference in their entireties).

Variants can be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art. As a non-limiting example, T7 RNA polymerase variants can be evolved using the continuous directed evolution system set out by Esvelt et al. (Nature 472:499-503 (2011); herein incorporated by reference in its entirety) where clones of T7 RNA polymerase can encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limiting example, T7 RNA polymerase variants can encode at least mutation as described in U.S. Pub. Nos.20100120024 and 20070117112; herein incorporated by reference in their entireties. Variants of RNA polymerase can also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.

In one aspect, the polynucleotide can be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the polynucleotide can be modified to contain sites or regions of sequence changes from the wild type or parent chimeric polynucleotide.

Polynucleotide or nucleic acid synthesis reactions can be carried out by enzymatic methods utilizing polymerases. Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. DNA polymerase I (pol I) or A polymerase family, including the Klenow fragments of E. coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families. Another large family is DNA polymerase α (pol α) or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analog-incorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.

DNA polymerases are also selected based on the optimum reaction conditions they require, such as reaction temperature, pH, and template and primer concentrations.

Sometimes a combination of more than one DNA polymerases is employed to achieve the desired DNA fragment size and synthesis efficiency. For example, Cheng et al. increase pH, add glycerol and dimethyl sulfoxide, decrease denaturation times, increase extension times, and utilize a secondary thermostable DNA polymerase that possesses a 3´ to 5´ exonuclease activity to effectively amplify long targets from cloned inserts and human genomic DNA. (Cheng et al., PNAS 91:5695-5699 (1994), the contents of which are incorporated herein by reference in their entirety). RNA polymerases from bacteriophage T3, T7, and SP6 have been widely used to prepare RNAs for biochemical and biophysical studies. RNA polymerases, capping enzymes, and poly-A polymerases are disclosed in the co-pending International Publication No. WO2014028429, the contents of which are incorporated herein by reference in their entirety.

In one aspect, the RNA polymerase which can be used in the synthesis of the polynucleotides of the present disclosure is a Syn5 RNA polymerase. (see Zhu et al. Nucleic Acids Research 2013, doi:10.1093/nar/gkt1193, which is herein incorporated by reference in its entirety). The Syn5 RNA polymerase was recently characterized from marine cyanophage Syn5 by Zhu et al. where they also identified the promoter sequence (see Zhu et al. Nucleic Acids Research 2013, the contents of which is herein incorporated by reference in its entirety). Zhu et al. found that Syn5 RNA polymerase catalyzed RNA synthesis over a wider range of temperatures and salinity as compared to T7 RNA polymerase. Additionally, the requirement for the initiating nucleotide at the promoter was found to be less stringent for Syn5 RNA polymerase as compared to the T7 RNA polymerase making Syn5 RNA polymerase promising for RNA synthesis.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis of the

polynucleotides described herein. As a non-limiting example, a Syn5 RNA polymerase can be used in the synthesis of the polynucleotide requiring a precise 3´-terminus.

In one aspect, a Syn5 promoter can be used in the synthesis of the polynucleotides. As a non-limiting example, the Syn5 promoter can be 5´-ATTGGGCACCCGTAAGGG-3´ (SEQ ID NO: 110). In one aspect, a Syn5 RNA polymerase can be used in the synthesis of polynucleotides comprising at least one chemical modification described herein and/or known in the art (see e.g., the incorporation of pseudo-UTP and 5Me-CTP.

In one aspect, the polynucleotides described herein can be synthesized using a Syn5 RNA polymerase which has been purified using modified and improved purification procedure described by Zhu et al. (Nucleic Acids Research 2013).

Various tools in genetic engineering are based on the enzymatic amplification of a target gene which acts as a template. For the study of sequences of individual genes or specific regions of interest and other research needs, it is necessary to generate multiple copies of a target gene from a small sample of polynucleotides or nucleic acids. Such methods can be applied in the manufacture of the polynucleotides of the present disclosure.

For example, polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), also called transcription mediated amplification (TMA), and rolling-circle amplification (RCA) can be utilized in the manufacture of one or more regions of the polynucleotides of the present disclosure.

Assembling polynucleotides or nucleic acids by a ligase is also widely used. DNA or RNA ligases promote intermolecular ligation of the 5´ and 3´ ends of polynucleotide chains through the formation of a phosphodiester bond. b. Chemical synthesis

Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest, such as a polynucleotide of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody). For example, a single DNA or RNA oligomer containing a codon-optimized nucleotide sequence coding for the particular isolated polypeptide can be synthesized. In other aspects, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. In some aspects, the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

A polynucleotide disclosed herein (e.g., a RNA, e.g., an mRNA) can be chemically synthesized using chemical synthesis methods and potential nucleobase substitutions known in the art. See, for example, International Publication Nos. WO2014/093924,

WO2013/052523; WO2013/039857, WO2012/135805, WO2013/151671; U.S. Publ. No. US2013/0115272; or U.S. Pat. Nos. US8999380 or US8710200, all of which are herein incorporated by reference in their entireties. c. Purification of Polynucleotides Encoding An Antibody

Purification of the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) can include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up can be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNA^TM oligo-T capture probes (EXIQON® Inc., Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

The term "purified" when used in relation to a polynucleotide such as a "purified polynucleotide" refers to one that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that makes another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

In some embodiments, purification of a polynucleotide of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody) removes impurities that can reduce or remove an unwanted immune response, e.g., reducing cytokine activity.

In some embodiments, the polynucleotide of the present disclosure (e.g., a

polynucleotide comprising a nucleotide sequence encoding an antibody) is purified prior to administration using column chromatography (e.g., strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)).

In some embodiments, the polynucleotide of the present disclosure (e.g., a

polynucleotide comprising a nucleotide sequence an antibody) purified using column chromatography (e.g., strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC, hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)) presents increased expression of the encoded antibody compared to the expression level obtained with the same polynucleotide of the present disclosure purified by a different purification method.

In some embodiments, a column chromatography (e.g., strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)) purified polynucleotide comprises a nucleotide sequence encoding an antibody comprising one or more of the point mutations known in the art. In some embodiments, the use of RP-HPLC purified polynucleotide increases antibody expression levels in cells when introduced into those cells, e.g., by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the expression levels of antibody in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotide increases functional antibody expression levels in cells when introduced into those cells, e.g., by 10- 100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the functional expression levels of antibody in the cells before the RP-HPLC purified

polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotide increases detectable antibody activity in cells when introduced into those cells, e.g., by 10-100%, i.e., at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% with respect to the activity levels of functional antibody in the cells before the RP-HPLC purified polynucleotide was introduced in the cells, or after a non-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the purified polynucleotide is at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, at least about 96% pure, at least about 97% pure, at least about 98% pure, at least about 99% pure, or about 100% pure.

A quality assurance and/or quality control check can be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In another embodiment, the polynucleotide can be sequenced by methods including, but not limited to reverse-transcriptase-PCR. d. Quantification of Expressed Polynucleotides Encoding Antibody

In some embodiments, the polynucleotides of the present disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an antibody), their expression products, as well as degradation products and metabolites can be quantified according to methods known in the art.

In some embodiments, the polynucleotides of the present disclosure can be quantified in exosomes or when derived from one or more bodily fluid. As used herein "bodily fluids" include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities,

bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes can be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, a sample of not more than 2mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration,

immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of a polynucleotide can be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker.

The assay can be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes can be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in real time, the level of polynucleotides remaining or delivered. This is possible because the polynucleotides of the present disclosure differ from the endogenous forms due to the structural or chemical modifications.

In some embodiments, the polynucleotide can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified polynucleotide can be analyzed in order to determine if the polynucleotide can be of proper size, check that no degradation of the polynucleotide has occurred. Degradation of the polynucleotide can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE). Pharmaceutical Compositions and Formulations

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention and/or treatment of disease in humans and other mammals. The antibodies can be used as therapeutic or prophylactic agents. For example when the antibody is an anti-influenza virus antibody the RNA encoding such an antibody is used to provide prophylactic or therapeutic protection from influenza virus infection. Prophylactic protection from influenza virus infection can be achieved following administration of a composition (e.g., a composition comprising one or more polynucleotides encoding one or more HCAbs) of the present disclosure. In some embodiments, the influenza virus is a type A influenza virus. In some embodiments, the influenza virus is any influenza virus comprising HA proteins of subtype H1 and/or H3 (e.g., H1N1, H3N2). Compositions can be administered once, twice, three times, four times or more. In some aspects, the compositions can be administered to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

It is envisioned that there may be situations where persons are at risk for infection with more than one strain of type of infectious agent. RNA (mRNA) therapeutic treatments are particularly amenable to combination vaccination approaches due to a number of factors including, but not limited to, speed of manufacture, ability to rapidly tailor treatments to accommodate perceived geographical threat, and the like. To protect against more than one strain of influenza virus, a combination treatment can be administered that includes RNA encoding at least one polypeptide (or portion thereof) of a first influenza HA-binding HCAb and further includes RNA encoding at least one polypeptide (or portion thereof) of a second influenza-HA binding HCAb. RNAs (mRNAs) can be co-formulated, for example, in a single lipid nanoparticle (LNP) or can be formulated in separate LNPs destined for co- administration. In some embodiments, the influenza virus is a type A influenza virus. In some embodiments, the influenza virus is any influenza virus comprising HA proteins of subtype H1 and/or H3 (e.g., H1N1, H3N2).

A prophylactically effective dose is a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the

therapeutically effective dose is a dose listed in a package insert for the treatment. A prophylactic therapy as used herein refers to a therapy that prevents, to some extent, the infection from increasing. The infection may be prevented completely or partially.

The methods of the inention involve, in some aspects, passively immunizing a mammalian subject against an influenza virus infection. The method involves administering to the subject a composition comprising at least one RNA polynucleotide having an open reading frame encoding at least one HCAb that targets (e.g., binds to) an influenza virus protein. In some aspects, methods of the present disclosure provide prophylactic treatments against an influenza virus infection. In some embodiments, the influenza virus is a type A influenza virus. In some embodiments, the influenza virus is any influenza virus comprising HA proteins of subtype H1 and/or H3 (e.g., H1N1, H3N2).

Therapeutic methods of treatment are also included within the invention. Methods of treating an influenza virus infection in a subject are provided in aspects of the disclosure. The method involves administering to the subject having an influenza virus infection a composition comprising at least one RNA polynucleotide having an open reading frame encoding at least one HCAb that targets (e.g., binds to) an influenza virus protein. In some embodiments, the influenza virus protein is an HA protein. In some embodiments, the influenza virus is a type A influenza virus. In some embodiments, the influenza virus is any influenza virus comprising HA proteins of subtype H1 and/or H3 (e.g., H1N1, H3N2).

In some embodiments, the polynucleotide encodes an amino acid sequence of an antibody that binds Ebola virus (EBOV) protein and comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity with any of the amino acid sequences provided in the Tables disclosing ebola antibodies.

Aspects of the disclosure provide compositions comprising RNA polynucleotides encoding single-domain antibodies. In some embodiments, the single domain antibody encoded by an RNA polynucleotide of the present application is a heavy chain antibody such as found in camelidae (e.g., camels and llamas). The binding elements of such heavy chain antibodies consist of a single polypeptide domain, known as the variable domain of heavy chain antibodies (V_HH). These antibody fragments are naturally devoid of light-chains, with the V_HH forming the entirety of the antigen-binding site. In contrast, classical antibodies (e.g., murine, human) have binding elements comprising two polypeptide domains: the variable regions of the heavy chain (V_H) and the light chain (V_L). The lack of dependence on interaction with a light chain variable domain for maintaining structural and functional integrity gives V_HH domains a substantial advantage over other small antibody fragments, in terms of ease of production and behavior in solution. For example, V_HH domains are the preferred types of molecules for immuno-affinity purification due to their high stability and ability to refold efficiently after complete denaturation, which frequently occurs during elution of antigen. Additionally, the smaller size and single domain make V_HH domains optimal for cellular transformation.

Exemplary polynucleotides, e.g., polynucleotide constructs, include antibody- encoding mRNA polynucleotides. In some embodiments, the RNA treatment of the disclosure is a polynucleotide encoding an antibody that binds to Ebola virus (EBOV) protein. There are five Ebola viruses within the genus Ebolavirus. Four of the five known ebolaviruses cause a severe and often fatal hemorrhagic fever in humans and other mammals, known as Ebola virus disease (EVD). The Ebola glycoprotein (GP) is the only virally expressed protein on the virion surface, where it is essential for the attachment to host cells and catalyzes membrane fusion. As a result, the Ebola GP is a critical component of vaccines, as well as a target of neutralizing antibodies and inhibitors of attachment and fusion. Pre-GP is cleaved by furin at a multi-basic motif into two subunits, GP1 and GP2, which remain associated through a disulfide linkage between Cys53 of GP1 and Cys609 of GP2. The heterodimer (GP1 and GP2) then assembles into a 450-kDa trimer (3 GP1 and 3 GP2) at the surface of nascent virions, where it exerts its functions.

In some embodiments, the single-domain antibody encoded by the nucleic acid composition targets (e.g., binds to) an EBOV glycoprotein (GP). In some embodiments, the single-domain antibody targets (e.g., binds to) surface GP. In some embodiments, the single- domain antibody targets (e.g., binds to) secreted GP (sGP). In some embodiments, the single-domain antibody targets (e.g., binds to) small sGP (ssGP). In some embodiments, the single-domain antibody targets (e.g., binds to) shed GP. In some embodiments, the single- domain antibody encoded by the nucleic acid composition targets (e.g., binds to) an EBOV nucleoprotein. In some embodiments, the single-domain antibody encoded by the nucleic acid composition targets (e.g., binds to) an EBOV matrix protein.

As used herein, the terms treat, treated, or treating when used with respect to a disorder such as a viral infection, refers to a treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease in response to infection with the virus as well as a treatment after the subject has developed the disease in order to fight the infection or prevent the infection from becoming worse.

An“effective amount” of an antibody RNA treatment is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides), and other components of the RNA treatment, and other determinants. Increased antibody production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA treatment), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered response of the host cell.

In some embodiments, RNA treatments (including polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment of the disease.

RNA treatments may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA treatments of the present disclosure provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

RNA treatments may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be a vaccine containing an virus treatment with or without an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a treatment or vaccine, the term “booster” refers to an extra administration of the prophylactic composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

In some embodiments, RNA treatments may be administered subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro- ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs.

Provided herein are pharmaceutical compositions including RNA treatments and RNA compositions and/or complexes optionally in combination with one or more

pharmaceutically acceptable excipients.

RNA treatments may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, compositions comprise at least one additional active substances, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Treatment compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as treatment compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, RNA treatments are administered to humans, human patients, or subjects. For the purposes of the present disclosure, the phrase“active ingredient” generally refers to the RNA treatments or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding HCAb polypeptides.

Formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

RNA treatments can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (e.g., HCAb) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with RNA treatments (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and

combinations thereof.

Naturally-occurring eukaryotic mRNA molecules have been found to contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), in addition to other structural features, such as a 5′- cap structure or a 3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5′-cap and the 3′-poly(A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretch of adenine nucleotides added to the 3′-end of the transcribed mRNA. It can comprise up to about 400 adenine nucleotides. In some embodiments the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.

In some embodiments, the RNA treatment may include one or more stabilizing elements. Stabilizing elements may include, for instance, a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3′-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it is peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3′-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5′ and two nucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA treatments include a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. It has been found that the synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

The invention provides compounds, compositions and methods of use thereof for reducing the effect of ABC on a repeatedly administered active agent such as a biologically active agent. As will be readily apparent, reducing or eliminating altogether the effect of ABC on an administered active agent effectively increases its half-life and thus its efficacy.

In some embodiments the term reducing ABC refers to any reduction in ABC in comparison to a positive reference control ABC inducing LNP such as an MC3 LNP. ABC inducing LNPs cause a reduction in circulating levels of an active agent upon a second or subsequent administration within a given time frame. Thus a reduction in ABC refers to less clearance of circulating agent upon a second or subsequent dose of agent, relative to a standard LNP. The reduction may be, for instance, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%. In some embodiments the reduction is 10-100%, 10-50%, 20-100%, 20-50%, 30-100%, 30-50%, 40%-100%, 40-80%, 50-90%, or 50-100%. Alternatively the reduction in ABC may be characterized as at least a detectable level of circulating agent following a second or subsequent administration or at least a 2 fold, 3 fold, 4 fold, 5 fold increase in circulating agent relative to circulating agent following administration of a standard LNP. In some embodiments the reduction is a 2-100 fold, 2-50 fold, 3-100 fold, 3-50 fold, 3-20 fold, 4-100 fold, 4-50 fold, 4-40 fold, 4-30 fold, 4-25 fold, 4-20 fold, 4-15 fold, 4-10 fold, 4-5 fold, 5 - 100 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold, 5-20 fold, 5-15 fold, 5-10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30 fold, 6-25 fold, 6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8- 50 fold, 8-40 fold, 8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10-100 fold, 10-50 fold, 10-40 fold, 10-30 fold, 10-25 fold, 10-20 fold, 10-15 fold, 20-100 fold, 20-50 fold, 20- 40 fold, 20-30 fold, or 20-25 fold.

The disclosure provides lipid-comprising compounds and compositions that are less susceptible to clearance and thus have a longer half-life in vivo. This is particularly the case where the compositions are intended for repeated including chronic administration, and even more particularly where such repeated administration occurs within days or weeks.

Significantly, these compositions are less susceptible or altogether circumvent the observed phenomenon of accelerated blood clearance (ABC). ABC is a phenomenon in which certain exogenously administered agents are rapidly cleared from the blood upon second and subsequent administrations. This phenomenon has been observed, in part, for a variety of lipid-containing compositions including but not limited to lipidated agents, liposomes or other lipid-based delivery vehicles, and lipid-encapsulated agents. Heretofore, the basis of ABC has been poorly understood and in some cases attributed to a humoral immune response and accordingly strategies for limiting its impact in vivo particularly in a clinical setting have remained elusive.

This disclosure provides compounds and compositions that are less susceptible, if at all susceptible, to ABC. In some important aspects, such compounds and compositions are lipid-comprising compounds or compositions. The lipid-containing compounds or compositions of this disclosure, surprisingly, do not experience ABC upon second and subsequent administration in vivo. This resistance to ABC renders these compounds and compositions particularly suitable for repeated use in vivo, including for repeated use within short periods of time, including days or 1-2 weeks. This enhanced stability and/or half-life is due, in part, to the inability of these compositions to activate B1a and/or B1b cells and/or conventional B cells, pDCs and/or platelets.

This disclosure therefore provides an elucidation of the mechanism underlying accelerated blood clearance (ABC). It has been found, in accordance with this disclosure and the inventions provided herein, that the ABC phenomenon at least as it relates to lipids and lipid nanoparticles is mediated, at least in part an innate immune response involving B1a and/or B1b cells, pDC and/or platelets. B1a cells are normally responsible for secreting natural antibody, in the form of circulating IgM. This IgM is poly-reactive, meaning that it is able to bind to a variety of antigens, albeit with a relatively low affinity for each.

It has been found in accordance with the invention that some lipidated agents or lipid- comprising formulations such as lipid nanoparticles administered in vivo trigger and are subject to ABC. It has now been found in accordance with the invention that upon administration of a first dose of the LNP, one or more cells involved in generating an innate immune response (referred to herein as sensors) bind such agent, are activated, and then initiate a cascade of immune factors (referred to herein as effectors) that promote ABC and toxicity. For instance, B1a and B1b cells may bind to LNP, become activated (alone or in the presence of other sensors such as pDC and/or effectors such as IL6) and secrete natural IgM that binds to the LNP. Pre-existing natural IgM in the subject may also recognize and bind to the LNP, thereby triggering complement fixation. After administration of the first dose, the production of natural IgM begins within 1-2 hours of administration of the LNP. Typically by about 2-3 weeks the natural IgM is cleared from the system due to the natural half-life of IgM. Natural IgG is produced beginning around 96 hours after administration of the LNP. The agent, when administered in a naïve setting, can exert its biological effects relatively unencumbered by the natural IgM produced post-activation of the B1a cells or B1b cells or natural IgG. The natural IgM and natural IgG are non-specific and thus are distinct from anti-PEG IgM and anti-PEG IgG.

Although Applicant is not bound by mechanism, it is proposed that LNPs trigger ABC and/or toxicity through the following mechanisms. It is believed that when an LNP is administered to a subject the LNP is rapidly transported through the blood to the spleen. The LNPs may encounter immune cells in the blood and/or the spleen. A rapid innate immune response is triggered in response to the presence of the LNP within the blood and/or spleen. Applicant has shown herein that within hours of administration of an LNP several immune sensors have reacted to the presence of the LNP. These sensors include but are not limited to immune cells involved in generating an immune response, such as B cells, pDC, and platelets. The sensors may be present in the spleen, such as in the marginal zone of the spleen and/or in the blood. The LNP may physically interact with one or more sensors, which may interact with other sensors. In such a case the LNP is directly or indirectly interacting with the sensors. The sensors may interact directly with one another in response to recognition of the LNP. For instance many sensors are located in the spleen and can easily interact with one another. Alternatively one or more of the sensors may interact with LNP in the blood and become activated. The activated sensor may then interact directly with other sensors or indirectly (e.g., through the stimulation or production of a messenger such as a cytokine e.g., IL6).

In some embodiments the LNP may interact directly with and activate each of the following sensors: pDC, B1a cells, B1b cells, and platelets. These cells may then interact directly or indirectly with one another to initiate the production of effectors which ultimately lead to the ABC and/or toxicity associated with repeated doses of LNP. For instance, Applicant has shown that LNP administration leads to pDC activation, platelet aggregation and activation and B cell activation. In response to LNP platelets also aggregate and are activated and aggregate with B cells. pDC cells are activated. LNP has been found to interact with the surface of platelets and B cells relatively quickly. Blocking the activation of any one or combination of these sensors in response to LNP is useful for dampening the immune response that would ordinarily occur. This dampening of the immune response results in the avoidance of ABC and/or toxicity.

The sensors once activated produce effectors. An effector, as used herein, is an immune molecule produced by an immune cell, such as a B cell. Effectors include but are not limited to immunoglobulin such as natural IgM and natural IgG and cytokines such as IL6. B1a and B1b cells stimulate the production of natural IgMs within 2-6 hours following administration of an LNP. Natural IgG can be detected within 96 hours. IL6 levels are increased within several hours. The natural IgM and IgG circulate in the body for several days to several weeks. During this time the circulating effectors can interact with newly administered LNPs, triggering those LNPs for clearance by the body. For instance, an effector may recognize and bind to an LNP. The Fc region of the effector may be recognized by and trigger uptake of the decorated LNP by macrophage. The macrophage are then transported to the spleen. The production of effectors by immune sensors is a transient response that correlates with the timing observed for ABC.

If the administered dose is the second or subsequent administered dose, and if such second or subsequent dose is administered before the previously induced natural IgM and/or IgG is cleared from the system (e.g., before the 2-3 window time period), then such second or subsequent dose is targeted by the circulating natural IgM and/or natural IgG or Fc which trigger alternative complement pathway activation and is itself rapidly cleared. When LNP are administered after the effectors have cleared from the body or are reduced in number, ABC is not observed.

Thus, it is useful according to aspects of the invention to inhibit the interaction between LNP and one or more sensors, to inhibit the activation of one or more sensors by LNP (direct or indirect), to inhibit the production of one or more effectors, and/or to inhibit the activity of one or more effectors. In some embodiments the LNP is designed to limit or block interaction of the LNP with a sensor. For instance the LNP may have an altered PC and/or PEG to prevent interactions with sensors. Alternatively or additionally an agent that inhibits immune responses induced by LNPs may be used to achieve any one or more of these effects.

It has also been determined that conventional B cells are also implicated in ABC. Specifically, upon first administration of an agent, conventional B cells, referred to herein as CD19(+), bind to and react against the agent. Unlike B1a and B1b cells though, conventional B cells are able to mount first an IgM response (beginning around 96 hours after

administration of the LNPs) followed by an IgG response (beginning around 14 days after administration of the LNPs) concomitant with a memory response. Thus conventional B cells react against the administered agent and contribute to IgM (and eventually IgG) that mediates ABC. The IgM and IgG are typically anti-PEG IgM and anti-PEG IgG.

It is contemplated that in some instances, the majority of the ABC response is mediated through B1a cells and B1a-mediated immune responses. It is further contemplated that in some instances, the ABC response is mediated by both IgM and IgG, with both conventional B cells and B1a cells mediating such effects. In yet still other instances, the ABC response is mediated by natural IgM molecules, some of which are capable of binding to natural IgM, which may be produced by activated B1a cells. The natural IgMs may bind to one or more components of the LNPs, e.g., binding to a phospholipid component of the LNPs (such as binding to the PC moiety of the phospholipid) and/or binding to a PEG-lipid component of the LNPs (such as binding to PEG-DMG, in particular, binding to the PEG moiety of PEG-DMG). Since B1a expresses CD36, to which phosphatidylcholine is a ligand, it is contemplated that the CD36 receptor may mediate the activation of B1a cells and thus production of natural IgM. In yet still other instances, the ABC response is mediated primarily by conventional B cells.

It has been found in accordance with the invention that the ABC phenomenon can be reduced or abrogated, at least in part, through the use of compounds and compositions (such as agents, delivery vehicles, and formulations) that do not activate B1a cells. Compounds and compositions that do not activate B1a cells may be referred to herein as B1a inert compounds and compositions. It has been further found in accordance with the invention that the ABC phenomenon can be reduced or abrogated, at least in part, through the use of compounds and compositions that do not activate conventional B cells. Compounds and compositions that do not activate conventional B cells may in some embodiments be referred to herein as CD19-inert compounds and compositions. Thus, in some embodiments provided herein, the compounds and compositions do not activate B1a cells and they do not activate conventional B cells. Compounds and compositions that do not activate B1a cells and conventional B cells may in some embodiments be referred to herein as B1a/CD19-inert compounds and compositions.

These underlying mechanisms were not heretofore understood, and the role of B1a and B1b cells and their interplay with conventional B cells in this phenomenon was also not appreciated.

Accordingly, this disclosure provides compounds and compositions that do not promote ABC. These may be further characterized as not capable of activating B1a and/or B1b cells, platelets and/or pDC, and optionally conventional B cells also. These compounds (e.g., agents, including biologically active agents such as prophylactic agents, therapeutic agents and diagnostic agents, delivery vehicles, including liposomes, lipid nanoparticles, and other lipid-based encapsulating structures, etc.) and compositions (e.g., formulations, etc.) are particularly desirable for applications requiring repeated administration, and in particular repeated administrations that occur within with short periods of time (e.g., within 1-2 weeks). This is the case, for example, if the agent is a nucleic acid based therapeutic that is provided to a subject at regular, closely-spaced intervals. The findings provided herein may be applied to these and other agents that are similarly administered and/or that are subject to ABC.

Of particular interest are lipid-comprising compounds, lipid-comprising particles, and lipid-comprising compositions as these are known to be susceptible to ABC. Such lipid- comprising compounds particles, and compositions have been used extensively as

biologically active agents or as delivery vehicles for such agents. Thus, the ability to improve their efficacy of such agents, whether by reducing the effect of ABC on the agent itself or on its delivery vehicle, is beneficial for a wide variety of active agents.

Also provided herein are compositions that do not stimulate or boost an acute phase response (ARP) associated with repeat dose administration of one or more biologically active agents. The composition, in some instances, may not bind to IgM, including but not limited to natural IgM.

The composition, in some instances, may not bind to an acute phase protein such as but not limited to C-reactive protein.

The composition, in some instances, may not trigger a CD5(+) mediated immune response. As used herein, a CD5(+) mediated immune response is an immune response that is mediated by B1a and/or B1b cells. Such a response may include an ABC response, an acute phase response, induction of natural IgM and/or IgG, and the like.

The composition, in some instances, may not trigger a CD19(+) mediated immune response. As used herein, a CD19(+) mediated immune response is an immune response that is mediated by conventional CD19(+), CD5(-) B cells. Such a response may include induction of IgM, induction of IgG, induction of memory B cells, an ABC response, an anti- drug antibody (ADA) response including an anti-protein response where the protein may be encapsulated within an LNP, and the like.

B1a cells are a subset of B cells involved in innate immunity. These cells are the source of circulating IgM, referred to as natural antibody or natural serum antibody. Natural IgM antibodies are characterized as having weak affinity for a number of antigens, and therefore they are referred to as“poly-specific” or“poly-reactive”, indicating their ability to bind to more than one antigen. B1a cells are not able to produce IgG. Additionally, they do not develop into memory cells and thus do not contribute to an adaptive immune response. However, they are able to secrete IgM upon activation. The secreted IgM is typically cleared within about 2-3 weeks, at which point the immune system is rendered relatively naïve to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 3 weeks after the initial exposure), the antigen is not rapidly cleared. However, significantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within 1 week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for use.

In humans, B1a cells are CD19(+), CD20(+), CD27(+), CD43(+), CD70(-) and CD5(+). In mice, B1a cells are CD19(+), CD5(+), and CD45 B cell isoform B220(+). It is the expression of CD5 which typically distinguishes B1a cells from other convention B cells. B1a cells may express high levels of CD5, and on this basis may be distinguished from other B-1 cells such as B-1b cells which express low or undetectable levels of CD5. CD5 is a pan- T cell surface glycoprotein. B1a cells also express CD36, also known as fatty acid translocase. CD36 is a member of the class B scavenger receptor family. CD36 can bind many ligands, including oxidized low density lipoproteins, native lipoproteins, oxidized phospholipids, and long-chain fatty acids.

B1b cells are another subset of B cells involved in innate immunity. These cells are another source of circulating natural IgM. Several antigens, including PS, are capable of inducing T cell independent immunity through B1b activation. CD27 is typically upregulated on B1b cells in response to antigen activation. Similar to B1a cells, the B1b cells are typically located in specific body locations such as the spleen and peritoneal cavity and are in very low abundance in the blood. The B1b secreted natural IgM is typically cleared within about 2-3 weeks, at which point the immune system is rendered relatively naïve to the previously administered antigen. If the same antigen is presented after this time period (e.g., at about 3 weeks after the initial exposure), the antigen is not rapidly cleared. However, significantly, if the antigen is presented within that time period (e.g., within 2 weeks, including within 1 week, or within days), then the antigen is rapidly cleared. This delay between consecutive doses has rendered certain lipid-containing therapeutic or diagnostic agents unsuitable for use.

In some embodiments it is desirable to block B1a and/or B1b cell activation. One strategy for blocking B1a and/or B1b cell activation involves determining which components of a lipid nanoparticle promote B cell activation and neutralizing those components. It has been discovered herein that at least PEG and phosphatidylcholine (PC) contribute to B1a and B1b cell interaction with other cells and/or activation. PEG may play a role in promoting aggregation between B1 cells and platelets, which may lead to activation. PC (a helper lipid in LNPs) is also involved in activating the B1 cells, likely through interaction with the CD36 receptor on the B cell surface. Numerous particles have PEG-lipid alternatives, PEG-less, and/or PC replacement lipids (e.g. oleic acid or analogs thereof) have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that otherwise would promote ABC upon repeat administration, is useful in preventing ABC by reducing the production of natural IgM and/or B cell activation. Thus, the invention encompasses LNPs that have reduced ABC as a result of a design which eliminates the inclusion of B cell triggers.

Another strategy for blocking B1a and/or B1b cell activation involves using an agent that inhibits immune responses induced by LNPs. These types of agents are discussed in more detail below. In some embodiments these agents block the interaction between B1a/B1b cells and the LNP or platelets or pDC. For instance the agent may be an antibody or other binding agent that physically blocks the interaction. An example of this is an antibody that binds to CD36 or CD6. The agent may also be a compound that prevents or disables the B1a/B1b cell from signaling once activated or prior to activation. For instance, it is possible to block one or more components in the B1a/B1b signaling cascade the results from B cell interaction with LNP or other immune cells. In other embodiments the agent may act one or more effectors produced by the B1a/B1b cells following activation. These effectors include for instance, natural IgM and cytokines.

It has been demonstrated according to aspects of the invention that when activation of pDC cells is blocked, B cell activation in response to LNP is decreased. Thus, in order to avoid ABC and/or toxicity, it may be desirable to prevent pDC activation. Similar to the strategies discussed above, pDC cell activation may be blocked by agents that interfere with the interaction between pDC and LNP and/or B cells/platelets. Alternatively agents that act on the pDC to block its ability to get activated or on its effectors can be used together with the LNP to avoid ABC.

Platelets may also play an important role in ABC and toxicity. Very quickly after a first dose of LNP is administered to a subject platelets associate with the LNP, aggregate and are activated. In some embodiments it is desirable to block platelet aggregation and/or activation. One strategy for blocking platelet aggregation and/or activation involves determining which components of a lipid nanoparticle promote platelet aggregation and/or activation and neutralizing those components. It has been discovered herein that at least PEG contribute to platelet aggregation, activation and/or interaction with other cells. Numerous particles have PEG-lipid alternatives and PEG-less have been designed and tested. Applicant has established that replacement of one or more of these components within an LNP that otherwise would promote ABC upon repeat administration, is useful in preventing ABC by reducing the production of natural IgM and/or platelet aggregation. Thus, the invention encompasses LNPs that have reduced ABC as a result of a design which eliminates the inclusion of platelet triggers. Alternatively agents that act on the platelets to block its activity once it is activated or on its effectors can be used together with the LNP to avoid ABC. Measuring ABC Activity and related activities

Various compounds and compositions provided herein, including LNPs, do not promote ABC activity upon administration in vivo. These LNPs may be characterized and/or identified through any of a number of assays, such as but not limited to those described below, as well as any of the assays disclosed in the Examples section, include the methods subsection of the Examples.

In some embodiments the methods involve administering an LNP without producing an immune response that promotes ABC. An immune response that promotes ABC involves activation of one or more sensors, such as B1 cells, pDC, or platelets, and one or more effectors, such as natural IgM, natural IgG or cytokines such as IL6. Thus administration of an LNP without producing an immune response that promotes ABC, at a minimum involves administration of an LNP without significant activation of one or more sensors and significant production of one or more effectors. Significant used in this context refers to an amount that would lead to the physiological consequence of accelerated blood clearance of all or part of a second dose with respect to the level of blood clearance expected for a second dose of an ABC triggering LNP. For instance, the immune response should be dampened such that the ABC observed after the second dose is lower than would have been expected for an ABC triggering LNP. B1a or B1b activation assay

Certain compositions provided in this disclosure do not activate B cells, such as B1a or B1b cells (CD19+ CD5+) and/or conventional B cells (CD19+ CD5-). Activation of B1a cells, B1b cells, or conventional B cells may be determined in a number of ways, some of which are provided below. B cell population may be provided as fractionated B cell populations or unfractionated populations of splenocytes or peripheral blood mononuclear cells (PBMC). If the latter, the cell population may be incubated with the LNP of choice for a period of time, and then harvested for further analysis. Alternatively, the supernatant may be harvested and analyzed. Upregulation of activation marker cell surface expression

Activation of B1a cells, B1b cells, or conventional B cells may be demonstrated as increased expression of B cell activation markers including late activation markers such as CD86. In an exemplary non-limiting assay, unfractionated B cells are provided as a splenocyte population or as a PBMC population, incubated with an LNP of choice for a particular period of time, and then stained for a standard B cell marker such as CD19 and for an activation marker such as CD86, and analyzed using for example flow cytometry. A suitable negative control involves incubating the same population with medium, and then performing the same staining and visualization steps. An increase in CD86 expression in the test population compared to the negative control indicates B cell activation. Pro-inflammatory cytokine release

B cell activation may also be assessed by cytokine release assay. For example, activation may be assessed through the production and/or secretion of cytokines such as IL-6 and/or TNF-alpha upon exposure with LNPs of interest.

Such assays may be performed using routine cytokine secretion assays well known in the art. An increase in cytokine secretion is indicative of B cell activation. LNP binding/association to and/or uptake by B cells

LNP association or binding to B cells may also be used to assess an LNP of interest and to further characterize such LNP. Association/binding and/or uptake/internalization may be assessed using a detectably labeled, such as fluorescently labeled, LNP and tracking the location of such LNP in or on B cells following various periods of incubation.

The invention further contemplates that the compositions provided herein may be capable of evading recognition or detection and optionally binding by downstream mediators of ABC such as circulating IgM and/or acute phase response mediators such as acute phase proteins (e.g., C-reactive protein (CRP). Methods of use for reducing ABC

Also provided herein are methods for delivering LNPs, which may encapsulate an agent such as a therapeutic agent, to a subject without promoting ABC.

In some embodiments, the method comprises administering any of the LNPs described herein, which do not promote ABC, for example, do not induce production of natural IgM binding to the LNPs, do not activate B1a and/or B1b cells. As used herein, an LNP that“does not promote ABC” refers to an LNP that induces no immune responses that would lead to substantial ABC or a substantially low level of immune responses that is not sufficient to lead to substantial ABC. An LNP that does not induce the production of natural IgMs binding to the LNP refers to LNPs that induce either no natural IgM binding to the LNPs or a substantially low level of the natural IgM molecules, which is insufficient to lead to substantial ABC. An LNP that does not activate B1a and/or B1b cells refer to LNPs that induce no response of B1a and/or B1b cells to produce natural IgM binding to the LNPs or a substantially low level of B1a and/or B1b responses, which is insufficient to lead to substantial ABC.

In some embodiments the terms do not activate and do not induce production are a relative reduction to a reference value or condition. In some embodiments the reference value or condition is the amount of activation or induction of production of a molecule such as IgM by a standard LNP such as an MC3 LNP. In some embodiments the relative reduction is a reduction of at least 30%, for example at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments the terms do not activate cells such as B cells and do not induce production of a protein such as IgM may refer to an undetectable amount of the active cells or the specific protein. Platelet effects and toxicity

The invention is further premised in part on the elucidation of the mechanism underlying dose-limiting toxicity associated with LNP administration. Such toxicity may involve coagulopathy, disseminated intravascular coagulation (DIC, also referred to as consumptive coagulopathy), whether acute or chronic, and/or vascular thrombosis. In some instances, the dose-limiting toxicity associated with LNPs is acute phase response (APR) or complement activation-related psudoallergy (CARPA).

As used herein, coagulopathy refers to increased coagulation (blood clotting) in vivo. The findings reported in this disclosure are consistent with such increased coagulation and significantly provide insight on the underlying mechanism. Coagulation is a process that involves a number of different factors and cell types, and heretofore the relationship between and interaction of LNPs and platelets has not been understood in this regard. This disclosure provides evidence of such interaction and also provides compounds and compositions that are modified to have reduced platelet effect, including reduced platelet association, reduced platelet aggregation, and/or reduced platelet aggregation. The ability to modulate, including preferably down-modulate, such platelet effects can reduce the incidence and/or severity of coagulopathy post-LNP administration. This in turn will reduce toxicity relating to such LNP, thereby allowing higher doses of LNPs and importantly their cargo to be administered to patients in need thereof.

CARPA is a class of acute immune toxicity manifested in hypersensitivity reactions (HSRs), which may be triggered by nanomedicines and biologicals. Unlike allergic reactions, CARPA typically does not involve IgE but arises as a consequence of activation of the complement system, which is part of the innate immune system that enhances the body’s abilities to clear pathogens. One or more of the following pathways, the classical

complement pathway (CP), the alternative pathway (AP), and the lectin pathway (LP), may be involved in CARPA. Szebeni, Molecular Immunology, 61:163-173 (2014).

The classical pathway is triggered by activation of the C1-complex, which contains. C1q, C1r, C1s, or C1qr2s2. Activation of the C1-complex occurs when C1q binds to IgM or IgG complexed with antigens, or when C1q binds directly to the surface of the pathogen. Such binding leads to conformational changes in the C1q molecule, which leads to the activation of C1r, which in turn, cleave C1s. The C1r2s2 component now splits C4 and then C2, producing C4a, C4b, C2a, and C2b. C4b and C2b bind to form the classical pathway C3- convertase (C4b2b complex), which promotes cleavage of C3 into C3a and C3b. C3b then binds the C3 convertase to from the C5 convertase (C4b2b3b complex). The alternative pathway is continuously activated as a result of spontaneous C3 hydrolysis. Factor P

(properdin) is a positive regulator of the alternative pathway. Oligomerization of properdin stabilizes the C3 convertase, which can then cleave much more C3. The C3 molecules can bind to surfaces and recruit more B, D, and P activity, leading to amplification of the complement activation.

Acute phase response (APR) is a complex systemic innate immune responses for preventing infection and clearing potential pathogens. Numerous proteins are involved in APR and C-reactive protein is a well-characterized one.

It has been found, in accordance with the invention, that certain LNP are able to associate physically with platelets almost immediately after administration in vivo, while other LNP do not associate with platelets at all or only at background levels. Significantly, those LNPs that associate with platelets also apparently stabilize the platelet aggregates that are formed thereafter. Physical contact of the platelets with certain LNPs correlates with the ability of such platelets to remain aggregated or to form aggregates continuously for an extended period of time after administration. Such aggregates comprise activated platelets and also innate immune cells such as macrophages and B cells. Delivery Agents

In one set of embodiments, lipid nanoparticles (LNPs) are provided. In one embodiment, a lipid nanoparticle comprises lipids including an ionizable lipid, a structural lipid, a phospholipid, and mRNA. Each of the LNPs described herein may be used as a formulation for the mRNA described herein. In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10%

phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol. In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of ionizable lipid:

cholesterol:DSPC: PEG2000-DMG. a. Ionizable Lipid

The present disclosure provides pharmaceutical compositions with advantageous properties. For example, the lipids described herein (e.g. those having any of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III), (IV), (V), or (VI) may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, the lipid compounds disclosed hereinhave a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA). For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent. In particular, the present application provides pharmaceutical

compositions comprising:

(a) a polynucleotide comprising a nucleotide sequence encoding an antibody; and

(b) a delivery agent. Lipid Nanoparticle Formulations

In some embodiments, nucleic acids of the invention (e.g. TARGET mRNA) are formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the invention can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and

PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.

Nucleic acids of the present disclosure (e.g. antibody mRNA) are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable cationic lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%, 30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%, 40%, 50, or 60% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% non- cationic lipid. For example, the lipid nanoparticle may comprise a molar ratio of 5-20%, 5- 15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or25% non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% sterol. For example, the lipid nanoparticle may comprise a molar ratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25%, 30%, 35%, 40%, 45%, 50%, or 55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG-modified lipid. For example, the lipid nanoparticle may comprise a molar ratio of 0.5- 10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. In some aspects, the ionizable lipids of the present disclosure may be one or more of compounds of Formula (I):

or their N-oxides, or salts or isomers thereof, wherein:

R₁ is selected from the group consisting of C_5-30 alkyl, C_5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

R₂ and R₃ are independently selected from the group consisting of H, C_1-14 alkyl, C_2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R₂ and R₃, together with the atom to which they are attached, form a heterocycle or carbocycle;

R₄ is selected from the group consisting of hydrogen, a C_3-6 carbocycle, -(CH₂)_nQ, -(CH₂)_nCHQR, -CHQR, -CQ(R)₂, and unsubstituted C_1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH₂)_nN(R)₂, -C(O)OR, -OC(O)R, -CX₃, -CX₂H, -CXH₂, -CN, -N(R)₂, -C(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)C(O)N(R)₂, -N(R)C(S)N(R)₂, -N(R)R₈, -N(R)S(O)₂R₈, -O(CH₂)_nOR, -N(R)C(=NR₉)N(R)₂, -N(R)C(=CHR₉)N(R)₂,

-OC(O)N(R)₂, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)₂R, -N(OR)C(O)OR,

-N(OR)C(O)N(R)₂, -N(OR)C(S)N(R)₂, -N(OR)C(=NR₉)N(R)₂, -N(OR)C(=CHR₉)N(R)₂, -C(=NR₉)N(R)₂, -C(=NR₉)R, -C(O)N(R)OR, and–C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

each R₆ is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)₂-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C_1-13 alkyl or C_2-13 alkenyl;

R₇ is selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

R₈ is selected from the group consisting of C_3-6 carbocycle and heterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C_1-6 alkyl, -OR, -S(O)₂R, -S(O)₂N(R)₂, C_2-6 alkenyl, C_3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C_1-3 alkyl, C_2-3 alkenyl, and H;

each R’ is independently selected from the group consisting of C_1-18 alkyl, C_2-18 alkenyl, -R*YR”, -YR”, and H;

each R” is independently selected from the group consisting of C_3-15 alkyl and C_3-15 alkenyl;

each R* is independently selected from the group consisting of C_1-12 alkyl and C_2-12 alkenyl;

each Y is independently a C_3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R₄

is -(CH₂)_nQ, -(CH₂)_nCHQR,–CHQR, or -CQ(R)₂, then (i) Q is not -N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (IA):

or its N-oxide, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M’; R₄ is hydrogen, unsubstituted C_1-3 alkyl, or -(CH₂)_nQ, in which Q is OH, -NHC(S)N(R)₂, -NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R₈, -NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -OC(O)N(R)₂,

-N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group,; and R₂ and R₃ are independently selected from the group consisting of H, C_1-14 alkyl, and C_2-14 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)₂, or -NHC(O)N(R)₂. For example, Q is -N(R)C(O)R, or -N(R)S(O)₂R.

In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (IB):

(IB), or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9; R₄ is hydrogen, unsubstituted C_1-3 alkyl, or -(CH₂)_nQ, in which Q is OH, -NHC(S)N(R)₂,

-NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R₈, -NHC(=NR₉)N(R)₂,

-NHC(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R₃ are independently selected from the group consisting of H, C_1-14 alkyl, and C_2-14 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)₂, or -NHC(O)N(R)₂. For example, Q is -N(R)C(O)R, or -N(R)S(O)₂R.

In certain embodiments, a subset of compounds of Formula (I) includes those of Formula (II):

(II), or its N-oxide, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M₁ is a bond or M’; R₄ is hydrogen, unsubstituted C_1-3 alkyl, or -(CH₂)_nQ, in which n is 2, 3, or 4, and Q is

OH, -NHC(S)N(R)₂, -NHC(O)N(R)₂, -N(R)C(O)R, -N(R)S(O)₂R, -N(R)R₈,

-NHC(=NR₉)N(R)₂, -NHC(=CHR₉)N(R)₂, -OC(O)N(R)₂, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected

from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R₃ are independently selected from the group consisting of H, C_1-14 alkyl, and C_2-14 alkenyl.

In one embodiment, the compounds of Formula (I) are of Formula (IIa),

or their N-oxides, or salts or isomers thereof, wherein R₄ is as described herein. In another embodiment, the compounds of Formula (I) are of Formula (IIb),

or their N-oxides, or salts or isomers thereof, wherein R₄ is as described herein.

In another embodiment, the compounds of Formula (I) are of Formula (IIc) or (IIe):

In another embodiment, the compounds of Formula (I) are of Formula (IIf):

(IIf) or their N-oxides, or salts or isomers thereof,

wherein M is -C(O)O- or–OC(O)-, M” is C_1-6 alkyl or C_2-6 alkenyl, R₂ and R₃ are independently selected from the group consisting of C_5-14 alkyl and C_5-14 alkenyl, and n is selected from 2, 3, and 4.

In a further embodiment, the compounds of Formula (I) are of Formula (IId),

(IId), or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R₂ through R₆ are as described herein. For example, each of R₂ and R₃ may be independently selected from the group consisting of C_5-14 alkyl and C_5-14 alkenyl.

In a further embodiment, the compounds of Formula (I) are of Formula (IIg),

(IIg), or their N-oxides, or salts or isomers thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M’; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R₂ and R₃ are independently selected from the group consisting of H, C_1-14 alkyl, and C_2-14 alkenyl. For example, M” is C_1-6 alkyl (e.g., C_1-4 alkyl) or C_2-6 alkenyl (e.g. C_2-4 alkenyl). For example, R₂ and R₃ are independently selected from the group consisting of C_5-14 alkyl and C_5-14 alkenyl.

In some embodiments, the ionizable lipids are one or more of the compounds described in U.S. Application Nos.62/220,091, 62/252,316, 62/253,433, 62/266,460, 62/333,557, 62/382,740, 62/393,940, 62/471,937, 62/471,949, 62/475,140, and 62/475,166, and PCT Application No. PCT/US2016/052352.

In some embodiments, the ionizable lipids are selected from Compounds 1-280 described in U.S. Application No.62/475,166.

In some embodiments, the ionizable lipid is

, or a salt thereof.

In some embodiments, the ionizable lipid is

, or a salt thereof.

In some embodiments, the ionizable lipid is

or a salt thereof. In some embodiments, the ionizable lipid is

, or a salt thereof.

The central amine moiety of a lipid according to Formula (I), (IA), (IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), or (IIg) may be protonated at a physiological pH. Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

In some aspects, the ionizable lipids of the present disclosure may be one or more of compounds of formula (III),

;

t is 1 or 2;

A₁ and A₂ are each independently selected from CH or N;

Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent; R₁, R₂, R₃, R₄, and R₅ are independently selected from the group consisting of C_5-20 alkyl, C_5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”;

R_X1 and R_X2 are each independently H or C₁-₃ alkyl;

each M is independently selected from the group consisting

of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S) -,

-CH(OH)-, -P(O)(OR’)O-, -S(O)₂-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;

M* is C₁-C₆ alkyl,

W¹ and W² are each independently selected from the group consisting

of -O- and -N(R₆)-;

each R₆ is independently selected from the group consisting of H and C_1-5 alkyl;

X¹, X², and X³ are independently selected from the group consisting of a bond, -CH₂-, -(CH₂)₂-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH₂)_n-C(O)-, -C(O)-(CH₂)_n-,

-(CH₂)_n-C(O)O-, -OC(O)-(CH₂)_n-, -(CH₂)_n-OC(O)-, -C(O)O-(CH₂)_n-, -CH(OH)-, -C(S)-, and -CH(SH)-;

each Y is independently a C_3-6 carbocycle;

each R is independently selected from the group consisting of C_1-3 alkyl and a C_3-6 carbocycle;

each R’ is independently selected from the group consisting of C_1-12 alkyl, C_2-12 alkenyl, and H;

each R” is independently selected from the group consisting of C_3-12 alkyl, C_3-12 alkenyl and -R*MR’; and

n is an integer from 1-6;

i) at least one of X¹, X², and X³ is not -CH₂-; and/or

ii) at least one of R₁, R₂, R₃, R₄, and R₅ is -R”MR’.

In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa8):

r

In some embodiments, the ionizable lipids are one or more of the compounds described in U.S. Application Nos.62/271,146, 62/338,474, 62/413,345, and 62/519,826, and PCT Application No. PCT/US2016/068300.

In some embodiments, the ionizable lipids are selected from Compounds 1-156 described in U.S. Application No.62/519,826.

In some embodiments, the ionizable lipids are selected from Compounds 1-16, 42-66, 68-76, and 78-156 described in U.S. Application No.62/519,826.

In some embodiments the ionizable li id is

, or a salt thereof.

The central amine moiety of a lipid according to Formula (III), (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), or (IIIa8) may be protonated at a physiological pH. Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. Phospholipids

The lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper- catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,

phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

In some embodiments, a phospholipid of the invention comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2

cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2- diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof.

In certain embodiments, a phospholipid useful or potentially useful in the present invention is an analog or variant of DSPC. In certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV):

(IV),

or a salt thereof, wherein:

each R¹ is independently optionally substituted alkyl; or optionally two R¹ are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R¹ are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl;

n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

;

each instance of L² is independently a bond or optionally substituted C_1-6 alkylene, wherein one methylene unit of the optionally substituted C_1-6 alkylene is optionally replaced with O, N(R^N), S, C(O), C(O)N(R^N), NR^NC(O), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), - NR^NC(O)O, or NR^NC(O)N(R^N);

each instance of R² is independently optionally substituted C_1-30 alkyl, optionally substituted C_1-30 alkenyl, or optionally substituted C_1-30 alkynyl; optionally wherein one or more methylene units of R² are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R^N), O, S, C(O), C(O)N(R^N), NR^NC(O),

NR^NC(O)N(R^N), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), NR^NC(O)O, C(O)S, SC(O), C(=NR^N), C(=NR^N)N(R^N), NR^NC(=NR^N), NR^NC(=NR^N)N(R^N), C(S), C(S)N(R^N), NR^NC(S), NR^NC(S)N(R^N), S(O), OS(O), S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^N)S(O), S(O)N(R^N), N(R^N)S(O)N(R^N), OS(O)N(R^N), N(R^N)S(O)O, S(O)₂, N(R^N)S(O)₂, S(O)₂N(R^N), N(R^N)S(O)₂N(R^N), OS(O)₂N(R^N), or N(R^N)S(O)₂O; each instance of R^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

p is 1 or 2;

provided that the compound is not of the formula:

,

wherein each instance of R² is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

In some embodiments, the phospholipids may be one or more of the phospholipids described in U.S. Application No.62/520,530. i) Phospholipid Head Modifications

In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phospholipid head (e.g., a modified choline group). In certain embodiments, a phospholipid with a modified head is DSPC, or analog thereof, with a modified quaternary amine. For example, in embodiments of Formula (IV), at least one of R¹ is not methyl. In certain embodiments, at least one of R¹ is not hydrogen or methyl. In certain embodiments the com ound of Formula IV is of one of the followin formulae:

,

or a salt thereof, wherein:

each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

each v is independently 1, 2, or 3.

In certain embodiments, a compound of Formula (IV) is of Formula (IV-a):

(IV-a),

or a salt thereof.

In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a cyclic moiety in place of the glyceride moiety. In certain embodiments, a phospholipid useful in the present invention is DSPC, or analog thereof, with a cyclic moiety in place of the glyceride moiety. In certain embodiments, the compound of Formula (IV) is of Formula (IV-b):

,

(IV-b),

or a salt thereof. (ii) Phospholipid Tail Modifications

In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified tail. In certain embodiments, a phospholipid useful or potentially useful in the present invention is DSPC, or analog thereof, with a modified tail. As described herein, a“modified tail” may be a tail with shorter or longer aliphatic chains, aliphatic chains with branching introduced, aliphatic chains with substituents introduced, aliphatic chains wherein one or more methylenes are replaced by cyclic or heteroatom groups, or any combination thereof. For example, in certain embodiments, the compound of (IV) is of Formula (IV-a), or a salt thereof, wherein at least one instance of R² is each instance of R² is optionally substituted C_1-30 alkyl, wherein one or more methylene units of R² are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R^N), - O, S, C(O), C(O)N(R^N), NR^NC(O), NR^NC(O)N(R^N), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), NR^NC(O)O, C(O)S, SC(O), C(=NR^N), C(=NR^N)N(R^N), NR^NC(=NR^N), NR^NC(=NR^N)N(R^N), - C(S), C(S)N(R^N), NR^NC(S), NR^NC(S)N(R^N), S(O), OS(O), S(O)O, OS(O)O, OS(O)₂, - S(O)₂O, OS(O)₂O, N(R^N)S(O), S(O)N(R^N), N(R^N)S(O)N(R^N), OS(O)N(R^N), N(R^N)S(O)O, - S(O)₂, N(R^N)S(O)₂, S(O)₂N(R^N), N(R^N)S(O)₂N(R^N), OS(O)₂N(R^N), or N(R^N)S(O)₂O.

In certain embodiments, the compound of Formula (IV) is of Formula (IV-c):

or a salt thereof, wherein:

each x is independently an integer between 0-30, inclusive; and

each instance is G is independently selected from the group consisting of optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R^N), O, S, C(O), C(O)N(R^N), NR^NC(O), NR^NC(O)N(R^N), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), NR^NC(O)O, C(O)S, SC(O), C(=NR^N), C(=NR^N)N(R^N), NR^NC(=NR^N), NR^NC(=NR^N)N(R^N), C(S), C(S)N(R^N), NR^NC(S), NR^NC(S)N(R^N), S(O), OS(O), S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^N)S(O), S(O)N(R^N), N(R^N)S(O)N(R^N), OS(O)N(R^N), N(R^N)S(O)O, S(O)₂, N(R^N)S(O)₂, S(O)₂N(R^N), N(R^N)S(O)₂N(R^N), OS(O)₂N(R^N), or N(R^N)S(O)₂O. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, a phospholipid useful or potentially useful in the present invention comprises a modified phosphocholine moiety, wherein the alkyl chain linking the quaternary amine to the phosphoryl group is not ethylene (e.g., n is not 2). Therefore, in certain embodiments, a phospholipid useful or potentially useful in the present invention is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in certain embodiments, a compound of Formula (IV) is of one of the following formulae:

,

or a salt thereof. Alternative lipids

In certain embodiments, an alternative lipid is used in place of a phospholipid of the present disclosure.

In certain embodiments, an alternative lipid of the invention is oleic acid.

In certain embodiments, the alternative lipid is one of the following:

Structural Lipids The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term“structural lipid” refers to sterols and also to lipids containing sterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein,“sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No.62/520,530. Polyethylene Glycol (PEG)-Lipids

The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term“PEG-lipid” refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG-lipids include PEG-modified

phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2- diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In one embodiment, the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C₁₄ to about C₂₂, preferably from about C₁₄ to about C₁₆. In some embodiments, a PEG moiety, for example an mPEG-NH₂, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In one embodiment, the PEG-lipid is PEG_2k-DMG.

In one embodiment, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) of various formulae, described herein may be synthesized as described International Patent Application No.

PCT/US2016/000129, filed December 10, 2016, entitled“Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG-DMG has the following structure:

In one embodiment, PEG lipids useful in the present invention can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a“PEG-OH lipid” (also referred to herein as“hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (–OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an–OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention.

In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (V). Provided herein are compounds of Formula (V):

or salts thereof, wherein:

R³ is–OR^O;

R^O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

L¹ is optionally substituted C_1-10 alkylene, wherein at least one methylene of the optionally substituted C_1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R^N), S, C(O), C(O)N(R^N), NR^NC(O), C(O)O, - OC(O), OC(O)O, OC(O)N(R^N), NR^NC(O)O, or NR^NC(O)N(R^N);

D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

;

each instance of R² is independently optionally substituted C_1-30 alkyl, optionally substituted C_1-30 alkenyl, or optionally substituted C_1-30 alkynyl; optionally wherein one or more methylene units of R² are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R^N), O, S, C(O), C(O)N(R^N), NR^NC(O), - NR^NC(O)N(R^N), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), NR^NC(O)O, C(O)S, SC(O), - C(=NR^N), C(=NR^N)N(R^N), NR^NC(=NR^N), NR^NC(=NR^N)N(R^N), C(S), C(S)N(R^N), NR^NC(S), NR^NC(S)N(R^N), S(O) , OS(O), S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^N)S(O), - S(O)N(R^N), N(R^N)S(O)N(R^N), OS(O)N(R^N), N(R^N)S(O)O, S(O)₂, N(R^N)S(O)₂, S(O)₂N(R^N), N(R^N)S(O)₂N(R^N), OS(O)₂N(R^N), or N(R^N)S(O)₂O;

each instance of R^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

p is 1 or 2.

In certain embodiments, the compound of Fomula (V) is a PEG-OH lipid (i.e., R³ is–OR^O, and R^O is hydrogen). In certain embodiments, the compound of Formula (V) is of Formula (V-OH): (V-OH),

or a salt thereof.

In certain embodiments, a PEG lipid useful in the present invention is a PEGylated fatty acid. In certain embodiments, a PEG lipid useful in the present invention is a compound of Formula (VI). Provided herein are compounds of Formula (VI):

or a salts thereof, wherein:

R³ is–OR^O;

R^O is hydrogen, optionally substituted alkyl or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

R⁵ is optionally substituted C_10-40 alkyl, optionally substituted C_10-40 alkenyl, or optionally substituted C_10-40 alkynyl; and optionally one or more methylene groups of R⁵ are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(R^N), O, S, C(O), - C(O)N(R^N), NR^NC(O), NR^NC(O)N(R^N), C(O)O, OC(O), OC(O)O, OC(O)N(R^N), - NR^NC(O)O, C(O)S, SC(O), C(=NR^N), C(=NR^N)N(R^N), NR^NC(=NR^N), NR^NC(=NR^N)N(R^N), - C(S), C(S)N(R^N), NR^NC(S), NR^NC(S)N(R^N), S(O), OS(O), S(O)O, OS(O)O, OS(O)₂, - S(O)₂O, OS(O)₂O, N(R^N)S(O), S(O)N(R^N), N(R^N)S(O)N(R^N), OS(O)N(R^N), N(R^N)S(O)O, - S(O)₂, N(R^N)S(O)₂, S(O)₂N(R^N), N(R^N)S(O)₂N(R^N), OS(O)₂N(R^N), or N(R^N)S(O)₂O; and each instance of R^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (VI) is of Formula (VI-OH):

(VI-OH),

or a salt thereof. In some embodiments, r is 45. In et other embodiments the com ound of Formula VI is:

In some aspects, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No.62/520,530.

In some embodiments, a PEG lipid of the invention comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified

dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG- DPG.In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of any of Formula I, II or III, a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula VI.

In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI.

In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid comprising a compound having Formula IV, a structural lipid, and the PEG lipid comprising a compound having Formula V or VI. In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of Formula I, II or III, a phospholipid having Formula IV, a structural lipid, and a PEG lipid comprising a compound having Formula VI.

In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

,

and a PEG lipid comprising Formula VI.

,

and an alternative lipid comprising oleic acid. In some embodiments, a LNP of the invention comprises an ionizable cationic lipid of

,

an alternative lipid comprising oleic acid, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.

In some embodiments a LNP of the invention com rises an ionizable cationic lipid of

a phospholipid comprising DOPE, a structural lipid comprising cholesterol, and a PEG lipid comprising a compound having Formula VI.

In some embodiments, a LNP of the invention comprises an N:P ratio of from about 2:1 to about 30:1.

In some embodiments, a LNP of the invention comprises an N:P ratio of about 6:1. In some embodiments, a LNP of the invention comprises an N:P ratio of about 3:1. In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of from about 10:1 to about 100:1.

In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the invention comprises a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the invention has a mean diameter from about 50nm to about 150nm.

In some embodiments, a LNP of the invention has a mean diameter from about 70nm to about 120nm.

As used herein, the term“alkyl”,“alkyl group”, or“alkylene” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation“C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups.

As used herein, the term“alkenyl”,“alkenyl group”, or“alkenylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. The notation“C2-14 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, C18 alkenyl may include one or more double bonds. A C18 alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

As used herein, the term“alkynyl”,“alkynyl group”, or“alkynylene” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The notation“C2-14 alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, C18 alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

As used herein, the term“carbocycle” or“carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation“C3-6 carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms.

Carbocycles may include one or more carbon-carbon double or triple bonds and may be non- aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term“cycloalkyl” as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.

As used herein, the term“heterocycle” or“heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triple bonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. The term“heterocycloalkyl” as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.

As used herein, the term“heteroalkyl”,“heteroalkenyl”, or“heteroalkynyl”, refers respectively to an alkyl, alkenyl, alkynyl group, as defined herein, which further comprises one or more (e.g., 1, 2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus) wherein the one or more heteroatoms is inserted between adjacent carbon atoms within the parent carbon chain and/or one or more heteroatoms is inserted between a carbon atom and the parent molecule, i.e., between the point of attachment. Unless otherwise specified, heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refers to both unsubstituted and substituted heteroalkyls, heteroalkenyls, or heteroalkynyls, i.e., optionally substituted heteroalkyls, heteroalkenyls, or heteroalkynyls.

As used herein, a“biodegradable group” is a group that may facilitate faster metabolism of a lipid in a mammalian entity. A biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group, and a heteroaryl group. As used herein, an“aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a“heteroaryl group” is an optionally substituted

heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. For example, M and M’ can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M’ can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a

hydroxyl, -OH), an ester (e.g., -C(O)OR -OC(O)R), an aldehyde (e.g.,-C(O)H), a carbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g.,-C(O)X, in which X is a halide selected from bromide, fluoride, chloride, and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., -OR), an acetal (e.g.,-C(OR)2R””, in which each OR are alkoxy groups that can be the same or different and R”” is an alkyl or alkenyl group), a phosphate (e.g., P(O)43- ), a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfinic acid (e.g., -S(O)OH), a sulfonic acid (e.g., -S(O)2OH), a thial (e.g., -C(S)H), a sulfate (e.g., S(O)42-), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2, or -N(R)C(O)R), an azido (e.g., -N3), a nitro (e.g., -NO2), a cyano (e.g., -CN), an isocyano (e.g., -NC), an acyloxy (e.g.,-OC(O)R), an amino (e.g., -NR2, -NRH, or -NH2), a carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH₂), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2, -N(R)S(O)₂R, -N(H)S(O)₂R, -N(R)S(O)₂H, or -N(H)S(O)2H), an alkyl group, an alkenyl group, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, a C_1-6 alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

Compounds of the disclosure that contain nitrogens can be converted to N-oxides by treatment with an oxidizing agent (e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to afford other compounds of the disclosure. Thus, all shown and claimed nitrogen-containing compounds are considered, when allowed by valency and structure, to include both the compound as shown and its N-oxide derivative (which can be designated as N+O or N+-O-). Furthermore, in other instances, the nitrogens in the compounds of the disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidation of the parent amine by an oxidizing agent such as m-CPBA. All shown and claimed nitrogen-containing compounds are also considered, when allowed by valency and structure, to cover both the compound as shown and its N-hydroxy (i.e., N-OH) and N-alkoxy (i.e., N-OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl, 3-14-membered carbocycle or 3-14- membered heterocycle) derivatives.

About, Approximately: As used herein, the terms“approximately” and“about,” as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). For example, when used in the context of an amount of a given compound in a lipid component of a nanoparticle composition,“about” may mean +/- 10% of the recited value. For instance, a nanoparticle composition including a lipid component having about 40% of a given compound may include 30-50% of the compound.

As used herein, the term“compound,” is meant to include all isomers and isotopes of the structure depicted.“Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. Further, a compound, salt, or complex of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

(Compound 25), Nanoparticle Compositions

The lipid composition of a pharmaceutical composition disclosed herein can include one or more components in addition to those described above. For example, the lipid composition can include one or more permeability enhancer molecules, carbohydrates, polymers, surface altering agents (e.g., surfactants), or other components. For example, a permeability enhancer molecule can be a molecule described by U.S. Patent Application Publication No.

2005/0222064. Carbohydrates can include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

A polymer can be included in and/or used to encapsulate or partially encapsulate a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in lipid nanoparticle form). A polymer can be biodegradable and/or biocompatible. A polymer can be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters,

polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones,

polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates,

polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

The ratio between the lipid composition and the polynucleotide range can be from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and the polynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In some embodiments, the wt/wt ratio of the lipid composition to the polynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

In one embodiment, the lipid nanoparticles described herein can comprise

polynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or 70:1, or a range or any of these ratios such as, but not limited to, 5:1 to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about 20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, from about 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 to about 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1, from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about 10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 to about 25:1, from about 10:1 to about 30:1, from about 10:1 to about 35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, from about 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1 to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about 20:1, from about 15:1 to about 25:1,from about 15:1 to about 30:1, from about 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1 to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about 55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one embodiment, the lipid nanoparticles described herein can comprise the polynucleotide in a concentration from approximately 0.1 mg/ml to 2 mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

In some embodiments, the pharmaceutical compositions disclosed herein are formulated as lipid nanoparticles (LNP). Accordingly, the present disclosure also provides nanoparticle compositions comprising (i) a lipid composition comprising a delivery agent such as a compound of Formula (I) or (III) as described herein, and (ii) a polynucleotide encoding an antibody. In such nanoparticle composition, the lipid composition disclosed herein can encapsulate the polynucleotide encoding an antibody.

Nanoparticle compositions are typically sized on the order of micrometers or smaller and can include a lipid bilayer. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes. For example, a nanoparticle composition can be a liposome having a lipid bilayer with a diameter of 500 nm or less.

Nanoparticle compositions include, for example, lipid nanoparticles (LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In certain embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers can be functionalized and/or crosslinked to one another. Lipid bilayers can include one or more ligands, proteins, or channels.

In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA. In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol.

In some embodiments, the LNP has a polydispersity value of less than 0.4. In some embodiments, the LNP has a net neutral charge at a neutral pH. In some embodiments, the LNP has a mean diameter of 50-150 nm. In some embodiments, the LNP has a mean diameter of 80-100 nm.

As generally defined herein, the term“lipid” refers to a small molecule that has hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic.

Examples of classes of lipids include, but are not limited to, fats, waxes, sterol-containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, and polyketides, and prenol lipids. In some instances, the amphiphilic properties of some lipids leads them to form liposomes, vesicles, or membranes in aqueous media.

In some embodiments, a lipid nanoparticle (LNP) may comprise an ionizable lipid. As used herein, the term“ionizable lipid” has its ordinary meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively charged or negatively charged. An ionizable lipid may be positively charged, in which case it can be referred to as“cationic lipid”. In certain embodiments, an ionizable lipid molecule may comprise an amine group, and can be referred to as an ionizable amino lipids. As used herein, a“charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and/or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule may be selected as desired.

It should be understood that the terms“charged” or“charged moiety” does not refer to a “partial negative charge" or“partial positive charge" on a molecule. The terms“partial negative charge" and“partial positive charge" are given its ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid, sometimes referred to in the art as an“ionizable cationic lipid”. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail that are connected via a linker structure.

In addition to these, an ionizable lipid may also be a lipid including a cyclic amine group.

In one embodiment, the ionizable lipid may be selected from, but not limited to, a ionizable lipid described in International Publication Nos. WO2013/086354 and

WO2013/116126; the contents of each of which are herein incorporated by reference in their entirety.

In yet another embodiment, the ionizable lipid may be selected from, but not limited to, formula CLI-CLXXXXII of US Patent No.7,404,969; each of which is herein incorporated by reference in their entirety.

In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012/170889, herein incorporated by reference in its entirety. In one embodiment, the lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2013/086354; the contents of each of which are herein incorporated by reference in their entirety.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition.

Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of compound of Formula (I) (e.g., Compounds 18, 25, 26 or 48). In some embodiments, the nanoparticle composition comprises a lipid composition consisting or consisting essentially of a compound of Formula (I) (e.g., Compounds 18, 25, 26 or 48) and a phospholipid (e.g., DSPC or MSPC).

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) can be used to measure zeta potentials. Dynamic light scattering can also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

The size of the nanoparticles can help counter biological reactions such as, but not limited to, inflammation, or can increase the biological effect of the polynucleotide.

As used herein,“size” or“mean size” in the context of nanoparticle compositions refers to the mean diameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding an antibody are formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100 nm. In one embodiment, the nanoparticles have a diameter from about 10 to 500 nm. In one embodiment, the nanoparticle has a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

In some embodiments, the largest dimension of a nanoparticle composition is 1 µm or shorter (e.g., 1 µm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter).

A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a

polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition disclosed herein can be from about 0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition disclosed herein can be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about 10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles can be from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, from about 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0 mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV to about 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20 mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV, from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, from about 10 mV to about 70 mV, from about 10 mV to about 60 mV, from about 10 mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV to about 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about 100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80 mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV, from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, from about 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about 30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mV to about 70 mV, from about 30 mV to about 60 mV, from about 30 mV to about 50 mV, from about 30 mV to about 40 mV, from about 40 mV to about 100 mV, from about 40 mV to about 90 mV, from about 40 mV to about 80 mV, from about 40 mV to about 70 mV, from about 40 mV to about 60 mV, and from about 40 mV to about 50 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be from about 10 mV to about 50 mV, from about 15 mV to about 45 mV, from about 20 mV to about 40 mV, and from about 25 mV to about 35 mV. In some embodiments, the zeta potential of the lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, and about 100 mV.

The term“encapsulation efficiency” of a polynucleotide describes the amount of the polynucleotide that is encapsulated by or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. As used herein,“encapsulation” can refer to complete, substantial, or partial enclosure, confinement, surrounding, or encasement.

Encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of the polynucleotide in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a polynucleotide can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical composition disclosed herein can depend on multiple factors such as the size of the polynucleotide, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the polynucleotide.

For example, the amount of an mRNA useful in a nanoparticle composition can depend on the size (expressed as length, or molecular mass), sequence, and other characteristics of the mRNA. The relative amounts of a polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotide present in a lipid nanoparticle composition of the present disclosure can be optimized according to considerations of efficacy and tolerability. For compositions including an mRNA as a polynucleotide, the N:P ratio can serve as a useful metric.

As the N:P ratio of a nanoparticle composition controls both expression and tolerability, nanoparticle compositions with low N:P ratios and strong expression are desirable. N:P ratios vary according to the ratio of lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids, and amounts thereof can be selected to provide an N:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. In certain embodiments, the N:P ratio is between 5:1 and 6:1. In one specific aspect, the N:P ratio is about is about 5.67:1.

In addition to providing nanoparticle compositions, the present disclosure also provides methods of producing lipid nanoparticles comprising encapsulating a polynucleotide. Such method comprises using any of the pharmaceutical compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art. See, e.g., Wang et al. (2015)“Delivery of oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68-80; Silva et al. (2015)“Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol.16: 940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: Structure, Preparation and Application” Adv. Pharm. Bull.5:305-13; Silva et al. (2015)“Lipid nanoparticles for the delivery of biopharmaceuticals” Curr. Pharm. Biotechnol.16:291-302, and references cited therein. b. Lipidoids

In some embodiments, the compositions or formulations of the present disclosure comprise a delivery agent, e.g., a lipidoid. The polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) can be formulated with lipidoids. Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore to achieve an effective delivery of the

polynucleotide, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration. Lipidoid complexes of polynucleotides can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.

The synthesis of lipidoids is described in literature (see Mahon et al., Bioconjug. Chem.201021:1448-1454; Schroeder et al., J Intern Med.2010267:9-21; Akinc et al., Nat Biotechnol.200826:561-569; Love et al., Proc Natl Acad Sci U S A.2010107:1864-1869; Siegwart et al., Proc Natl Acad Sci U S A.2011108:12996-3001; all of which are

incorporated herein in their entireties).

Formulations with the different lipidoids, including, but not limited to penta[3-(1- laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010)), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity. The lipidoid "98N12- 5" is disclosed by Akinc et al., Mol Ther.200917:872-879. The lipidoid "C12-200" is disclosed by Love et al., Proc Natl Acad Sci U S A.2010107:1864-1869 and Liu and Huang, Molecular Therapy.2010669-670. Each of the references is herein incorporated by reference in its entirety.

In one embodiment, the polynucleotides described herein can be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids can be prepared by the methods described in U.S. Patent No.8,450,298 (herein incorporated by reference in its entirety).

The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotides. Lipidoids and polynucleotide formulations comprising lipidoids are described in Intl. Pub. No. WO 2015/051214 (herein incorporated by reference in its entirety. c. Hyaluronidase

In some embodiments, the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) and hyaluronidase for injection (e.g., intramuscular or subcutaneous injection). Hyaluronidase catalyzes the hydrolysis of hyaluronan, which is a constituent of the interstitial barrier. Hyaluronidase lowers the viscosity of hyaluronan, thereby increases tissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440). Alternatively, the hyaluronidase can be used to increase the number of cells exposed to the polynucleotides administered intramuscularly, intratumorally, or subcutaneously. d. Nanoparticle Mimics

In some embodiments, the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) is encapsulated within and/or absorbed to a nanoparticle mimic. A nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells. As a non-limiting example, the polynucleotides described herein can be encapsulated in a non-viron particle that can mimic the delivery function of a virus (see e.g., Intl. Pub. No. WO2012/006376 and U.S. Pub. Nos. US2013/0171241 and US2013/0195968, each of which is herein incorporated by reference in its entirety). e. Self-Assembled Nanoparticles, or Self-Assembled Macromolecules

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) in self-assembled nanoparticles, or amphiphilic macromolecules (AMs) for delivery. AMs comprise biocompatible amphiphilic polymers that have an alkylated sugar backbone covalently linked to poly(ethylene glycol). In aqueous solution, the AMs self-assemble to form micelles. Nucleic acid self-assembled nanoparticles are described in Intl. Appl. No. PCT/US2014/027077, and AMs and methods of forming AMs are described in U.S. Pub. No. US2013/0217753, each of which is herein incorporated by reference in its entirety. f. Cations and Anions

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) and a cation or anion, such as Zn2+, Ca2+, Cu2+, Mg2+ and combinations thereof. Exemplary formulations can include polymers and a polynucleotide complexed with a metal cation as described in, e.g., U.S. Pat. Nos.6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety. In some embodiments, cationic nanoparticles can contain a combination of divalent and monovalent cations. The delivery of polynucleotides in cationic nanoparticles or in one or more depot comprising cationic nanoparticles can improve polynucleotide bioavailability by acting as a long-acting depot and/or reducing the rate of degradation by nucleases. g Amino Acid Lipids

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) that is formulation with an amino acid lipid. Amino acid lipids are lipophilic compounds comprising an amino acid residue and one or more lipophilic tails. Non-limiting examples of amino acid lipids and methods of making amino acid lipids are described in U.S. Pat. No.8,501,824. The amino acid lipid

formulations can deliver a polynucleotide in releasable form that comprises an amino acid lipid that binds and releases the polynucleotides. As a non-limiting example, the release of the polynucleotides described herein can be provided by an acid-labile linker as described in, e.g., U.S. Pat. Nos.7,098,032, 6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931, each of which is herein incorporated by reference in its entirety. h. Interpolyelectrolyte Complexes

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) in an interpolyelectrolyte complex.

Interpolyelectrolyte complexes are formed when charge-dynamic polymers are complexed with one or more anionic molecules. Non-limiting examples of charge-dynamic polymers and interpolyelectrolyte complexes and methods of making interpolyelectrolyte complexes are described in U.S. Pat. No.8,524,368, herein incorporated by reference in its entirety. i. Crystalline Polymeric Systems

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) in crystalline polymeric systems. Crystalline polymeric systems are polymers with crystalline moieties and/or terminal units comprising crystalline moieties. Exemplary polymers are described in U.S. Pat. No.8,524,259 (herein incorporated by reference in its entirety). j Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) and a natural and/or synthetic polymer. The polymers include, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l- lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, elastic biodegradable polymer, biodegradable copolymer, biodegradable polyester copolymer, biodegradable polyester copolymer, multiblock copolymers, poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), amine-containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof.

Exemplary polymers include, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDELTM (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as PHASERX® (Seattle, WA).

The polymer formulations allow a sustained or delayed release of the polynucleotide (e.g., following intramuscular or subcutaneous injection). The altered release profile for the polynucleotide can result in, for example, translation of an encoded protein over an extended period of time. The polymer formulation can also be used to increase the stability of the polynucleotide. Sustained release formulations can include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc. Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc. Deerfield, IL).

As a non-limiting example modified mRNA can be formulated in PLGA

microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process. EVAc are non- biodegradable, biocompatible polymers that are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device;

transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F- 407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene- polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5ºC and forms a solid gel at temperatures greater than 15ºC.

As a non-limiting example, the polynucleotides described herein can be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No.

6,177,274. As another non-limiting example, the polynucleotides described herein can be formulated with a block copolymer such as a PLGA-PEG block copolymer (see e.g., U.S. Pub. No. US20120004293 and U.S. Pat. Nos.8,236,330 and 8,246,968), or a PLGA-PEG- PLGA block copolymer (see e.g., U.S. Pat. No.6,004,573). Each of the references is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can be formulated with at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(amine-co-esters) or combinations thereof.

Exemplary polyamine polymers and their use as delivery agents are described in, e.g., U.S. Pat. Nos.8,460,696, 8,236,280, each of which is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can be formulated in a biodegradable cationic lipopolymer, a biodegradable polymer, or a biodegradable copolymer, a biodegradable polyester copolymer, a biodegradable polyester polymer, a linear

biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof as described in, e.g., U.S. Pat. Nos.6,696,038, 6,517,869, 6,267,987, 6,217,912, 6,652,886, 8,057,821, and 8,444,992; U.S. Pub. Nos. US20030073619, US2004/0142474, US2010/0004315, US2012/0009145 and US2013/0195920; and Intl Pub. Nos. WO2006/063249 and WO2013/086322, each of which is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can be formulated in or with at least one cyclodextrin polymer as described in U.S. Pub. No. US20130184453. In some embodiments, the polynucleotides described herein can be formulated in or with at least one crosslinked cation-binding polymers as described in Intl. Pub. Nos. WO2013/106072, WO2013/106073 and WO2013/106086. In some embodiments, the polynucleotides described herein can be formulated in or with at least PEGylated albumin polymer as described in U.S. Pub. No. US2013/0231287. Each of the references is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides disclosed herein can be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate. Components can be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle for delivery (Wang et al., Nat Mater.20065:791-796; Fuller et al., Biomaterials.200829:1526- 1532; DeKoker et al., Adv Drug Deliv Rev.201163:748-761; Endres et al., Biomaterials. 201132:7721-7731; Su et al., Mol Pharm.2011 Jun 6;8(3):774-87; herein incorporated by reference in their entireties). As a non-limiting example, the nanoparticle can comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (Intl. Pub. No. WO2012/0225129, herein incorporated by reference in its entirety).

The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci U S A.2011108:12996-13001; herein incorporated by reference in its entirety). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles can efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.

In some embodiments, a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG can be used to delivery of the polynucleotides as described herein. In some embodiments, the lipid nanoparticles can comprise a core of the polynucleotides disclosed herein and a polymer shell, which is used to protect the

polynucleotides in the core. The polymer shell can be any of the polymers described herein and are known in the art., the polymer shell can be used to protect the polynucleotides in the core.

Core–shell nanoparticles for use with the polynucleotides described herein are described in U.S. Pat. No.8,313,777 or Intl. Pub. No. WO2013/124867, each of which is herein incorporated by reference in their entirety. k. Peptides and Proteins

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) that is formulated with peptides and/or proteins to increase transfection of cells by the polynucleotide, and/or to alter the biodistribution of the polynucleotide (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein (e.g., Intl. Pub. Nos. WO2012/110636 and WO2013/123298. In some embodiments, the peptides can be those described in U.S. Pub. Nos.

US2013/0129726, US2013/0137644 and US2013/0164219. Each of the references is herein incorporated by reference in its entirety. l. Conjugates

In some embodiments, the compositions or formulations of the present disclosure comprise the polynucleotides described herein (e.g., a polynucleotide comprising a nucleotide sequence encoding a Factor VIII polypeptide) that is covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide) as a conjugate. The conjugate can be a peptide that selectively directs the nanoparticle to neurons in a tissue or organism, or assists in crossing the blood-brain barrier.

The conjugates include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co- glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

In some embodiments, the conjugate can function as a carrier for the polynucleotide disclosed herein. The conjugate can comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine that can be grafted to with poly(ethylene glycol). Exemplary conjugates and their preparations are described in U.S. Pat. No.6,586,524 and U.S. Pub. No. US2013/0211249, each of which herein is incorporated by reference in its entirety.

The conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Targeting groups can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent frucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In particular embodiments, the targeting group is an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein. As a non-limiting example, the targeting group can be a glutathione receptor (GR)-binding conjugate for targeted delivery across the blood-central nervous system barrier as described in, e.g., U.S. Pub. No.

US2013/0216612 (herein incorporated by reference in its entirety).

In some embodiments, the conjugate can be a synergistic biomolecule-polymer conjugate, which comprises a long-acting continuous-release system to provide a greater therapeutic efficacy. The synergistic biomolecule-polymer conjugate can be those described in U.S. Pub. No. US2013/0195799. In some embodiments, the conjugate can be an aptamer conjugate as described in Intl. Pat. Pub. No. WO2012/040524. In some embodiments, the conjugate can be an amine containing polymer conjugate as described in U.S. Pat. No.

8,507,653. Each of the references is herein incorporated by reference in its entirety. In some embodiments, the polynucleotides can be conjugated to SMARTT POLYMER

TECHNOLOGY® (PHASERX®, Inc. Seattle, WA).

In some embodiments, the polynucleotides described herein are covalently conjugated to a cell penetrating polypeptide, which can also include a signal sequence or a targeting sequence. The conjugates can be designed to have increased stability, and/or increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).

In some embodiments, the polynucleotides described herein can be conjugated to an agent to enhance delivery. In some embodiments, the agent can be a monomer or polymer such as a targeting monomer or a polymer having targeting blocks as described in Intl. Pub. No. WO2011/062965. In some embodiments, the agent can be a transport agent covalently coupled to a polynucleotide as described in, e.g., U.S. Pat. Nos.6,835.393 and 7,374,778. In some embodiments, the agent can be a membrane barrier transport enhancing agent such as those described in U.S. Pat. Nos.7,737,108 and 8,003,129. Each of the references is herein incorporated by reference in its entirety.

The structural lipids include sterols and lipids containing sterol moieties. In some embodiments, the structural lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the amount of the structural lipids (e.g., cholesterol) in the lipid composition ranges from about 20 mol% to about 60 mol%.

The PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG- modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEG-lipid are 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the amount of PEG-lipid in the lipid composition ranges from about 0.1 mol% to about 5 mol%.

In some embodiments, the LNP formulations described herein can additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in U.S. Pub. No. US2005/0222064, herein incorporated by reference in its entirety. Methods of Use

The polynucleotides, pharmaceutical compositions and formulations described herein are used in the preparation, manufacture and therapeutic use of to treat and/or prevent diseases, disorders or conditions. In some embodiments, the polynucleotides, compositions and formulations of the present disclosure are used to treat and/or prevent infectious disease such as HIV, Ebola and influenza.

RNA treatments may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA treatments to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. RNA treatments compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of RNA treatments compositions may be decided by the attending physician within the scope of sound medical judgment. The specific

therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or

coincidental with the specific compound employed; and like factors well known in the medical arts.

In some embodiments, RNA treatments compositions may be administered at dosage levels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013/078199, herein incorporated by reference in its entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc.. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In exemplary embodiments, RNA treatments compositions may be

administered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, RNA treatment compositions may be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, RNA treatment compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency of administration are encompassed by the present disclosure. For example, an RNA treatment composition may be administered three or four times.

In some embodiments, RNA treatment compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments, the RNA for use in a method of treating a subject is administered to the subject in a single dosage of between 10 µg/kg and 400 µg/kg of the nucleic acid treatment in an effective amount to treat the subject. In some embodiments, the RNA treatment for use in a method of treating a subject is administered to the subject in a single dosage of between 10 µg and 400 µg of the nucleic acid treatment in an effective amount to treat the subject.

An RNA pharmaceutical composition described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

This disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of“including,” “comprising,” or“having,”“containing,”“involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. EXAMPLES

Example 1. Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotides and or parts or regions thereof may be accomplished utilizing the methods taught in International Application WO2014/152027 entitled“Manufacturing Methods for Production of RNA Transcripts”, the contents of which is incorporated herein by reference in its entirety. Purification methods may include those taught in International Application

WO2014/152030 and WO2014/152031, each of which is incorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may be performed as taught in WO2014/144039, which is incorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may be accomplished using a procedure selected from the group consisting of polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis, and detection of RNA impurities, wherein characterizing comprises determining the RNA transcript sequence, determining the purity of the RNA transcript, or determining the charge heterogeneity of the RNA transcript. Such methods are taught in, for example, WO2014/144711 and WO2014/144767, the contents of each of which is incorporated herein by reference in its entirety. Example 2. Chimeric polynucleotide synthesis

Introduction

According to the present disclosure, two regions or parts of a chimeric polynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a first region or part of 100 nucleotides or less is chemically synthesized with a 5′ monophosphate and terminal 3′desOH or blocked OH. If the region is longer than 80 nucleotides, it may be synthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionally modified region or part using in vitro transcription (IVT), conversion the 5′monophosphate with subsequent capping of the 3′ terminus may follow.

Monophosphate protecting groups may be selected from any of those known in the art.

The second region or part of the chimeric polynucleotide may be synthesized using either chemical synthesis or IVT methods. IVT methods may include an RNA polymerase that can utilize a primer with a modified cap. Alternatively, a cap of up to 130 nucleotides may be chemically synthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase, followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with a phosphate-sugar backbone. If one of the regions or parts encodes a polypeptide, then it is preferable that such region or part comprise a phosphate-sugar backbone. Ligation is then performed using any known click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art.

Synthetic route

The chimeric polynucleotide is made using a series of starting segments. Such segments include:

(a) Capped and protected 5′ segment comprising a normal 3′OH (SEG.1)

(b) 5′ triphosphate segment which may include the coding region of a polypeptide and comprising a normal 3′OH (SEG.2)

(c) 5′ monophosphate segment for the 3′ end of the chimeric polynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG.3) is treated with cordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG.2) is then ligated to SEG.3 using RNA ligase. The ligated polynucleotide is then purified and treated with pyrophosphatase to cleave the diphosphate. The treated SEG.2-SEG.3 construct is then purified and SEG.1 is ligated to the 5′ terminus. A further purification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated or joined segments may be represented as: 5′UTR (SEG.1), open reading frame or ORF (SEG.2) and 3′UTR+PolyA (SEG.3).

The yields of each step may be as much as 90-95%. Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2x KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, MA). This system includes 2x KAPA ReadyMix12.5 µl; Forward Primer (10 uM) 0.75 µl; Reverse Primer (10 uM) 0.75 µl;

Template cDNA -100 ng; and dH₂0 diluted to 25.0 µl. The reaction conditions are at 95° C for 5 min. and 25 cycles of 98° C for 20 sec, then 58° C for 15 sec, then 72° C for 45 sec, then 72° C for 5 min. then 4° C to termination.

The reaction is cleaned up using Invitrogen’s PURELINK™ PCR Micro Kit

(Carlsbad, CA) per manufacturer’s instructions (up to 5 µg). Larger reactions will require a cleanup using a product with a larger capacity. Following the cleanup, the cDNA is quantified using the NANODROP^TM and analyzed by agarose gel electrophoresis to confirm the cDNA is the expected size. The cDNA is then submitted for sequencing analysis before proceeding to the in vitro transcription reaction. Example 4. In vitro Transcription (IVT)

The in vitro transcription reaction generates polynucleotides containing uniformly modified polynucleotides. Such uniformly modified polynucleotides may comprise a region or part of the polynucleotides of the disclosure. The input nucleotide triphosphate (NTP) mix is made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1 Template cDNA 1.0 µg

2 10x transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 2.0 µl

3 Custom NTPs (25mM each) 7.2 µl

4 RNase Inhibitor 20 U

5 T7 RNA polymerase 3000 U

6 dH₂0 Up to 20.0 µl. and

7 Incubation at 37° C for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C overnight for cleanup the next day.1 U of RNase-free DNase is then used to digest the original template. After 15 minutes of incubation at 37° C, the mRNA is purified using Ambion’s MEGACLEAR™ Kit (Austin, TX) following the manufacturer’s instructions. This kit can purify up to 500 µg of RNA.

Following the cleanup, the RNA is quantified using the NanoDrop and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred. Example 5: Enzymatic Capping

Capping of a polynucleotide is performed as follows where the mixture includes: IVT RNA 60 µg-180µg and dH₂0 up to 72 µl. The mixture is incubated at 65° C for 5 minutes to denature RNA, and then is transferred immediately to ice.

The protocol then involves the mixing of 10x Capping Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 µl); 20 mM GTP (5.0 µl); 20 mM S-Adenosyl Methionine (2.5 µl); RNase Inhibitor (100 U); 2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U); dH₂0 (Up to 28 µl); and incubation at 37° C for 30 minutes for 60 µg RNA or up to 2 hours for 180 µg of RNA.

The polynucleotide is then purified using Ambion’s MEGACLEAR™ Kit (Austin, TX) following the manufacturer’s instructions. Following the cleanup, the RNA is quantified using the NANODROP™ (ThermoFisher, Waltham, MA) and analyzed by agarose gel electrophoresis to confirm the RNA is the proper size and that no degradation of the RNA has occurred. The RNA product may also be sequenced by running a reverse-transcription-PCR to generate the cDNA for sequencing. Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must be performed before cleaning the final product. This is done by mixing Capped IVT RNA (100 µl); RNase Inhibitor (20 U); 10x Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM

MgCl₂)(12.0 µl); 20 mM ATP (6.0 µl); Poly-A Polymerase (20 U); dH₂0 up to 123.5 µl and incubation at 37° C for 30 min. If the poly-A tail is already in the transcript, then the tailing reaction may be skipped and proceed directly to cleanup with Ambion’s MEGACLEAR™ kit (Austin, TX) (up to 500 µg). Poly-A Polymerase is preferably a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyA tailing reaction may not always result in an exact size polyA tail. Hence polyA tails of approximately between 40-200 nucleotides, e.g., about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope of the invention. Example 7: Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5′- guanosine cap structure according to manufacturer protocols: 3´-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, MA). 5′-capping of modified RNA may be completed post- transcriptionally using a Vaccinia Virus Capping Enzyme to generate the“Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate:

m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl- transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O- methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase.

Enzymes are preferably derived from a recombinant source. When transfected into mammalian cells, the modified mRNAs have a stability of between 12-18 hours or more than 18 hours, e.g., 24, 36, 48, 60, 72 or greater than 72 hours. Example 8: Capping Assays

A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be transfected into cells at equal concentrations. 6, 12, 24 and 36 hours post-transfection the amount of protein secreted into the culture medium can be assayed by ELISA. Synthetic polynucleotides that secrete higher levels of protein into the medium would correspond to a synthetic polynucleotide with a higher translationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be compared for purity using denaturing Agarose-Urea gel electrophoresis or HPLC analysis. Polynucleotides with a single, consolidated band by electrophoresis correspond to the higher purity product compared to polynucleotides with multiple bands or streaking bands.

Synthetic polynucleotides with a single HPLC peak would also correspond to a higher purity product. The capping reaction with a higher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be transfected into cells at multiple concentrations. 6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted into the culture medium can be assayed by ELISA. Polynucleotides resulting in the secretion of higher levels of pro-inflammatory cytokines into the medium would correspond to a polynucleotides containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the caps taught herein can be analyzed for capping reaction efficiency by LC-MS after nuclease treatment. Nuclease treatment of capped polynucleotides would yield a mixture of free nucleotides and the capped 5′-5-triphosphate cap structure detectable by LC-MS. The amount of capped product on the LC-MS spectra can be expressed as a percent of total polynucleotide from the reaction and would correspond to capping reaction efficiency. The cap structure with higher capping reaction efficiency would have a higher amount of capped product by LC-MS. Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual polynucleotides (200-400 ng in a 20 µl volume) or reverse transcribed PCR products (200-400 ng) are loaded into a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, CA) and run for 12-15 minutes according to the manufacturer protocol. Example 10: Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 µl) are used for Nanodrop UV absorbance readings to quantitate the yield of each polynucleotide from an chemical synthesis or in vitro transcription reaction. Example 11: Formulation of Modified mRNA Using Lipidoids

Polynucleotides are formulated for in vitro experiments by mixing the polynucleotides with the lipidoid at a set ratio prior to addition to cells. In vivo formulation may require the addition of extra ingredients to facilitate circulation throughout the body. To test the ability of these lipidoids to form particles suitable for in vivo work, a standard formulation process used for siRNA-lipidoid formulations may used as a starting point. After formation of the particle, polynucleotide is added and allowed to integrate with the complex. The

encapsulation efficiency is determined using a standard dye exclusion assays. Example 12: Influenza HA Binding Antibodies

A phage library was constructed from the RNA of immunized Harbour mice and screened for phage binding to H1 HA, H3 HA, and EBOV protein. Nine anti-H3N2 (Table 1, SEQ ID NOs: 1-9) and one anti-H1N1 (Table 1, SEQ ID NO: 10) influenza binding HCAbs were selected based on specificities determined via an enzyme-linked immunosorbent assay (ELISA). As shown in FIG.1, the selected H1 and H3 binders did not appear to cross-bind the HA subtypes.

Binding of the selected HCAbs to HA was further analyzed using bi-layer

interferometry on OctetRed96. FIG.2 shows the results of this analysis, where binding of the selected HCAbs to HA of different strains of H3N2 and H1N1 was assessed. The results appeared consistent with the soluble ELISA data. The H1 clone, H1_20 (SEQ ID NO: 10), showed binding to H1N1/PR8, but not to a few other strains of H1N1. It was further affirmed that the H3 and H1 binding HCAbs did not cross bind to H1 and H3, respectively. To further characterize the binding properties of the selected HCAbs, epitope binning was assessed, the results of which are reported in FIG.3. BNINFAB-1 and BNINFAB-2 were included in the study as known HA stem binding benchmark antibodies. A competing antibody which does not bind (×) shares (e.g., competes for) the same epitope with the first antibody. A competing antibody which binds (√) does not compete (e.g., does not share) for the same epitope as the first antibody. As shown in FIG.3, all seven of the H3 binding HCAbs bind to the same epitope on H3N2, which is different from the epitopes of

BNINFAB-1 and BNINFAB-2.

Next, the binding kinetics of the selected HCAbs binding to H3 were measured. In these experiments, VH-Fc antibodies were captured on a ProA chip and H3 hemagglutinin was flown over the flow cells of the chip. Kinetic traces for increasing analyte concentrations are shown in FIG.4A with the resulting derived kinetic parameters reported in FIG.4B. As shown by the reported K_D values (K_D = k_off/k_on), H3_18 and H3_1 bind HA with

approximately 10-fold greater affinity than the remaining clones tested.

Selected H3 binding HCAbs were tested for the ability of each to neutralize H3N2 Brisbane/2007 virus in vitro. As shown by the results reported in Table 7, H3_1, H3_14, and H3_18 neutralized H3N2 Brisbane/2007 virus, with H3_18 being approximately 30-fold more potent against the virus strain as compared to the benchmark antibody BNINFAB-1. Table 7. In vitro neutralization of H3N2 Brisbane 2007

Table 8 reports data obtained from additional neutralization experiments investigating the effects of H3_14, H3_18, and H3_50 on a variety of H3N2 strains in vitro. BNINFAB-1 was included in the study as a benchmark antibody, as in the above experiments. Table 8. In vitro neutralization of H3N2 strains

Selected mRNA encoding anti-influenzan antibody were tested for the ability to treat influenza in vivo. As shown by the results presented in Figure 5 IM injection of anti-flu mRNA leads to robust protection in lethal challenge model. The methods enable higher expressing Abs and novel formulations by IM dosing. Example 13: Binding Site Identification

In order to identify an epitope of an antibody of the invention, HDX and epitope mapping studies were performed with H318. The results demonstrated the binding profile of the antibody. Hydrogen deuterium exchange analysis of H318 HCAb and H3N2

Brisbane/2007 HA predicted several two putative binding sites for H318.

The shaded N is a predicted glycosylation site. Sequences correspond to SEQ ID NOs: 111- 126 from top to bottom. An alignment of influenza HA sequences from major antigenic clusters along with the binding data to these antigens (shown above) confirmed that these sites were crucial for the binding of H318. No binding occurred for HA sequences in which specific mutations (L155S, K157G, F158S, K159T) or glycosylation sites were present (bottom two of the three boxes presented above). Alanine scanning within this region helps provide information about critical binding residues which can then be used for maturation of affinity or breadth to cover all H3N2 variants.

Thus, H318 antibody recognizes a conserved epitope on HA1 domain and cross neutralizes multiple H3N2 strains. Example 14. Pharmacokinetic and Tolerability Studies of CR6261 in Cynomolgus monkey

This study aimed at testing the pharmacokinetics of CR6261 expressed from therapeutic mRNA delivered via various routes in Cynomolgus monkey. The monkeys (n=3) were injected either intravenously (IV) with 0.5 mg/kg or 2 mg/kg, subcutaneously (SC) with 2 mg/kg or via intramuscular injection (IM) with 0.5 mg/kg, 1 mg/kg or 2 mg/kg of mRNA. mRNAs were formulated in MC3 LNPs prior to administration. PBS-formulated controls were also tested for the 0.5 mg/kg IV and IM doses.

Protein was detected using LC/MS detection of tryptic peptides specific to CR6261, following Protein A capture of IgG antibodies from serum.

Results for dosing with 1 mg/kg or 2/mg/kg are described in Table 9. As used in the table, half-life means the amount of time it takes for the serum concentration of a the mRNA to fall 50%, Tmax means the time after administration of the mRNA when the maximum plasma concentration is reached, and Cmax means the maximum plasma concentration of the mRNA. AUC means area under the concentration curve (the integral of the concentration- time curve, a method of measurement of the bioavailability of the mRNA) and CL stands for clearance (the removal of a substance from the blood. Vdss is Volume of Distribution at Steady State (an estimate of drug distribution independent of elimination processes). Table 9. Mean Pharmacokinetic Values after IV, SC, or IM Injection

After a 2 mg/kg intravenous administration of CR6261-encoding mRNA, the protein expression peaked at 48 hr post-dose with a mean value of 4.54 µg/mL. The mean elimination half-life was 111 hr. The clearance of the expressed antibody was slow, 2.39 mL/hr/kg. The volume of distribution, 456 mL/kg, was moderate.

After a 2 mg/kg subcutaneous administration of CR6261-encoding mRNA, the protein expression peaked at 48-96 hr post-dose with a mean value of 2.97 ug/mL. The mean elimination half-life was 105 hr.

After intramuscular administration of the CR6261-encoding mRNA, the protein expression peaked at 48 hr post-dose with mean values of 2.67 and 4.05 ug/mL after 1 and 2 mg/kg, respectively. The mean elimination half-life was 82 and 107 hr, respectively.

Results for dosing with 0.5 mg/kg are set forth in Table 10. For this dosing regimen, data were collected only for an initial 72 hour period. For comparison purposes, data from Table 4 (above) were recalculated based on the 72 hour time window. T l 1 mm r f m x n A - 2

These data show that dose-normalized Cmax and AUC are higher at the 0.5 mg/kg dose consistent with the absence of an inflammatory response.

The data in total (all doses and administration routes tested) further show that peak expression in the 2-4 ug/ml mean range occurs at 48 hr post-injection for all routes. The mean elimination half-life was 111 hr. (Mean elimination half-lives may be under-estimated due to fewer time points collected in the terminal phase for certain doses.) The clearance was slow (2.39 mL/hr/kg) and the volume of distribution was moderate (456 mL/kg).

Next, quantitative PCR was used to quantify CR6261 mRNA in serum from animals dosed with 2 mg/kg mRNA. The data are set forth in Table 11. Table 11. PK parameters of CR6261 mRNA

These data show half-life for therapeutic mRNA of abot 40-45 hours, demonstrating good therapeutic applicability. Example 15: Expression of peptides in LNPs of the invention

An RNA polynucleotide encoding antibody 9114 was administered to non-human primates at doses of 0.1 mg/kg (Fig.7A) or 0.3 mg/kg (Fig.7B) in standard lipids and the LNPs of the invention. Peak expression data is shown in Fig 7. Based on this data it is expected to achieve between 1 and 10 microgram per ml peak exposure in human subjects.

The tolerability of an RNA polynucleotide encoding antibody 9114 delivered in an LNP of the invention at doses up to 1 mg/kg weekly was assessed in non-human primates. The data shown in Figs.8 and 9 demonstrate that the lipid formulation at such high doses was well tolerated. The parameters tested included body weight (8A), aspartate aminotransferase levels (8B), alanine aminotransferase levels (8C), complement activation (9A), IL6 levels (9B) and MCP-1 levels (9C).

The effect of the unique lipids of the invention was assessed in another study in which mRNA encoding EPO was administered to non-human primates.6 weekly doses of hEPO mRNA LNPs were administered at 0.2 mg/kg by IV infusion. The data is shown in Fig.10. In the control panel (Fig.10A) 2 animals showed significant increases in expression while 2 animals lost expression by week 6.1 of the animals that lost expression had a significant spike in anti-PEG IgM and IgG. In contrast in the animals administered the lipid formulation of the invention (compound 18 and PEG stearate, Fig.10B) all animals maintained expression out to 6 weeks. No animals demonstrated a signifcant spike in IgM or IgG. No stimulation of IL-6 and no activation of complement were observed, in contrast ot the control group.

A repeat dose of hEPO mRNA in Cmpd18-containing LNPs with modified PEG lipids was next performed in non-human primates. Briefly, cynomolgus monkeys were treated with LNP-encapsulated mRNA administered by intravenous (IV) infusion. Oleic acid- and Cmpd428-containing LNPs were analyzed to determine their effect on accelerated blood clearance (ABC). In particular, it was an objective to determine if the DSPC and PEG- lipid replacements demonstrate low B cell activation, low anti-PEG IgM, and maintenance of protein expression in higher species. The following four groups were compared:

MC3/DSPC/Chol/PEG lipid (control), Cmpd18/DSPC/Chol/ PEG lipid (control),

Cmpd18/DSPC/Chol/Cmpd428 (test group), and Cmpd18/Oleic Acid/Chol/ PEG lipid (test group). The monkeys were administered a pseudouridine modified hEPO mRNA (0.2 mg/kg) weekly dosing in the foregoing LNPs for 6 weeks.

Pharmokinetic parameters including AUC were determined for Cmpd 18: Cmpd 428 combinations. Contsitutive AUC was seen over time with baseline complement and cytokine levels and low to no levels of IgM observed.

The particle characteristics for each of the formulations are shown in Table 12 below. mRNA used for this study was m1ψ-modified mRNA encoding EPO. Formulations includes components at the following mol % - 50:10:38.5:1.5. Table 12 shows size, encapsulation efficiency (EE) and polydispersity index (PDI). Particles had characteristics within acceptable limits for in vivo testing. Table 12

Cmpd428 is

wherein r is 45.

Throughout the 6 weeks of experimentation, hEPO protein expression, anti-PEG IgM and IgG levels, IgM, IgG and cytokine and complement levels were measured for the novel LNPs. FIG.15 depicts hEPO expression (ng/mL) at predose, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, and 72 hours following once weekly IV administration of the hEPO mRNA – LNP formulations at day 1, day 8, day 15, day 22, day 29, day 36, and day 43.

In FIG.16, levels of anti-PEG IgM (U/mL) were measured following once weekly IV administration of the hEPO mRNA– LNP formulations at day 1, day 8, day 15, day 22, day 29, day 36, and day 43. The Cmpd428 group demonstrated maintenance of protein expression across the 6 weeks, maintenance of baseline levels of anti-PEG IgM and anti-PEG IgG, no complement activation, and minimal cytokine activation. Levels of anti-PEG IgM and IgG, complement, and cytokine elevations appeared to inversely correlate with a loss of protein expression. The MC3 and Cmpd18 controls and oleic acid group all showed reduced hEPO protein expression at week 6. The oleic acid group demonstrated the highest levels of initial of protein production. The MC3 and Cmpd18 controls and Cmpd428 groups showed similar levels of hEPO expression. Cmpd18/DSPC at 0.2 mg/kg did not show increased hEPO expression relative to MC3/DSPC; however, Cmpd18/oleic acid did show increased protein expression (3-fold) relative to MC3/DSPC and Cmpd18/DSPC. Table 13 shows hEPO expression as measured by AUC of the four LNP groups. In particular, the hEPO AUC (ng/mL*h) was measured each week of the 6 week study. Table 13

Particles formulated with Cmpd18 as the cationic amino lipid outperformed standard MC3-based LNPs in terms of protein expression throughout repeated dosing in primates. Oleic acid as a DSPC replacement resulted in initially high protein expression but did not fully ameliorate ABC over the course of the study. Compound428 as a PEG-lipid

replacement resulted in reduced ABC as evidenced by maintenance of hEPO AUC over the course of the study. Taken together, these data show that deletion of B cells leads to absence of to ABC after 5 injections. In particular, a single injection of anti CD20, 7 days before LNP injection lead to (1) low/no IgM/IgG to PEG; (2) no clearance of RNA or protein; and (3) low/no cytokines/complement activation, making this an attractive candidate for co- administration regimens featuring LNP-encapsulated mRNAs. Example 16: Polyclonal Phage ELISA and Panning

A phage library was screened for phage binding to EBOV. Three panning cycles were performed to amplify the phages with stronger EBOV biding. The specificity of the polyclonal phages generated after each round of panning was determined via an enzyme- linked immunosorbent assay (ELISA) using EBOV and an irrelevant protein (H1N1). For the ELISA, the wells were coated with 100ug of the His-tagged protein (EBOV or H1N1). Then, the test phages were incubated overnight at 4^oC and washed three times following the ELISA protocol. Binding was detected using M13 pAB-HRP. The polyclonal ELISA was performed three times to test the phage pools from each round. The phage did not bind to H1N1 or to the His-tag. The binding that occurred was specific to the EBOV protein. As shown in FIG.11, phage binders to EBOV were enriched with each round of panning.

The resulting polyclonal phages that exhibited the strongest EBOV binding were then subjected to a monoclonal phage ELISA. H1N1 was again used as a control. Nine phages were further selected for use in a soluable VHH ELISA. Their binding to EBOV was further confirmed via Western blot. The sequences are provided in Table 14.

Table 14: Sequences of selected clones used in soluble VHH ELISA (sequences, from top to bottom, correspond to SEQ ID NOs: 127-135)

Table 15: EBOV VHH sequences from the naïve library

Example 17: Cross-Binding with the Zaire and Sudan Strains

A polyclonal ELISA was also performed to find Zaire and Sudan strain cross-binders. Four screens, each subjected to a different combination of Zaire and Sudan strains during the panning process (FIG.12A-12B) were tested. The results of the polyclonal ELISA following Round 3 are shown in FIG.12A. Screen 1 was then selected to undergo a monoclonal phage ELISA to test individual clones’ specificity for both the Zaire and the Sudan strains. The results are also shown in FIG.12B. All three rounds of the four screens were sequenced and Next Generation Sequencing (NGS) was used to identify the enriched sequences. The results are shown in FIG.13. NGS yielded two dominan VHH. Both were from Round 2, Screen 2 and their sequences and Western blot are provided in FIG.14A-14B.

The two dominant VHHs, ZSZ01 and ZSZ02, were further tested and showed a higher binding affinity to the Sudan strain, as shown in Table 16. Furthermore, two epitope bins were identified. 11E (from the naïve library) has a fast K_off, meaning that ZSZ01 and ZSZ02 do not bind to captured 11E, but 11E does bind to captured ZSZ01 and ZSZ02 (Table 17). Table 16: VHH Binding Affinities

Table 17: Epitope Bins

Example 18. HIV Antibodies and Combinations thereof

Therapeutic and prophylactic combinations of HIV antibodies have been studied for many years. However, the delivery of antibodies using mRNA therapeutics has posed a number of challenges. The expression of complex proteins such as antibodies and scFv is difficult. Also antibodies delivered as combinations of proteins are challenging to mimic using mRNA therapeutics.

Experiments were set up to identify optimal combinations of antibodies with a broad spectrum of neutralization expressed in vivo from mRNA. The methods set forth in Examples 18 and 19 describe the creation and optimizing of antibodies and single chain versions to enable co-expression in a therapeutically effective formulation. The antibodies are characterized for pre-clinical activity, in vitro and in vivo.

Several effective combinations of HIV antibodies have been described in the art and include PGT12110-1074, PGDM1400, and 3BNC117 NIH 45-46 N6 (Scharf et al. Cell Reports 20147, 785-795). An effective combination involves the optimal convergence of the spectrum of neutralizing activity across many HIV strains/clades, optimized level of expression, and protein stability contributing to half-life in vivo. Not all antibodies are amenable to conversion to scFv format. In vivo expression of antibodies and scFv from mRNA is challenging. Additionally, co-expression of combinations of IgGs may form non- functional pairings. An exemplary target product of a combination of HIV antibodies of the invention was designed and developed. The target product is a combination of a class IgG and two scFvFcs, which yields IgGs, mono- and bispecific scFvFcs, and IgG/sc“hybrids.” The product includes four mRNAs total, which produce an IgG (one mRNA encoding the heavy chain and one mRNA encoding the light chain) and two scFV-FCs (each encoded by one mRNA).

The scFV-FC synthesis from a typical antibody format involved the addition of linkers. Initially, constant domains were removed. Two linkers were added to connect the FV region which binds the antigen, and the FC region which binds and recycles to FcRN. The ability to develop functional scFv based on a functional antibody (also referred to as tolerance of conversion) is antibody-dependent.

The method for conversion of HIV bnAbs (neutralizing antibodies) was as follows. Domain orientation was selected, linker length was optimized, and antibodies were chosen with the most favorable biophysical properties. Breadth and potency of neutralization was maintained and heterogeneity and aggregation were minimized. The methods involved conversion of each IgG into an scFV-FC with neutralization potency and breadth comparable to the IgG parent.

Three antibodies were converted to scFv, one V1/V2 binding antibody (PGDM1400) and two V3 base binders (PGT121 and 10-1074). In a first round of modifications to the antibodies to generate scFv, a set of scFv having a (G₄S)₃ linker between the VL and VH domains was generated. These Round 1 variants are referred to herein as variant 1 or Var1 versions of each antibody. A second round of modifications was made to the scFv to produce a second set of variants, culminating in creation of variants referred to herein as variant 7 or Var7. The changes made to the variant 7 scFv involved increasing the length of the linker between the VL and VH domains to a (G₄S)₄ linker and replacing native glycan sites for stabilization in one of the constructs (PGT-121).

The ability of the RNA constructs developed for these variants to express the scFV- FC protein in vitro was assayed. These data are shown in Figure 17. In contrast to intact antibodies (IgG) and round 1 scFv variants, the round 2 scFv variants of PGT121 and PGDM1400 showed significantly increased expression levels. The PGDM1400 scFv construct having the longer linker had significantly higher expression levels (340 mg/L) than the same construct with a shorter linker (243 mg/L). The PGT121 round 2 construct having 2 native glycan site replaced and a longer linker also had significantly higher expression levels (248 mg/L) than the same construct with a shorter linker and no glycans replaced (170 mg/L) as well as a construct with a shorter linker and 1 glycan replaced (143 mg/L). Although the round 210-1074 construct showed better expression levels than the round 1 construct, expression levels were significantly lower than IgG. However, the round 2 construct showed a reduction in aggregation relative to IgG.

Thus, each scFV-FCs has distinct issues which were optimized. The tolerance to conversion to an scFV-FC format varied among the tested bnAbs (Table 18 and Figure 18). Neutralization and thermostability of the round 1 and 2 variants in comparison to intact IgG was examined. Removing VH-VL strain by increasing the length of the linker between these two regions of the molecule and adding glycan stability improved PGT-121 (to within 2 fold IgG) and an improved thermostability. These modification allowed a native-like

neutralization to be achieved. A longer linker for the PGDM1400 scFv resulted in a level of neutralization within 2 fold of IgG and no loss in thermostability. Although a longer linker for the 10-1074 construct had not improved expression levels, the modified construct demonstrated a significant level of neutralization (within 2 fold of IgG) and no loss in thermostability.

CD4 binding site anti-HIV antibodies were more challenging to convert to the scFv- FC format. After synthesis of a number of scFvFc constructs, expression and stability were optimized for the V3 and V1/V2 binders. In view of the challenges associated with CD4 binding site antibodies, it was decided to maintain the intact IgG structure for these antibodies and an mRNA composition was assembled which included an intact CD4 binding site-specific IgG and two scFv molecules, one specific for V3 and one for V1/V2. Table 18. Expression/purity: +/- indicates poor, + indicates moderate, and ++ indicates good. Relative neutralization potency: bold indicates good (within 2 fold) and italics indicates average/poor (3-10 fold worse).

In view of these data, the target product was prepared with two mRNAs encoding an intact antibody (CD4 binding site specific) as IgG in combination with 2 mRNAs encoding scFv-Fcs. The data in Figure 18 demonstrates the significant neutralization capabilities of the scFv, in particular PGDM-1400_Var7 (SEQ ID NO.185) and PGT-121_Var7b (SEQ ID NO. 184). Expressing scFV-FCs and IgG HIV bNAbs in mRNA format in vivo

mRNA encoding scFV-FC’s and IgGs were administered to mice in 3 separate experiments and expression and activity levels were measured (data represented in Figures 19- 21). The data demonstrate that each ofthe tested proteins were expressed and functional. The data of Figures 19-21 demonstrate that the expressed antibodies can generate neutralizing titers When the concentration was increased 3-fold (to 0.5mg/kg), variant 7 scFV-FCs of PGDM1400 and PGT121 and IgGs showed strong ID50 neutralization titers (data not shown). At 0.25mg/kg these Var7 titers remained strong (data not shown). The presence of two glycans (PGT-121 increased titers about 2 fold.

A combination of 2scFV’s and one IgG was produced and found to generate enhanced neutralizing titers. Further Optimization of scFv for maximal expression, thermostability and half-life:

12 disulfide variant constructs were generated: five PGDM1400, two PGT121, and five 10-1074 to test for altered stability. Three PDGM1400 disulfide constructs expressed at greater than appreciable levels in vitro (>50 mg/L) and two constructs (DS3 and DS4) showed added stability. The remaining constructs likely displayed a non-native/misfolded disulfide formation.

Eight isoelectric variants were produced to be tested for increased half-life. The eight constructs included two each of PGDM-1400, two each of 10-1074 and four of PGT-121. Of the eight, four isolectric variants (two for PGT-121 and two for PGDM-1400) expressed at levels comparable to their parental IgGs and showed favorable thermostability (relative to variant 1)

Six VH3-23 CDR grafts (stable framework antibody, VH3-23, and HIV bnAb) were attempted, two each for PGDM-1400, two for PGT-121 and two for 10-1074. Three were shown to have expression (>50mg/L) for further characterization and two had increased thermosatability. The CDRs were grafted onto the framework.

There is evidence in the literature that antibody clearance can be influenced by electrostatics. Ustekinumab has a median terminal half-life of 22 days, whereas briakinumab has a terminal half-life of only 8–9 days (Schoch et al.2015, PNAS, vol.112 no.19 p.5997). A second approach looks at stability of the VH3-23 germline. A study of a general approach to antibody thermostabilization successfully grafted ~10 Abs onto the VH3-23 germline and saw an increase in thermostability (McConnell et al. MAbs.2014 Sep-Oct; 6(5): 1274– 1282.). The VH3-23 germline was used for synthetic libraries due to stability and ease of pairing with many differing light chains.

A non-human primate study combination study will be conducted on the following combination: N6 (IgG), PDGM_1400 (scFV-FC_var7), and PGT_121 (scFV-FC_var7b). Table 19. Evaluation of HIV single chain C serum kinetics in vivo combination study.5 ug of individual antibody Gr1-5 and 10ug total for combination.

In order to design constructs having enhanced thermostability, improved hydrophobic packing, optimized VH-VL interface packing, optimized surface residues, and engineered disulfides further designs of scFV-FCs onto (G4S)₄ were generated.

A summary of the effects of modifying the scFv (in comparison with IgG) on thermostability, neutralization, in vitro expression and aggregation is shown in Table 20. Table 20:

The optimized constructs were tested. for increased thermostability, enhanced neutralization, expression and/or half-life. One modification involved CDR grafting onto a stable VH3-23 framework and engineering disulfides. Table 21.

Glycan analysis was performed on IgGs expressed from primary hepatocytes or primary muscle cells. Antibodies were expressed in 3 different cell lines: HEK, primary mouse muscle and liver cell line. Glycans were collected by enzyme digestion. Analysis was by LC-MS. The results are shown in Table 21. No significant differences were observed in the total glycan content. HEK is IgG expressed in Human embryonic kidney cells (HEK293), MsPMC was IgG expressed in primary mouse muscle cells (ABM, T4990), and MsPLC was IgG expressed in primary mouse liver cells (ABM, T2010).

A combination of 2 scFv’s and one IgG was produced and tested for expression and activity in vivo. The combination was found to generate enhanced neutralizing titers. The data is shown in Table 22 and compared with the activity of each component alone. The evaluation of HIV scFv serum kinetics in vivo involved the administration of 5 ug of individual antibody (Groups 1-5) and 10ug total for the combined antibodies. The data presented in the table for pre-bleeds and 24 hour is average over 5 animals. Day 5 and day 8 are pooled serum. Table 22

Example 19: BMGF Single Chain and IgG Expression PGDM1400 scFV-FC Expression

scFV-FCs of PGDM1400 (0.5 mpk) were administered to Balb/C mice by IV.

Expression levels PGDM1400 scFV-FCs were measured by ELISA to total human IgG in mouse serum at 24 hours and 5 days. For some single chain constructs these values were independently measured twice (noted in tables). Table 23. Expression levels of Var7 with differing orientations and signal peptides were measured. Italicized text indicates the variation selected for further in vivo studies. Bold text (top three rows) indicates VH-VL orientation and plain text (bottom two rows) indicates VL- VH orientation.

The scFv having a VL-VH orientation was expressed at higher levels at 24 hours and five days (40-60 ug/mL at 24 hours and 30-40 ug/mL at five days) than the scFv having a VH-VL orientation. The best expression under the testing conditions was from the VL-VH IgKappa8 signal peptide. For scFV-FCs tested on two independent days, both values are given.

Two disulfide variants and a point mutant versions were designed and tested for expression levels. The data is shown in Table 24. Disulfide variants 3 and 4 and point mutant V10 were designed for increased thermostability and/or half life (30-40 ug/mL at 24 hours and 30-50 ug/mL at five days).

Table 24.

All three constructs demonstrated high expression levels and were selected for further in vivo studies. PGT121 scFV-FC Expression

scFV-FCs of PGT121 (0.5 mpk) were administered to Balb/C mice by IV. Expression levels PGT121 scFV-FCs were measured by ELISA to total human IgG in mouse serum at 24 hours and 5 days. The data is shown in Tables 25 and 26. Table 25. Expression levels of Var7A/B with differing orientations and signal peptides were measured. Italicized text indicates the variation selected for further in vivo studies. Bold and italicized text indicates VH-VL orientation and plain text indicates VL-VH orientation.

The best VL-VH and VH-VL orientations were expressed in the range 100-200 ug/mL at 24 hours and 30-60 ug/mL at 5 days. The best expression observed was from the VL-VH IgKappa8 signal peptide.

Table 26. Expression levels of Var7A/B with differing orientations and signal peptides. Bold and italicized text indicates VH-VL orientation and plain text indicates VL-VH orientation.

Electrostatic and point mutants were designed to increase thermostability and/or receptor recycling. These different constructs (0.5 mpk) were administered to Balb/C mice by IV. Expression levels of total human IgG were measured by ELISA at 24 hours and 5 days. The data is shown in Figure 22. VL-VH for PI1, Var13, and PI2 express the range 150-200 ug/mL at 24 hours and 30-40 ug/mL at five days. For scFV-FCs tested on two independent days, both values are given.

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.” The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e.,“one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list,“or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of” or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as

“comprising,”“including,”“carrying,”“having,”“containing,”“involving,”“holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of” and“consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g.,“comprising”) are also contemplated, in alternative embodiments, as“consisting of” and“consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes“a composition comprising A and B”, the disclosure also contemplates the alternative embodiments“a composition consisting of A and B” and“a composition consisting essentially of A and B”.

Claims

1. A composition, comprising:

a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or antigen-binding fragment thereof, wherein the composition when administered to a subject in need thereof as a single intravenous dose is sufficient to

(i) maintain antibody levels at a normal physiological level or a supraphysiological level for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours post-administration, and/or

(ii) maintain antibody levels at 50% or more of the normal antibody level for at least 24 hours, at least 48 hours, at least 72 hours, or at least 96 hours post-administration. 2. The pharmaceutical composition of claim 1, further comprising a delivery agent. 3. A pharmaceutical composition, comprising:

a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or antigen-binding fragment thereof and a lipid nanocarrier. 4. The pharmaceutical composition of any one of claims 1-3, wherein the lipid nanoparticle or the delivery agent comprises Compound 18, a salt or a stereoisomer thereof, or any combination thereof. 5. The pharmaceutical composition of any one of claims 1-3, wherein the lipid nanoparticle or the delivery agent comprises Compound 25, salts and stereoisomers thereof, and any combination thereof. 6. The pharmaceutical composition of any one of claims 1-5, wherein the lipid nanoparticle or the delivery agent further comprises a PEG lipid. 7. The pharmaceutical composition of claim 6, wherein the PEG lipid has the Formula (VII):

or a salt thereof, wherein:

R³ is–OR^O;

R^O is hydrogen, optionally substituted alkyl, or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

L¹ is optionally substituted C_1-10 alkylene, wherein at least one methylene of the optionally substituted C_1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene,–O–,–N(R^N)–,–S–,–C(O)–,–C(O)N(R^N)–, –NR^NC(O)–,–C(O)O–,–OC(O)–,–OC(O)O–,–OC(O)N(R^N)–,–NR^NC(O)O–, or –NR^NC(O)N(R^N)–;

m is 0, 1,

2,

3,

4,

5,

6,

7, 8, 9, or 10;

A is of the formula:

or ;

each instance of L² is independently a bond or optionally substituted C_1-6 alkylene, wherein one methylene unit of the optionally substituted C_1-6 alkylene is optionally replaced with–O–,–N(R^N)–,–S–,–C(O)–,–C(O)N(R^N)–,–NR^NC(O)–,–C(O)O–,–OC(O)–, –OC(O)O–,–OC(O)N(R^N)–,–NR^NC(O)O–, or–NR^NC(O)N(R^N)–;

each instance of R² is independently optionally substituted C_1-30 alkyl, optionally substituted C_1-30 alkenyl, or optionally substituted C_1-30 alkynyl; optionally wherein one or more methylene units of R² are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene,–N(R^N)–,–O–,–S–,–C(O)–,–C(O)N(R^N)–, –NR^NC(O)–,–NR^NC(O)N(R^N)–,–C(O)O–,–OC(O)–,–OC(O)O–,–OC(O)N(R^N)–, –NR^NC(O)O–,–C(O)S–,–SC(O)–,–C(=NR^N)–,–C(=NR^N)N(R^N)–,–NR^NC(=NR^N)–, –NR^NC(=NR^N)N(R^N)–,–C(S)–,–C(S)N(R^N)–,–NR^NC(S)–,–NR^NC(S)N(R^N)–,–S(O)–, –OS(O)–,–S(O)O–,–OS(O)O–,–OS(O)₂–,–S(O)₂O–,–OS(O)₂O–,–N(R^N)S(O)–, –S(O)N(R^N)–,–N(R^N)S(O)N(R^N)–,–OS(O)N(R^N)–,–N(R^N)S(O)O–,–S(O)₂–,

–N(R^N)S(O)₂–,–S(O)₂N(R^N)–,–N(R^N)S(O)₂N(R^N)–,–OS(O)₂N(R^N)–, or–N(R^N)S(O)₂O–; each instance of R^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.

8. The pharmaceutical composition of claim 7, wherein the PEG lipid has the Formula (VIII):

(VIII),

or a salts thereof, wherein:

R³ is–OR^O;

R^O is hydrogen, optionally substituted alkyl or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

R⁵ is optionally substituted C_10-40 alkyl, optionally substituted C_10-40 alkenyl, or optionally substituted C_10-40 alkynyl; and optionally one or more methylene groups of R⁵ are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene,–N(R^N)–,–O–,–S–, –C(O)–,–C(O)N(R^N)–,–NR^NC(O)–,–NR^NC(O)N(R^N)–,–C(O)O–,–OC(O)–,–OC(O)O–, –OC(O)N(R^N)–,–NR^NC(O)O–,–C(O)S–,–SC(O)–,–C(=NR^N)–,–C(=NR^N)N(R^N)–, –NR^NC(=NR^N)–,–NR^NC(=NR^N)N(R^N)–,–C(S)–,–C(S)N(R^N)–,–NR^NC(S)–,

–NR^NC(S)N(R^N)–,–S(O)–,–OS(O)–,–S(O)O–,–OS(O)O–,–OS(O)₂–,–S(O)₂O–, –OS(O)₂O–,–N(R^N)S(O)–,–S(O)N(R^N)–,–N(R^N)S(O)N(R^N)–,–OS(O)N(R^N)–, –N(R^N)S(O)O–,–S(O)₂–,–N(R^N)S(O)₂–,–S(O)₂N(R^N)–,–N(R^N)S(O)₂N(R^N)–, –OS(O)₂N(R^N)–, or–N(R^N)S(O)₂O–; and

each instance of R^N is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

9. The pharmaceutical composition of claim 8, wherein the PEG lipid has the formula:

, wherein r is an integer between 1 and 100.

10. The pharmaceutical composition of claim 9, wherein r is 45.

11. The pharmaceutical composition of any one of claims 1-10, wherein the lipid nanoparticle or the delivery agent further comprises a phospholipid.

12. The pharmaceutical composition of any one of claims 1-11, wherein the RNA comprises a microRNA (miR) binding site.

13. The pharmaceutical composition of any one of claims 1-12, wherein the open reading frame is codon-optimized.

14. The pharmaceutical composition of any one of claims 1-13, wherein the RNA polynucleotide comprises at least one chemical modification.

15. The pharmaceutical composition of claim 14, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1- ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine , 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine,), 5-methoxyuridine and 2′-O-methyl uridine.

16. The pharmaceutical composition of any one of claims 1-15, wherein the RNA polynucleotide comprises at least one 5′ terminal cap.

17. The pharmaceutical composition of any one of claims 1-16, wherein the lipid nanoparticle comprises an ionizable cationic lipid, a PEG-modified lipid, a structural lipid and a phospholipid.

18. The pharmaceutical composition of any one of claims 1-17, wherein the antibody is specific for a viral antigen.

19. The pharmaceutical composition of claim 18, wherein the viral antigen is from HIV, Ebola virus, CHIKV or influenza virus.

20. The pharmaceutical composition of any one of claims 1-17, wherein the antibody is specific for a cancer antigen.

21. The pharmaceutical composition of any one of claims 1-17, wherein the antibody is specific for an immune cell antigen.

22. The pharmaceutical composition of any one of claims 1-17, wherein the antibody is a checkpoint inhibitor.

23. The pharmaceutical composition of any one of claims 1-17, wherein the antibody is specific for a cell surface antigen.

24. A composition, comprising:

a ribonucleic acid (RNA) polynucleotide having an open reading frame (ORF) encoding an antibody or single-chain variable fragment (scFv) having specificity for a human immunodeficiency virus (HIV) and a pharmaceutically acceptable carrier or excipient.

25. The composition of claim 24, wherein the scFv comprises variable regions of heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide of about 4 to about 30 amino acids.

26. The composition of claim 24, wherein the scFv comprises variable regions of heavy (VH) and light chains (VL) of immunoglobulins, connected with a linker peptide of about 20 amino acids.

27. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence of any of SEQ ID NOs: 182-198.

28. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 182-198.

29. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 182-198.

30. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 182-198.

31. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence having at least 98% sequence identity to any one of SEQ ID NOs: 182-198.

32. The composition of any one of claims 24-25, wherein the antibody or scFv comprises a polypeptide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 182-198.

33. The composition of any one of claims 24-25, wherein the ORF comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 166-171.

34. The composition of any one of claims 24-25, wherein the RNA comprises a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 174-179.

35. The composition of any one of claims 24-31, wherein the antibody is a full length antibody or an antigen-binding fragment thereof.

36. The composition of any one of claims 24-35, formulated in a lipid nanocarrier.

37. The composition of any one of claims 24-36, wherein the ORF is codon- optimized.

38. The composition of any one of claims 24-37, wherein the RNA polynucleotide comprises at least one chemical modification.

39. The composition of claim 38, wherein the chemical modification is selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′- thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl- pseudouridine, 2-thio-5-aza-uridine , 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5- methyluridine,), 5-methoxyuridine and 2′-O-methyl uridine.

40. The composition of any one of claims 24-39, wherein the RNA polynucleotide comprises at least one 5′ terminal cap.

41. The composition of claim 40, wherein the at least one 5′ terminal cap comprises 7mG(5′)ppp(5′)NlmpNp.

42. The composition of claim 36, wherein the lipid nanoparticle comprises an ionizable amino lipid, a PEG-modified lipid, a sterol and a non-cationic lipid.

43. The composition of claim 42, wherein the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% non-cationic lipid:25-55% sterol:0.5-15% PEG-modified lipid.

44. The composition of any one of claims 24-43, wherein at least 80% or 100% of the uracil in the open reading frame have a chemical modification.

45. The composition of claim 44, wherein the chemical modification is in the 5- position of the uracil.

46. The composition of any one of claims 44-45, wherein the chemical modification is a 5-methoxyuracil.

47. A composition, comprising:

a ribonucleic acid (RNA) polynucleotide having an open reading frame encoding an antibody or single-chain variable fragment (scFv) that specifically binds to human

immunodeficiency virus (HIV), wherein the antibody has two or more of the following characteristics: (a) demonstrates neutralization of HIV, with an ID₅₀ of within 20% of a control neutralization activity, wherein the control is a corresponding protein antibody; (b) is an scFv that demonstrates protection, as measured by decreased plasma viremia relative to baseline prior to administration of the scFv in an animal model of HIV infection when administered either before or after virus challenge; (c) is an scFv having a (G4S)₄ linker; or (d) wherein the antibody or scFv comprises three complementarity determining regions (CDRs) contained within any one of the variable region sequences listed in SEQ ID NO.200- 217 and a pharmaceutically acceptable carrier or excipient.

48. The composition of claim 47, wherein the antibody is a full length antibody or an antigen-binding fragment thereof.

49. A composition, comprising:

three or more ribonucleic acid (RNA) polynucleotides each having an open reading frame (ORF), wherein a first ORF encodes a full length antibody or antigen binding fragment thereof that specifically binds to HIV, wherein a second ORF encodes an scFv that specifically binds to HIV, and wherein a third ORF encodes an scFv that specifically binds to HIV.

50. The composition of claim 49, wherein the first ORF encodes an antibody that specifically interacts with a CD4 binding site.

51. The composition of claim 49, wherein the first ORF encodes an N6 IgG.

52. The composition of claim 49, wherein the composition includes four RNA polynucleotides.

53. The composition of claim 52, wherein two of the four RNA polynucleotides comprise ORFs encoding a light chain and a heavy chain of an N6 IgG.

54. The composition of claim 53, wherein the ORFs encoding a light chain and a heavy chain of an N6 IgG have a nucleic acid sequence of SEQ ID NO.195 and 194 respectively.

55. The composition of claim 53, wherein the ORFs encoding a light chain and a heavy chain of an N6 IgG have a nucleic acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO.171 and 170 respectively.

56. The composition of claim 50, wherein the first ORF encodes a polypeptide having at least 80% sequence identity to SEQ ID NO.186 and/or 187.

57. The composition of claim 50, wherein the first ORF encodes a polypeptide having at least 90% sequence identity to SEQ ID NO.186 and/or 187.

58. The composition of claim 49, wherein second ORF encodes an scFv that specifically interacts with a V1/V2 region of HIV.

59. The composition of claim 49, wherein the second ORF encodes a functional variant of PDGM1400.

60. The composition of claim 59, wherein the second ORF encodes a polypeptide having at least 80% sequence identity to SEQ ID NO.185, 188-192.

61. The composition of claim 59, wherein the second ORF encodes a polypeptide having at least 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO.209, 212-216.

62. The composition of claim 59, wherein the second ORF has at least 80% sequence identity to SEQ ID NO.169.

63. The composition of claim 59, wherein the second ORF has at least 90%, 95%, 985, OR 100% sequence identity to SEQ ID NO.169.

64. The composition of claim 49, wherein the RNA polynucleotide having the second ORF has a nucleic acid sequence of SEQ ID NO.177.

65. The composition of claim 49, wherein the RNA polynucleotide having the second ORF has at least 80%, 85%, 90%, 95%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO.177.

66. The composition of any one of claims 49-65, wherein the scFv that specifically interacts with a V1/V2 region of HIV demonstrates at least 2 times higher neutralization of HIV than an scFv that specifically interacts with a V1/V2 region of HIV and includes a (GGGS)₃ (SEQ ID NO: 199) linker.

67. The composition of claim 49, wherein third ORF encodes an scFv that specifically interacts with a V3 base region.

68. The composition of claim 67, wherein the third ORF encodes a functional variant of PGT-121.

69. The composition of claim 68, wherein the third ORF encodes a polypeptide having at least 80% sequence identity to SEQ ID NO.182-184, 193-194, 197-198.

70. The composition of claim 68, wherein the third ORF encodes a polypeptide having at least 90%, 95%, 98% or 100% sequence identity to SEQ ID NO.182-184, 193-194, 197-198.

71. The composition of claim 68, wherein the third ORF has at least 80% sequence identity to SEQ ID NO.166-168.

72. The composition of claim 68, wherein the third ORF has at least 90%, 95%, 985, or 100% sequence identity to SEQ ID NO.166-168.

73. The composition of claim 68, wherein the RNA polynucleotide having the third ORF has a nucleic acid sequence of SEQ ID NO.174-176.

74. The composition of claim 68, wherein the RNA polynucleotide having the third ORF has at least 80%, 85%, 90%, 95%, or 99% sequence identity to a nucleic acid sequence of SEQ ID NO.174-176.

75. The composition of claim 68, wherein the third ORF encodes a functional variant of a polypeptide having at least 80%, 85%, 90%, 95%, 98% or 100% sequence identity to SEQ ID NO.195-196.

76. The composition of any one of claims 68-75, wherein the scFv that specifically interacts with a V3 region of HIV demonstrates at least 2 times higher neutralization of HIV than an scFv that specifically interacts with a V3 region of HIV and includes a (GGGS)₃ (SEQ ID NO: 199) linker.

77. The composition of any one of claims 49-76, wherein the composition is formulated in a lipid nanoparticle (LNP) comprising an ionizable amino lipid, a structural lipid, a PEG lipid and a non-cationic lipid.

78. The composition of claim 77, wherein each RNA polynucleotide is formulated in a separate LNP.

79. The composition of claim 77, wherein all the RNA polynucleotides are formulated in a separate LNP.

80. A method of treating an HIV infection in a subject, comprising administering to a subject the composition of any one of claims 24-79 in a therapeutically effective amount to treat the HIV infection.

81. The method of claim 80, wherein the subject is a human.

82. The method of claim 80, wherein the method of treating is a method of passively immunizing a mammalian subject against an HIV infection by administering to the subject the composition, wherein the subject is at risk of having or being exposed to an influenza virus infection.

83. A method of treating an HIV infection in a subject, comprising administering to a subject the composition of any one of claims 49-79, wherein each of the RNA polynucleotides in the composition is administered separately to the subject in a

therapeutically effective amount to treat the HIV infection.

84. A method of treating an HIV infection in a subject, comprising administering to a subject the composition of any one of claims 49-79, wherein each of the RNA polynucleotides in the composition is administered together in a single LNP mixture in a therapeutically effective amount to treat the HIV infection.