WO2016201377A1 - Targeted adaptive vaccines - Google Patents

Targeted adaptive vaccines Download PDF

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Publication number
WO2016201377A1
WO2016201377A1 PCT/US2016/037095 US2016037095W WO2016201377A1 WO 2016201377 A1 WO2016201377 A1 WO 2016201377A1 US 2016037095 W US2016037095 W US 2016037095W WO 2016201377 A1 WO2016201377 A1 WO 2016201377A1
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Prior art keywords
polypeptide
mrna
polynucleotides
polynucleotide
pcsk9
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PCT/US2016/037095
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French (fr)
Inventor
Eric YI-CHUN HUANG
Stephen Hoge
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Moderna Therapeutics, Inc.
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Priority to US62/173,783 priority
Application filed by Moderna Therapeutics, Inc. filed Critical Moderna Therapeutics, Inc.
Publication of WO2016201377A1 publication Critical patent/WO2016201377A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0008Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • A61P3/06Antihyperlipidemics
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424Serine endopeptidases (3.4.21)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53DNA (RNA) vaccination
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/545Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55516Proteins; Peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55544Bacterial toxins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55555Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/60Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031Proteins
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/40Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotide molecules encoding targeted adaptive vaccines (TAVs), such as PCSK9 TAVs, TNF alpha TAVs, IL-17A TAVs, or GDF8 TAVs.

Description

TARGETED ADAPTIVE VACCINES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.

62/173,783, filed on June 10, 2015; which is incorporated by reference herein in its entirety. STATEMENT REGARDING SEQUENCE LISTIN

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is

MRNA_012_01WO_ST25.txt. The text file is 1,333 KB, was created on June 10, 2016, and is being submitted electronically via EFS-Web. FIELD OF THE INVENTION

[0003] The present invention relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture, formulation and use of polynucleotides encoding targeted adaptive vaccines (TAVs). BACKGROUND OF THE INVENTION

[0004] Vaccination is an effective way to provide prophylactic protection against infectious diseases, such as influenza, AIDS, hepatotisis virus infection, cholera, malaria and tuberculosis, and many other diseases. Vaccines may also be used to treat diseases, such as cancers. For example, the first cancer treatment vaccine, sipuleucel- T (Provenge®, manufactured by Dendreon) is used for certain men with metastatic prostate cancer. It is designed to stimulate an immune response to prostatic acid phosphatase (PAP), an antigen that is found on most prostate cancer cells. (Kantoff PW, et al, 2010, New England Journal of Medicine, 365, 411-422, which is incorporated herein by reference in its entirety).

[0005] A typical vaccine contains an antigenic target molecule, e.g., an agent that resembles a weakened or dead form of the disease-causing agent, which could be a microorganism, such as bacteria, virus, fungi, parasite and prion, or the toxins or one or more surface proteins (often called antigens) of such a microorganism. The antigenic target molecule may alternatively be an endogenous polypeptide, e.g., an antigenic polypeptide or fragment thereof expressed or cancer cells or associated with a disease. The antigen or agent in the vaccine can stimulate the body's immune system to recognize the agent as a foreign invader, generate antibodies against it, destroy it and develop a memory of it. The vaccine-induced memory enables the immune system to act quickly to protect the body from any of these agents that it later encounters.

[0006] Vaccine production used in the art has several stages, including the generation of antigens, antigen purification and inactivation, and vaccine formulation. First, the antigen is generated through culturing viruses in cell lines, growing bacteria in bioreactors, or producing recombinant proteins derived from viruses and bacteria in cell cultures, yeast or bacteria. Recombinant proteins are then purified and the viruses and bacteria are inactivated before they are formulated with adjuvants in vaccines. It has been a challenge to drastically reduce the time and expense associated with current technologies in vaccine development.

[0007] It is of great interest to develop new approaches for developing new vaccines, not only for infectious agents and cancer but for other therapeutic indications. SUMMARY OF THE INVENTION

[0008] Described herein are compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of polynucleotides encoding targeted adaptive vaccines (TAVs).

[0009] In one embodiment, the present invention comprises an mRNA comprising: (a) a first region comprising a sequence encoding one or more polypeptide (e.g., an immunogenic polypeptide); and, optionally, (b) a second region comprising a sequence encoding one or more immunomodulatory polypeptide, wherein the mRNA comprises a modified nucleobase. In particular embodiments, the mRNA comprises the second region. In particular embodiments, the mRNA further comprises a third region between the first region and the second region, wherein the third region comprises a sequence encoding a spacer or a cleavage site. In certain embodiments, the spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site. In particular embodiments, first region comprises two or more sequences encoding polypeptides, which may comprise the same or different amino acid sequences. In particular embodiments, the first region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more polypeptides. In certain embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site. In particular embodiments, the one or more polypeptide comprises a human or murine polypeptide or fragment or variant thereof. In particular embodiments, the one or more polypeptides is proprotein convertase subtilisin/kexin type 9 (PCSK9), interleukin-17A (IL-17A), tumor necrosis factor alpha (TNF alpha) or growth differentiation factor 8 (GDF8).

[00010] In particular embodiments, the second region of the mRNA comprises two or more sequences encoding immunomodulatory polypeptides, which may be the same or different. In some embodiments, the second region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides.

[00011] In particular embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site. In certain embodiments, the immunomodulatory polypeptide is selected from the group consisting of: GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti-PD1, anti-PDL1/2, anti-OX40/OX40L, anti- GITR/GITRL, and anti-TIM3. In particular embodiments, the one or more immunomodulatory polypeptide is an immune enhancing polypeptide. In particular embodiments, the immune enhancing polypeptide is selected from the group consisting of: mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, and a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid. In particular embodiments, the immune enhancing polypeptide is T cell epitope of M2 protein of H1N1 Puerto Rico/8 or mannose binding protein. In certain embodiments, the mRNA encodes a fusion polypeptide comprising a scaffold polypeptide, the first region, and the second region, wherein the first region and the second region are inserted into the scaffold polypeptide. In certain embodiments, the mRNA further comprises a fourth region comprising a sequence encoding a dendritic cell targeting polypeptide. In particular embodiments, the dendritic cell targeting polypeptide is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In certain embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00012] As used herein, DEC205 refers to CD-205; DC-SIGN refers to Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), DCIR2 refers to dendritic cell inhibitory receptor 2, Dectin-1/2 refers to Dectin 1 (aslo known as C-type lectin domain family 7 member A or CLEC7A) or Dectin 2 (also known as C-type lectin-2), CD80/86 refers to CD80 (also known as B7-1) or CD 86 (also known as B7-2), GM-CSF refers to Granulocyte macrophage colony-stimulating factor; soluble LAG3 refers to CD 223; PD1 refers to programmed cell death protein 1; PDL1/2 refers to programmed cell death-ligand 1/2 (also known as CD 274 or B7 homolog 1 (B7-H1)); GITR refers to glucocorticoid-induced TNFR family related gene; GITRL refers to GITR ligand; and TIM3 refers to T cell immunoglobulin and mucin domain. One of skill in the art will appreciate that CD11c is also known as Integrin, alpha X (complement component 3 receptor 4 subunit) or ITGAX; F4/80- like receptor is also known as FIRE; CIRE is also known as CD209, CD209 antigen- like protein A, and CDSIGN; CD36 is also known as FAT (fatty acid translocase), FAT/CD36, (FAT)/CD36, SCARB3, GP88, glycoprotein IV (gpIV), and glycoprotein IIIb (gpIIIb); OX40 is also known as CD134; OX40L is also known as CD252.

[00013] In a related embodiment, the present invention includes a lipid

nanoparticle comprising any of the aforementioned mRNAs. In particular

embodiments, the lipid nanoparticle further comprises an immunomodulatory agent or moiety, including any of those described above. In particular embodiments, the immunomodulatory agent or moiety enhances an immune response. In particular embodiments, the lipid nanoparticle further comprising a dendritic cell targeting agent or moiety. In certain embodiments, the dendritic cell targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In certain embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00014] In a further related embodiment, the present invention includes a pharmaceutical composition comprising any of the aforementioned mRNAs or lipid nanoparticles, and a pharmaceutically acceptable carrier, diluent or excipient. In particular embodiments, the pharmaceutical composition further comprises an immunomodulatory agent or moiety. In particular embodiments, the

immunomodulatory agent or moiety enhances the immune response. In some embodiments, the pharmaceutical composition comprises a dendritic cell targeting agent or moiety. In certain embodiments, the dendritic cell targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In particular embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00015] In another related embodiments, the present invention includes a method of inducing an immune response to a PCSK9 polypeptide in a cell, tissue or organism, comprising contacting said cell, tissue or organism with any of the aforementioned mRNAs, lipid nanoparticles, or pharmaceutical compositions. In particular embodiment, the PCSK9 polypeptide is endogenous to the cell, tissue or organism.

[00016] In a further related embodiment, the present invention provides a method of treating or preventing hypercholesterolaemia or atherosclerosis in a subject in need thereof, comprising providing to the subject an effective amount of any of the aforementioned pharmaceutical compositions. In certain embodiments, the subject is treated for familial hypercholesterolaemia.

[00017] Particular embodiments are directed to mRNA comprising: (a) a first region comprising a sequence encoding one or more of an antigen polypeptide; and (b) a second region comprising a sequence encoding one or more immunomodulatory polypeptide, wherein the mRNA comprises a modified nucleobase. In some embodiments, the mRNA further comprises a third region between the first region and the second region, wherein the third region comprises a sequence encoding a spacer or a cleavage site. In certain embodiments, the spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site.

[00018] In particular embodiments, the first region comprises two or more sequences encoding antigen polypeptides. In some embodiments, the two or more antigen polypeptides comprise the same amino acid sequences. In particular embodiments, the two or more antigen polypeptides comprise different amino acid sequences. In certain embodiments, the first region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more antigen polypeptides. In particular embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.

[00019] In particular embodiments, the second region comprises two or more sequences encoding immunomodulatory polypeptides. In some embodiments, the two or more immunomodulatory polypeptides are the same. In certain embodiments, the two or more immunomodulatory polypeptides are different. In particular

embodiments, the second region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides. In some embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.

[00020] In some embodiments, the immunomodulatory polypeptide is selected from the group consisting of: GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti-PD1, anti-PDL1/2, anti-OX40/OX40L, anti-GITR/GITRL, and anti-TIM3. In certain embodiments, the one or more immunomodulatory polypeptide is an immune enhancing polypeptide. In particular embodiments, the immune enhancing polypeptide is selected from the group consisting of: mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, and a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid. In some embodiments, the immune enhancing polypeptide is T cell epitope of M2 protein of H1N1 Puerto Rico/8 or mannose binding protein.

[00021] In various embodiments, the mRNA encodes a fusion polypeptide comprising a scaffold polypeptide, the first region, and the second region, wherein the first region and the second region are inserted into the scaffold polypeptide. In particular embodiments, the mRNA further comprises a fourth region comprising a sequence encoding a dendritic cell targeting polypeptide. In some embodiments, the dendritic cell targeting polypeptide is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers.

[00022] In some embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36. In particular embodiments, the antigen polypeptide is a PCSK9 polypeptide. In some embodiments, the one or more PCSK9 polypeptide comprises a human or murine PCSK9 polypeptide or fragment or variant thereof. In certain embodiments, the one or more PCSK9 polypeptide comprises a PCSK9 polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables or Figure 14.

[00023] In some embodiments, the antigen polypeptide is a PCSK9 polypeptide. In particular embodiments, the one or more PCSK9 polypeptide comprises a PCSK9 epitope comprising an amino acid sequence selected from the group consisting of: FSAKDVINEAWFPED; KDVINEAWFPEDQRV; DVINMAWFPEDQQVL; and NMAWFPEDQQVLTPN, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In certain embodiments, the one or more polypeptide comprises a PCSK9 polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 7-9 or Figure 14. In various

embodiments, the one or more PCSK9 polypeptide comprises a PCSK9 epitope comprising an amino acid sequence selected from the group consisting of:

FSAKDVINEAWFPED; KDVINEAWFPEDQRV; DVINMAWFPEDQQVL; and NMAWFPEDQQVLTPN, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In particular embodiments, the PCSK9 epitope comprises an amino acid sequence of any one of the peptides shown in Figure 14, e.g., any one of Peptides 10-15, 21-22, 24-28, 33-36, 62-63, 70-74, 85-92, 94, 106- 109, 126, 129-133, 144-152, 157-164, 174-175, 177, 197-198, 207-209, 220, 222-224, 226, 230-236, and 243-249 and 265-268, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In particular embodiments, the mRNA encodes a fusion protein comprising the one or more PCSK9 polypepide or fragment or variant thereof and an N-terminal IM selected from the group consisting of Tet, PR8M2x3, and TpDx3.

[00024] In some embodiments, the antigen polypeptide is a tumor necrosis factor alpha (TNF alpha) polypeptide. In certain embodiments, the one or more TNF alpha polypeptide comprises a human or murine TNF alpha polypeptide or fragment or variant thereof. In particular embodiments, the one or more TNF alpha polypeptide comprises a TNF alpha polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 3 or 4, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In certain embodiments, mRNA encodes a fusion protein comprising the one or more TNF alpha polypepide or fragment or variant thereof and a C-terminal IM selected from the group consisting of PR8M2x3, HA307-318x3, and MBP.

[00025] In particular embodiments, the antigen polypeptide is an interleukin-17A (IL-17A) polypeptide. In some embodiments, one or more IL-17A polypeptide comprises a human or murine IL-17A polypeptide or fragment or variant thereof. In some embodiments, the one or more IL-17A polypeptide comprises a IL-17A polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 5 or 6, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In certain embodiments, mRNA encodes a fusion protein comprising the at least one IL-17A polypepide or fragment or variant thereof and a C-terminal IM selected from the group consisting of PR8M2x3, STF2D, HA307-318x3, and MBP.

[00026] In particular embodiments, the antigen polypeptide is a growth

differentiation factor 8 (GDF8) polypeptide. In some embodiments, the one or more GDF8 polypeptide comprises a human or murine GDF8 polypeptide or fragment or variant thereof. In particular embodiments, the one or more GDF8 polypeptide comprises a GDF8 polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 7 or 8, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity. In various embodiments, the mRNA encodes a fusion protein comprising the one or more GDF8 polypepide or fragment or variant thereof and a C-terminal IM comprising tet.

[00027] Some embodiments are directed to lipid nanoparticle comprising the mRNA described herein. In some embodiments, the lipid nanoparticle further comprising an immunomodulatory agent or moiety. In particular embodiments, the immunomodulatory agent or moiety enhances an immune response. In certain embodiments, the lipid nanoparticle comprises a dendritic cell targeting agent or moiety. In some embodiments, the dendritic cell targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In certain embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00028] Some embodiments are directed to a pharmaceutical composition comprising an mRNA or a lipid nanoparticle described herein, and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the composition comprises an immunomodulatory agent or moiety. In certain embodiments, the immunomodulatory agent or moiety enhances the immune response. In certain embodiments, the composition comprises a dendritic cell targeting agent or moiety. In some embodiments, the dendritic cell targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In some embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00029] Particular embodiments are directed to a method of inducing an immune response to an antigen polypeptide in a cell, tissue or organism, comprising contacting said cell, tissue or organism with mRNAs, lipid nanoparticles, or pharmaceutical compositions of the present invention. In some embodiments, the antigen polypeptide is endogenous to the cell, tissue or organism. In certain embodiments, the antigen is a PCSK9 polypeptide. In particular embodiments, the antigen is a TNF alpha polypeptide. In some embodiments, the antigen is an IL-17A polypeptide. In certain embodiments, the antigen is a GDF8 polypeptide.

[00030] Certain embodiments are directed to a method of treating or preventing hypercholesterolaemia or atherosclerosis in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition wherein the at least one polypeptide is a PCSK9 polypeptide. In some embodiments, the hypercholesterolaemia is a familial hypercholesterolaemia.

[00031] Some embodiments are directed to a method of treating or preventing an inflammatory disease, an autoimmune disease, or a cancer in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition described herein, wherein the at least one polypeptide is a TNF alpha polypeptide. [00032] Some embodiments are directed to a method of treating or preventing an inflammatory disease, an autoimmune disease, or a cancer in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition described herein, wherein the at least one polypeptide is an IL-17A polypeptide.

[00033] Particular embodiments are directed to a method of treating or preventing a disease associated with muscle loss in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition described herein, wherein the at least one polypeptide is a GDF8 polypeptide.

[00034] In particular embodiments, a polynucleotide of the present invention comprises a PCSK9 polynucleotide sequence or encodes a polypeptide sequence set forth in any of Tables 12, 13, 17, 18 or 19, or Figure 14, or a fragment or variant thereof. In particular embodiments, a polynucleotide of the present invention comprises an IL-17A polynucleotide sequence or encodes a polypeptide sequence set forth in any of Tables 5, 6, 18 or 19, or a fragment or variant thereof. In particular embodiments, a polynucleotide of the present invention comprises a TNFalpha polynucleotide sequence or encodes a polypeptide sequence set forth in any of Tables 3, 4, 18 or 19, or a fragment or variant thereof. In particular embodiments, a polynucleotide of the present invention comprises a GDF8 polynucleotide sequence or encodes a polypeptide sequence set forth in any of Tables 7, 8 or 20, or a fragment or variant thereof.

[00035] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[00036] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[00037] FIG.1 comprises Figure 1A and Figure 1B showing a schematic of an IVT polynucleotide construct. Figure 1A is a schematic of an IVT polynucleotide construct taught in commonly owned US Patent No.8,999,380, the contents of which are incorporated herein by reference. Figure 1B is a schematic of an IVT polynucleotide construct.

[00038] FIG.2 is a schematic of a series of chimeric polynucleotides of the present invention which may be used as mRNA. A and B represent independent regions of linked nucleosides, and C represents an optional region of linked nucleosides.

[00039] FIG.3 is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications and showing regions analogous to those regions of an mRNA polynucleotide.

[00040] FIG.4 is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications based on the formula: 5 ' [An]x_Ll- [B0]y_L2-[Cp]z-L33 ' (Formula I), where each of A and B independently comprise a region of linked nucleosides; C is an optional region of linked nucleosides; at least one of regions A, B, or C is positionally modified, wherein said positionally modified region comprises at least two chemically modified nucleosides of one or more of the same nucleoside type of adenosine, thymidine, guanosine, cytidine, or uridine, and wherein at least two of the chemical modifications of nucleosides of the same type are different chemical modifications; n, o and p are independenty an integer between 15- 1000; x and y are independently 1-20; z is 0-5; LI and L2 are independently optional linker moieties, said linker moieties being either nucleic acid based or non-nucleic acid based; and L3 is an optional conjugate or an optional linker moiety, said linker moiety being either nucleic acid based or non-nucleic acid based.

[00041] FIG.5 is a is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications based on Formula I and further illustrating a blocked or structured 3’ terminus.

[00042] FIG.6 is a schematic of a circular polynucleotide construct of the present invention.

[00043] FIG.7 is a schematic of a circular polynucleotide construct of the present invention.

[00044] FIG.8 is a schematic of a circular polynucleotide construct of the present invention comprising at least one spacer region.

[00045] FIG.9 is a PRIOR ART figure showing certain gain of function mutations. The figure is from the publication by Fasano, et al., 2009; Atherosclerosis; Mar; 203(1):166-71. [00046] FIG.10 is a schematic showing the components of one illustrative embodiment of targeted adaptive vaccines of the present invention.

[00047] FIGS.11A and 11B are graphs showing anti-human PCSK9 antibody levels (FIG.11A) or anti-mouse antibody levels (FIG.11B) in mouse serum at the indicated time points following administration of an mRNA encoding a human PCSK9 TAV, an mRNA encoding a mouse PCSK9 TAV, or PBS. For FIG.11A, the top line at day 40 corresponds to human (PCSK9) TAV, and the bottom line corresponds to the PBS control. For FIG.11B, the top line at day 40 corresponds to human (PCSK9) TAV, the middle line corresponds to the murine (PCSK9) control, and the bottom line corresponds to the PBS control.

[00048] FIG.12 is a graph showing endogenous PCSK9 levels in mouse serum at the indicated time points following administration of an mRNA encoding a human PCSK9 TAV, an mRNA encoding a mouse PCSK9 TAV, or PBS. At day 40, the top line corresponds to the PBS control, the middle line corresponds to the murine (PCSK9) control, and the bottom line corresponds to the human (PCSK9) TAV.

[00049] FIGS.13A and 13B are graphs showing total lipid (LDL/VLDL/HDL) levels in mouse serum at the indicated time points following administration of an mRNA encoding a TAV comprising a human PCSK9 antigenic polypeptide, an mRNA encoding a TAV comprising a mouse PCSK9 antigen polypeptide, or PBS (FIG.13A) and an internal group comparison of total serum lipid levels normalized to Day -12 to Day 0 average for each group (FIG.13B). For FIG.13A, the top line at day 40 corresponds to the murine (PCSK9) control, the middle line corresponds to the PBS control, and the bottom line corresponds to human (PCSK9) TAV.

[00050] FIG.14 provides the identification number and amino acid sequences of the overlapping human PCSK9 and mouse PCSK9 peptides used for mapping immunogenic epitopes.

[00051] FIGS.15A-15I are graphs showing the concentration of anti-PCSK9 antibodies (µg/ml) in mouse serum before, during and after treatment regimens with PBS, PCSK9 polypeptide, or mRNA at day 0, day 7, day 14, and day 28. FIG.15A shows the serum concentrations of anti-PCSK9 antibodies taken from PBS-injected control mice. FIG.15B shows anti-PCSK9 antibody concentrations measured in mice administered with recombinant wild-type mouse PCSK9 polypeptide. FIGS.15C-15F show the concentrations of anti-PCSK9 antibodies in mouse serum during and after treatment with mRNA encoding PCSK9 fused with an N-terminal (top panel) or a C- terminal (bottom panel) immunogenicity enhancing polypeptide (IM; also referred to herein as an immune enhancing polypeptide, immunopotentiation motifs or immunogenicity enhancing motif or polypeptide) fusion polypeptides. The mRNA encoding PCSK9-IM fusion polypeptides included PCSK9 and either Tetanus (nTetanus and cTetanus; FIG.15C), T cell epitope of M2 protein of H1N1 Puerto Rico/8 (nPR8M2 and cPR8M2; FIG.15D), mannose binding protein (nMBP and cMBP; FIG.15E), three copies of universal T helper epitope (nUnivThEpitopeX3 and cUnivThEpitopeX3; FIG.15F), chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid (nTpD and cTpD; FIG.15G), three copies of T cell epitopes from Influenza HA antigen at the C-terminal (cHA307-318x3; FIG. 15H), or C-terminal flagellin derived immunogen (cSTF2d; FIG.15I).

[00052] FIGS.16A-16J are graphs showing the concentration of anti-TNF alpha antibodies (µg/ml) in mice before, during and after treatment regimens with PBS, TNF alpha polypeptide, or mRNA at day 0, day 7, day 14, and day 28. FIG.16A shows the serum concentrations of anti-TNF alpha antibodies taken from PBS- injected control mice. FIG.16 B shows anti-TNF alpha antibody concentrations measured in mice administered with mRNA encoding wild-type mouse TNF-alpha (muTNFa). FIG.16C shows anti-TNF alpha antibody concentrations measured in mice administered with recombinant wild-type mouse TNF alpha polypeptide. FIGS. 16D-16J show the concentrations of anti-TNF alpha antibodies in miceduring and after treatment with mRNA encoding fusion polypeptides of mouse TNF alpha with an N-terminal (top panel) or a C-terminal (bottom panel) IM. The mRNA encoding TNF alpha-IM fusion polypeptides included TNF alpha and either Tetanus (nTetanus and cTetanus; FIG.16D), T cell epitope of M2 protein of H1N1 Puerto Rico/8 (nPR8M2x3 and cPR8M2x3; FIG.16E), mannose binding protein (FIG. nMBP and cMBP; 16F), three copies of T cell epitopes from Influenza HA antigen (nHA307- 318x3 and cHA307-318x3; FIG.16G), chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid (nTpD and cTpD; FIG.16H), three copies of universal T helper epitope (nUnivThEpitopeX3 and cUnivThEpitopeX3; FIG.16I), or C-terminal flagellin derived immunogen (nSTF2d and cSTF2d; FIG.16J).

[00053] FIGS.17A-17J are graphs showing the concentration of anti-IL-17A antibodies (µg/ml) in mice before, during and after treatment regimens with PBS, IL- 17A polypeptide, or mRNA at day 0, day 7, day 14, and day 28. FIG.17A shows the serum concentrations of anti-IL-17A antibodies taken from PBS-injected control mice. FIG.17 B shows anti- IL-17A antibody concentrations measured in mice administered with mRNA encoding wild-type mouse IL-17A. FIG.17C shows anti- IL-17A antibody concentrations measured in mice administered with recombinant wild-type mouse IL-17A polypeptide. FIGS.17D-17E show the concentrations of anti- IL-17A antibodies from mice treated with mRNA encoding fusion polypeptides of mouse IL-17A with an N-terminal (top panel) or a C-terminal (bottom panel) IM. The mRNA encoding IL-17A-IM fusion polypeptides included IL-17A and three copies of T cell epitopes from Influenza HA antigen (nHA307-318x3 and cHA307- 318; FIG.17D) and T cell epitope of M2 protein of H1N1 Puerto Rico/8 (nPR8M2x3 and cPR8M2x3; FIG.17E). Additional mRNAs encoding IL-17A fusion proteins with C-terminals were also tested, which included C-terminal mannose binding protein (cMBP; FIG.17F), C-terminal flagellin derived immunogen (cSTF2d; FIG. 17G), C-terminal tetanus (cTetanus; FIG.17H), three copies of chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid at the C-terminal (cTpD; FIG.17I), and three copies of universal T helper epitope at the C-terminal (cUnivThEpitopeX3; FIG.17J).

[00054] FIGS.18A-18J shows the relative strength of the antibody as measured by relative strength (titer/100) in mice of the antibody response at day 0, day 14, and day 28 before, during, and after treatment regimens with PBS, GDF8 polypeptide, and mRNA encoding GDF8 fusion polypeptides. FIG.18A shows the relative antibody strength of anti-GDF8 antibodies taken from PBS-injected control mice. FIGS.18B- 18J shows the relative antibody strength of anti-GDF8 antibody from mice treated with multiple (top panels) or single (bottom panels) administrations on treatment days. FIG.18B shows the relative antibody strength of anti-GDF8 from mice treated with mRNA encoding mouse wild-type GDF8. FIG.18C shows the relative antibody strength of anti-GDF8 antibody from mice treated with recombinant mouse wild-type GDF8 polypeptide. FIGS.18D-18J show the relative strength of anti-GDF8 antibodies from mice treated with mRNA encoding fusion polypeptides of mouse GDF8with C-terminal or N-terminal IMs. IMs include C-terminal flagellin derived immunogen (FIG.18D), N-terminal flagellin derived immunogen (FIG.18E), N- terminal tetanus epitope (FIG.18F), C-terminal mannose binding protein (FIG.18G), N-terminal mannose binding protein (FIG.18H), N-terminal Toll like receptor 2 (FIG. 18I), and N-terminal T cell epitope of M2 protein of H1N1 Puerto Rico/8 (FIG.18J). DETAILED DESCRIPTION

[00055] The present invention is directed to polynucleotides encoding targeted adaptive vaccines (TAVs). The present invention provides compositions, methods, devices and kits that may rapidly provide vaccines for prophylactic protection and treatment by providing an“antigen-like character” (e.g., via chemical or structural modification of the antigen or a polynucleotide encoding the antigen) to a normally non-antigenic molecule, for example an endogenous protein or fragment thereof. The TAVs may also increase or boost the immune response of antigenic molecules, whether endogenous or not, and may result in the production of antibodies against undesirable or pathological, endogenous“self” proteins, including, for example, overexpressed endogenous PSCK9 as well as gain-of-function PCSK9 mutants.

Certain embodiments are directed to polynucleotides encoding TAVs for endogenous proteins, e.g., proprotein convertase subtilisn kexin type 9 (PCSK9), tumor necrosis factor alpha (TNF alpha), interleukin-17A (IL-17A), and Growth/differentiation factor 8 (GDF8). Particular embodiments are directed to polynucleotides encoding PCSK9 targeted adaptive vaccines (TAVs), e.g., mRNAs encoding PCSK9 TAVs.

[00056] Methods are also provided to reverse or turn off the immune response at the time at which the desired outcome is achieved and the TAV effect is no longer desired.

[00057] To this end, TAVs may be utilized to treat or prevent a variety of indications, including but not limited to those associated with overexpressed PCSK9 or PCSK9 gain-of-function mutations, such as hypercholesterolaemia, e.g., FH, and atherosclerosis; diseases or disorders associated with elevated IL-17A levels or activity, such as diseases associated with inflammation, autoimmune diseases, and cancer; diseases or disorders associated with elevated TNF alpha levels or activity, such as diseases associated with inflammation, autoimmune disease, or cancer; and diseases or conditions that may be treated by reducing endogenous GDF8, such as muscular dystrophy.

[00058] It is of great interest in the fields of therapeutics, diagnostics, reagents and 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 that 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.

[00059] In one embodiment, the present invention utilizes mRNA technology to induce auto-antibodies against, for example an endogenous protein like PCSK9, by immunizing patients who have high serum cholesterol and/or who are at risk of developing coronary heart disease with an mRNA encoding a TAV comprising a PCSK polypeptide, or a fragment or variant thereof.

[00060] In particular embodiments, the present invention provides compositions comprising polynucleotides encoding one or more targeted adaptive vaccines (TAVs). In certain embodiments, the encoded TAVs comprise: (a) an antigen; and, optionally, (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. In certain embodiments, the encoded TAVs comprise: (a) an antigen; and (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. In particular embodiments, the antigen and the dendritic cell targeting agent or moiety and/or immunomodulatory agent or moiety are encoded by the same polynucleotide. In particular embodiments, the antigen is a PCSK9 polypeptide or a fragment or variant thereof.

[00061] In certain embodiments, the present invention provides polynucleotides, e.g., mRNAs, that encode: (a) an antigen; and, optionally, (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. In certain

embodiments, the polynucleotides encode: (a) an antigen; and (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. These polynucleotides or mRNAs may be referred to herein as a TAV polynucleotides or TAV mRNAs, respectively, although it is understood that TAV mRNAs are TAV polynucleotides. TAV polynucleotides and TAV mRNAs may also be referred to herein collectively as TAV compositions. In particular embodiments, the TAV polynucleotide, e.g., mRNA, encodes the antigen and the immunomodulatory agent or moiety. In particular embodiments, the TAV polynucleotides comprise one or more chemically modified nucleosides. mRNAs comprising one or more chemically modified nucleoside are referred to herein as modified mRNAs or mmRNAs. In particular embodiments, the antigen is a PCSK9 polypeptide or a fragment or variant thereof.

[00062] In particular embodiments, the TAV compositions may be formulated in any suitable delivery formulation or in simple saline. In certain embodiments, the TAV compositions, e.g., TAV mRNAs, are encapsulated in a microparticle or nanoparticle, e.g., a lipid nanoparticle (LNP). In particular embodiments, the TAV composition or the LNP comprising a TAV composition is present in a

pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients. In particular embodiments, the pharmaceutical composition comprises an adjuvant.

[00063] In certain embodiments, the TAV compositions are designed to encode an endogenous human PCSK9 protein or an endogenous mouse PCSK9 protein, or a homolog, ortholog, fragment or variant of either. In particular embodiments, a TAV composition of the invention encodes one or more PCSK9 epitopes, e.g., antigenic epitopes. In particular embodiments, the TAV compositions are designed to encode an endogenous human TNF alpha protein or an endogenous mouse TNF alpha protein, or a homolog, ortholog, fragment or variant of either. In particular embodiments, a TAV composition of the invention encodes one or more TNF alpha epitopes, e.g., antigenic epitopes. In some embodiments, the TAV compositions are designed to encode an endogenous human IL-17A protein or an endogenous mouse IL-17A protein, or a homolog, ortholog, fragment or variant of either. In particular embodiments, a TAV composition of the invention encodes one or more IL-17A epitopes, e.g., antigenic epitopes. In various embodiments, the TAV compositions are designed to encode an endogenous human GDF8 protein or an endogenous mouse GDF8 protein, or a homolog, ortholog, fragment or variant of either. In particular embodiments, a TAV composition of the invention encodes one or more GDF8 epitopes, e.g., antigenic epitopes. In exemplary embodiments, the TAV compositions of the invention are polynucleotides. In some embodiments, the polynucleotide is an mRNA encoding the TAV. In some embodiments the TAV-encoding mRNAs are chemically modified. mRNAs comprising one or more chemical modifications may be referred to herein as modified mRNAs (mmRNAs).

[00064] In certain embodiments, the dendritic cell targeting agent or moiety of the present invention may be selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, an engineered protein scaffold and a peptide that targets one or more dendritic cell surface markers, and the like. Engineered protein scaffolds may be selected from the group consisting of fibronectin, transferrin, and a Kunitz domain or any such protein-based scaffold or structural protein. In certain embodiments, the cell surface markers for the dendritic cells may be selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.

[00065] In some embodiments, the immunomodulatory agent or moiety is encoded on the same polynucleotide as the antigen. In particular embodiments, the immunomodulatory agent or moiety is located N- or C-terminal to the antigen. In certain embodiments, two or more immunomodulatory agents or moieties are included, which may be the same or different.

[00066] In a non-limiting example, the immunomodulatory agent or moiety may be selected from GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti-PD1, anti-PDL1/2, anti-OX40/OX40L, anti-GITR/GITRL, and anti-TIM3.

[00067] In certain embodiments, the immunomodulatory agent or moiety is an immune enhancing polypeptide, which may also be referred to herein as an immune enhancing polypeptide, an immunopotentiating polypeptide, or an

immunopotentiation motif (IM). Non-limiting examples of immune enhancing polypeptides include, e.g., mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, or a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid. The polypeptide sequences of these immune enhancing polypeptide are provided in the accompanying Examples. In particular embodiments, a TAV comprises two or more immune enhancing polypeptides, which may optionally be separated by a linker sequence or a cleavage site, e.g., a cathepsin cleavage site (VVR).

[00068] In another non-limiting example, the immunomodulatory agent or moiety may comprise a second antigen, such as a bacterial or viral protein or fragment thereof to enhance antigenicity. In some embodiments, the bacterial or viral protein fragment is encoded on the same polynucleotide as the TAV antigen.

[00069] In some embodiments, reversing agents and methods are provided which function to turn off the TAV once the desired effect is achieved. In some

embodiments, these reversing agents may be selected from Bortezomib

(VELCADE®) or Rituximab (RITUXAN®), and a polynucleotide encoding

Rituximab.

[00070] In some embodiments are provided methods of inducing, eliciting, boosting or triggering an immune response in a cell, tissue or organism, comprising contacting said cell, tissue or organism with any of the TAV compositions described or taught herein.

[00071] Described herein are compositions (including pharmaceutical

compositions) and methods for the design, preparation, manufacture and/or formulation of TAV compositions where at least one component of the TAV is encoded by a polynucleotide. As such the present invention is directed, in part, to polynucleotides, specifically mRNA or IVT polynucleotides, chimeric

polynucleotides and/or circular polynucleotides encoding one or more targeted adaptive vaccines or components thereof.

[00072] Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the TAV compositions described herein.

[00073] According to one aspect of the present invention, the polynucleotides are preferably modified in a manner as to avoid the deficiencies of or provide improvements over other molecules of the art. I. Compositions of the Invention

Targeted Adaptive Vaccines (TAVs)

[00074] TAV polynucleotides are designed to provide to a cell, tissue or subject: (1) a polypeptide; and, optionally, (2) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. In particular embodiments, the polypeptide is an antigen, e.g., a vaccine antigen. In some embodiments, the TAV polynucleotides are designed to provide to a cell, tissue or subject a PCSK9, a TNF alpha, an IL-17A, or a GDF8 polypeptide. In particular embodiments, the encoded PCSK9, TNF alpha, IL-17A, or GDF8 polypeptide is immunogenic, e.g., it induces a T cell or B cell immune response when administered to a mammal. Immunogenic PCSK9, TNF alpha, IL-17A, or GDF8 polypeptides may be referred to herein as PCSK9 antigens, TNF alpha antigens, IL-17A antigens, or GDF8 antigens, respectively.

[00075] Polynucleotides of the present invention may encode at least one polypeptide of interest, e.g., an antigen. In addition to those identified herein, a selection of polypeptides of interest or "Targets" ofthe present invention are listed in Table 16 below.

Table 16: Targets

Figure imgf000020_0001

Figure imgf000021_0001

Figure imgf000022_0001

[00076] In certain embodiments, the encoded PCSK9 polypeptide comprises a polypeptide sequence endogenous to a particular species of mammal, e.g., a human, and is capable of inducing an immune response to the endogenous PCSK9 polypeptide in the same species of mammal, e.g., a human. In particular

embodiments, the encoded PCSK9 polypeptide comprises a polypeptide sequence endogenous to one species of mammal, e.g., a mouse, and is capable of inducing an immune response to the endogenous PCSK9 polypeptide in a different species of mammal, e.g., a human. Optionally, TAVs may further comprise a reversing agent to turn off the TAV.

[00077] In particular embodiments, TAV polynucleotides are used to deliver an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen, and, optionally, either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety to a cell, tissue or subject. In particular embodiments, the TAV polynucleotides deliver an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen, and an immunomodulatory agent or moiety to the cell, tissue or subject. In certain embodiments, TAV polynucleotides comprise a polynucleotide that encodes an antigen described herein and, optionally, also encodes either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety. In particular

embodiments, TAV polynucleotides comprise a polynucleotide encoding a PCSK9 antigen and an immunomodulatory agent or moiety. In some embodiments, TAV polynucleotides comprise a polynucleotide encoding a TNF alpha antigen and an immunomodulatory agent or moiety. In certain embodiments, TAV polynucleotides comprise a polynucleotide encoding an IL-17A antigen and an immunomodulatory agent or moiety. In various embodiments, TAV polynucleotides comprise a polynucleotide encoding a GDF8 antigen and an immunomodulatory agent or moiety. In particular embodiments, the polynucleotide is an mRNA, such as a modified mRNA.

[00078] Various components of the TAV may be delivered to the cell, tissue, or subject as: a polynucleotide encoding a polypeptide component of the TAV; a polypeptide component of the TAV; or another type of chemical agent or moiety (i.e., a non-proteinaceous agent or moiety) of the TAV. Accordingly, in certain embodiments, TAVs comprise a formulation comprising different types of components, e.g., a TAV may comprise an mRNA encoding an antigen(such as a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen), together with a polypeptide or non-proteinaceous immune modulating agent or moiety and/or a polypeptide dendritic cell targeting agent. In certain embodiments, the various components of a TAV are present in the same formulation or pharmaceutical composition. In particular embodiments, one or more component is present in a lipid nanoparticle (LNP) or other microparticle or nanoparticle.

[00079] In certain embodiments, a TAV polynucleotide comprises a

polynucleotide, e.g., an mRNA encoding both aan antigen (e.g., a PCSK9, TNF alpha, IL-17A, or GDF8 polypeptide) and, optionally, an immunomodulatory polypeptide. In particular embodiments, the immunomodulatory polypeptide enhances an immune response, e.g., to the encoded antigen.

[00080] In certain embodiments, a TAV polynucleotide is an mRNA comprising: (a) a first region comprising a sequence encoding one or more polypeptide or antigen (e.g., one or more PCSK9, TNF alpha, IL-17A or GDF8 polypeptide antigen); and (b) a second region comprising a sequence encoding one or more immunomodulatory polypeptide. In certain embodiments, the mRNA comprises a modified nucleobase. The mRNA may also comprise a third region between the first region and the second region, wherein the third region comprises a sequence encoding a linker, spacer or a cleavage site. In certain instances, the linker, spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site, which allows cleavage and separation of the antigen and the immunomodulatory polypeptide following translation in a cell.

[00081] In particular embodiments, TAV polynucleotides of the invention encode two or more polypeptides or antigens, which may be the same or different. In certain embodiments, the two or more polypetides or antigens are derived from the same polypeptide. For example, they may be two or more different antigenic fragments of a polypeptide. In certain embodiments, the TAV polynucleotides encode concatamers or a single polypeptide antigen. The two or more polypeptides or antigens may be encoded by or translated from the same polynucleotide, e.g., mRNA. In particular embodiments, the first region of the mRNA comprises two or more sequences encoding antigens. The first region may also comprise a sequence encoding a spacer, linker or a cleavage site between the sequences encoding the two or more antigens. In certain embodiments, the spacer or cleavage site is a 2A peptide or a cathepsin cleavage site. In some embodiments, the TAV polynucleotides encode two or more PCSK9 polypeptides or PCSK9 antigens, which may be the same or different. In certain embodiments, the TAV polynucleotides encode two or more TNF alpha polypeptides or TNF alpha antigens, which may be the same or different. In particular embodiments, the TAV polynucleotides encode two or more IL-17A polypeptides or IL-17A antigens, which may be the same or different. In some embodiments, the TAV polynucleotides encode two or more GDF8 polypeptides or GDF8 antigens, which may be the same or different. In particular embodiments, the first region of the mRNA comprises two or more sequences encoding PCSK9 antigens. In some embodiments, the first region of the mRNA comprises two or more sequences encoding TNF alpha antigens. In certain embodiments, the first region of the mRNA comprises two or more sequences encoding IL-17A antigens. In various embodiments, the first region of the mRNA comprises two or more sequences encoding GDF8 antigens. In particular embodiments, the two or more sequences are both antigens that induce an immune response. In particular embodiments, the mRNA encodes two or more different antigenic regions or epitopes within a target polypeptide, such as, e.g., PCSK9, TNF alpha, IL-17A, GDF8, or those listed in Table 1. In particular embodiments, the polynucleotide encodes two, three or more peptides described herein, e.g., those shown in any of Figure 14, or Tables 4, 6 or 8, or portions thereof. For example, a polypeptide may include portions of two or more overlapping peptides depicted in any of these tables or described elsewhere herein. In particular embodiments, a polypeptide comprises 15-25 or 18-20 amino acid residues, comprising overlapping regions of two, three or four peptides described herein.

[00082] Similarly, in certain embodiments, the second region comprises two or more sequences encoding immunomodulatory polypeptides, which may be the same or different. The second region may comprise a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides. In particular embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site. In certain embodiments, the immunomodulatory polypeptide is selected from the group consisting of: GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti-PD1, anti-PDL1/2, anti-OX40/OX40L, anti- GITR/GITRL, and anti-TIM3.

[00083] In certain embodiments, the one or more immunomodulatory polypeptide is an immune enhancing polypeptide. In particular embodiments, the immune enhancing polypeptide is selected from the group consisting of: mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, and a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid.

[00084] In certain embodiments, the mRNA comprises a further or alternative, e.g., fourth, region comprising a sequence encoding a dendritic cell targeting polypeptide. In particular embodiment, the polynucleotide, e.g., mRNA, also comprises a sequence encoding a linker or a cleavage site adjacent to one or both of the 5’ and 3’ of the sequence encoding the dendritic cell targeting polypeptide or the fourth region. In particular embodiments, the linker or cleavage site is a 2A peptide or a cathepsin cleavage site. In particular embodiments, the dendritic cell targeting polypeptide is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers. In certain embodiments, the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36. [00085] In certain embodiments, a TAV polynucleotide is an mRNA comprising: (a) a first region comprising a sequence encoding one or more polypeptide or antigen (e.g., one or more PCSK9, TNF alpha, IL-17A or GDF8 polypeptide or antigen); and (b) a second region comprising a sequence encoding a dendritic cell targeting polypeptide. The mRNA may also comprise a third region between the first region and the second region, wherein the third region comprises a sequence encoding a linker, spacer or a cleavage site. In certain instances, the linker, spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site, which allows cleavage and separation of the antigen and the dendritic cell targeting polypeptide following translation in a cell.

[00086] In certain embodiments, the TAV polynucleotide or TAV mRNA encodes a fusion polypeptide comprising a scaffold polypeptide, the first region, and, optionally, the second region, wherein the first region and, optionally, the second region are inserted into the scaffold polypeptide. In one embodiment, the

polynucleotide or mRNA encodes a fusion polypeptide comprising a scaffold polypeptide into which are inserted one or more antigens and/or immune enhancing polypeptides. In another embodiment, a polynucleotide or mRNA encodes a fusion polypeptide comprising a scaffold polypeptide into which are inserted one or more antigens, wherein the scaffold polypeptide is fused to an immune enhancing polypeptide. In some embodiments, the one or more antigens are one or more PCSK9 antigens. In particular embodiments, the one or more antigens are one or more TNF alpha antigens. In certain embodiments, the one or more antigens are one or more IL- 17A antigens. In various embodiments, the one or more antigens are one or more GDF8 antigens. In particular embodiments, the third region and or the fourth region are also inserted into the scaffold or fused to the scaffold polypeptide. Examples of scaffold polypeptides that may be used in this manner are known in the art.

[00087] Various other TAV embodiments contemplate the use of pharmaceutical compositions or microparticles or nanoparticles, which comprise the various components of the TAV compositions of the invention. For example, in one embodiment, a TAV composition comprises an mRNA comprising a first sequence encoding a PCSK9 antigen and, optionally, a second sequence encoding an immune enhancing polypeptide, as well as a dendritic target agent or moiety. The mRNA may be present within a lipid nanoparticle, and the dendritic targeting agent may be present within the lipid nanoparticle or displayed on its surface. Cell targeting agents may be associated with or on the surface of particles containing mRNAs or TAV

polynucleotides of the present invention.

[00088] When present in the same polynucleotide, e.g., mRNA, the various components of the TAV (e.g., the polynucleotides encoding the antigen and the immune enhancing polypeptide) may be located in various positions upstream or downstream of each other. In certain embodiments, the sequence encoding the antigen is located upstream of or 5’ to the sequence encoding the immune modulating polypeptide. In other embodiments, the sequence encoding the antigen is located downstream of or 3’ to the sequence encoding the immune modulating polypeptide. The present invention further contemplates polynucleotides encoding multiple antigens and/or multiple immune modulation polypeptides, wherein the various antigens and immune modulating polypeptides are present in any order. For example, sequences encoding antigens may be present both upstream and downstream of one or more sequence encoding an immune modulating polypeptide. Similarly, sequences encoding immune modulating polypeptides may be present both upstream and downstream of one or more sequence encoding an antigen. Polynucleotides Encoding PCSK9 Polypeptides

[00089] Familial hypercholesterolaemia (FH) is one of the most common inherited disorders of human metabolism, affecting approximately 1 in 500 individuals in most populations. The disorder is characterized by markedly increased low-density lipoprotein (LDL) cholesterol in serum, causing deposition of cholesterol in peripheral tissues to form tendon and skin xanthomas. Accumulation of cholesterol in the arterial wall results in accelerated atherosclerosis and premature coronary heart disease. FH is generally accepted to be an autosomal dominant disorder with a gene dosage effect. Most FH results from inheritance of a defective parental allele, as the effects of the disease do not occur early enough to affect reproductive capacity, but some de novo mutations have been reported.

[00090] Heterozygous mutations in the PCSK9 gene, which encodes a protein named proprotein convertase subtilisn kexin type 9 because of its similarity to the proprotein convertase family of proteins, were first identified in two French families following genetic linkage studies. Patients with PCSK9 mutations have the severest and clearest symptoms of FH. These mutations cause hypercholesterolaemia mainly by reducing the number of LDL receptors on liver cells. At least two studies confirmed PCSK9’s role in the maintenance of LDL homeostasis. The first of these findings was that adenoviral-mediated expression of the normal gene in mice caused LDL-receptor protein essentially to disappear from the liver without any change in LDLR mRNA, and the second was that knocking out PCSK9 in mice increased hepatic LDL-receptor protein levels and reduced serum cholesterol. Another observation was the discovery that being heterozygous for a null variant of PCSK9 significantly reduced both serum cholesterol and the risk of coronary heart disease.

[00091] PCSK9 polynucleotides of the present invention may encode any PCSK9 polypeptide, or fragment or variant thereof, including antigenic fragments or epitopes of a PCSK9 polypeptide. In particular embodiments, the encoded PCSK9 polypeptides are capable of eliciting an immune response to a PCSK9 polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as PCSK9 antigens. In certain embodiments, the encoded PCSK9 polypeptides are capable of eliciting an immune response to an endogenout PCSK9 protein in a mammal, e.g. a human. In particular embodiments, a PCSK9 polynucleotide of the present invention may encode any of the PCSK9 polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof. In certain embodiments, a PCSK9 polynucleotide encodes a PCSK9 polypeptide and an immunomodulatory moiety, such as an immune enhancing polypeptide polypeptide, e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8), T cell epitope from tetanus toxin, chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid, flagellin derived immunogen, T cell epitopes from Influenza HA antigen, Universal T helper epitope, or Mannose binding protein.

[00092] Particular embodiments contemplate that administration of mRNA encoding a fusion peptide comprising an antigen and a mannose binding protein to a subject will illicit an enhanced immune response, e.g., greater antigen-specific antibody production in the subject, as compared to an mRNA that encodes the same antigen alone.

[00093] Certain embodiments contemplate that administration of mRNA encoding a fusion peptide comprising an antigen and PR8M2 will illicit an enhanced immune response, e.g., greater antigen-specific antibody production in the subject, as compared to an mRNA that encodes the same antigen alone. [00094] In some embodiments, the mRNA of the present invention encodes two or more different epitopes. Particular embodiment contemplate that the identification of the epitope as the smallest immunogenic subunit derived from antigenic proteins has promoted the development of epitope-based vaccines. These prevent the danger of administering whole proteins or genes that have unknown and possibly dangerous properties. Certain embodiments contemplate that DNA encoded epitopes in synthetic constructs can be processed and presented to CD8+ T-lymphocytes despite unnatural flanking amino acid sequences, allowing for the development of polyepitope vaccines for cancer and infectious disease which induce multiple cytotoxic T-lymphocyte responses. The resultant immunity may be restricted to various HLA alleles and targeted to numerous antigens to avoid escape from immune detection by antigen loss variants.

[00095] Some embodiments contemplate that the evolution of vaccine strategies has seen a move from whole organisms to recombinant proteins, and further towards the ultimate in minimalist vaccinology, the epitope. Such embodiments contempleat that epitope-based approach allows for only a relatively tiny, but immunologically relevant, sequence can be capable of inducing protective immunity against a large and complex pathogen. The post-reductionist era in epitope-based vaccinology has seen a quest to re-construct complexity and design vaccines containing many epitopes. The hope is that such multi-epitope vaccines might induce immunity against multiple antigenic targets, multiple strain variants, and/or even multiple pathogens. The ability of DNA vaccination to co-deliver a series of antibody and/or CD4 T cell epitopes remains largely unexplored. Successful viral vector and DNA-based experimental vaccines coding for multiple contiguous CD8 CTL epitopes have, however, recently been described. This simple CTL poly-epitope (or polytope) strategy may find application in the design of vaccines against several diseases including EBV, HIV and cancer.

[00096] Illustrative wild-type PCSK9 polynucleotide and polypeptide sequences are shown in Table 1 below. In particular embodiments, a PCSK9 TAV RNA comprises any of these polynucleotide sequences, or fragments or variants thereof, or encodes any of the polypeptides, or fragments or variants thereof. Table 1: Illustrative PCSK9 sequences

Figure imgf000030_0001
Figure imgf000031_0001

Figure imgf000032_0001

[00097] The polypeptide sequences of full length human PCSK9 and full length mouse PCKS9 are provided herein, together with the amino acid sequences of various fragments of human PCSK9 and mouse PCSK9. In addition, immunogenic epitopes of human PCSK9 and mouse PCSK9 are described herein. The present invention contemplates the use of polynucleotides encoding any of these polypeptides, and fragments and variants thereof, as PCKS9 polypeptides or antigens. In addition, the present invention contemplates the use of polynucleotides encoding PCSK9 homologs, including PCSK9 polypeptides from other animals, as well as variants and fragments thereof. In some embodiments, the PCSK9 polynucleotide encodes a PCSK9 antigen that is a C terminal catalytic domain fragment of a PCSK9 polypeptide. In some embodiments, a PCSK9 polynucleotide encodes a fragment of a PCSK9 polypeptide; in particular embodiments, the PCSK9 fragment is 1-60, 60-80 amino acids, 30-70 amino acids, greater than 60, 70, or 80, amino acids, in length.

[00098] Particular embodiments of the present invention include one or more polynucleotides encoding antigenic PCSK9 peptides identified herein and the encoded peptides, which include but are not limited to those described in Tables 1 and 2 and in Example 4 as Peptide 10: HGTTATFHRCAKDPW; Peptide 11:

TATFHRCAKDPWRLP; Peptide 12:FHRCAKDPWRLPGTY; Peptide 13:

CAKDPWRLPGTYVVV; Peptide 14: DPWRLPGTYVVVLKE; Peptide 15:

RLPGTYVVVLKEETH; Peptide 129: LIHFSAKDVINEAWF; Peptide 130:

FSAKDVINEAWFPED; Peptide 131: KDVINEAWFPEDQRV; Peptide 132:

INEAWFPEDQRVLTP; Peptide 133: AWFPEDQRVLTPNLV; Peptide 265:

IHFSTKDVINMAWFP; Peptide 266: STKDVINMAWFPEDQ; Peptide 267: DVINMAWFPEDQQVL; Peptide 268: NMAWFPEDQQVLTPN;

Peptide 129-133 (human PCSK9): LIHFSAKDVINEAWFPEDQRVLTPNLV; or Peptide 265-268 (mouse PCSK9): IHFSTKDVINMAWFPEDQQVLTPN; variants thereof having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of these PCSK9 peptides; variants thereof having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitution, deletions, or insertions as compared to any one of these PCSK9 peptides; and fragments thereof having at least 6, at least 8, at least 10, or at least 12 contiguous amino acid residues of any of these PCSK9 peptides. Related

embodiments of the present invention include polynucleotides, e.g., mRNAs, encoding any of these PCSK9 peptides or variants or fragments thereof;

pharmaceutical compositions comprising any of these PCSK9 peptides or variants or fragments thereof or any polynucleotides encoding them and a pharmaceutically acceptable diluent, excipient, or carrier; and vaccines comprising any of any of these PCSK9 peptides or variants or fragments thereof or any polynucleotides encoding them and an adjuvant. Related embodiments include methods of stimulating an immune response to a PCSK9 polypeptide comprising contacting a cell, tissue or subject with a PCSK9 polynucleotide encoding any of these PCSK9 polypeptides, pharmaceutical compositions comprising a polynucleotide encoding any of these PCSK9 polypeptides, or vaccines comprising a polynucleotide encoding any of these PCSK9 polypeptides. In particular embodiments, the method is practiced in vitro or in vivo, e.g., in a mammal. In particular embodiments, the present invention includes a method for treating or preventing hypercholesterolemia or atherosclerosis in subject in need thereof, comprising providing to the subject an effective amount of any of these PCSK9 polynucleotides, pharmaceutical compositions comprising them, or vaccines comprising them.

Table 2: Illustrative PCSK9 polypeptides:

Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001

Figure imgf000050_0001
[00099] In particular embodiments, multiple PCSK9 antigens can be encoded by a single TAV polynucleotide. For example, a TAV polynucleotide may encode one or more, two or more, or three or more antigenic epitopes of a PCSK9 polypeptide. These antigenic epitopes may be the same or different. In particular embodiments, they are separated from each other by a linker sequence or a cleavage sequence, such as, e.g., a cathepsin S cleavage site sequence, e.g., VVR.

[000100] The present invention further contemplates the use of polynucleotides encoding variants of any of the PCSK9 polypeptides described herein, including variants having one or more amino acid substitutions, insertions or deletions, as compared to a wild type PCSK9 polypeptide or fragment thereof. In particular embodiments, a variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference wild type PCSK9 polypeptide or fragment thereof, including any of those described herein. Polynucleotides Encoding TNF alpha Polypeptides

[000101] Tumor Necrosis Factor alpha (TNF alpha), is an inflammatory cytokine that is produced from various kinds of cells such as lymphoid cells, cardiac myocytes, activated macrophages, endothelial cells, mast cells, fibroblasts, neurons and adipose tissue, and is responsible for a diverse range of signaling events within cells. TNF alpha is primarily secreted as a 212-amino acid-long type II trans-membrane protein arranged in stable homotrimers. From these trans-membrane TNF-alpha

homotrimers, a combined form the soluble homotrimeric TNF alpha (sTNF) is released through proteolytic cleavage by the metalloprotease TNF-α converting enzyme (TACE).

[000102] In the immune system, TNF alpha is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation. TNF alpha synthesis occurs in many cell types as a response to an external stimulus, such as, for example, a mitogen, an infectious organism, or trauma. Over production of TNF alpha is associated with numerous pathologies, including diseases associated with chronic inflammation, autoimmune disorders, and cancer.

[000103] Illustrative wild-type TNF alpha polynucleotide and polypeptide sequences are shown in Table 3 below. In particular embodiments, a TNF alpha TAV RNA comprises any of these polynucleotide sequences, or fragments or variants thereof, or encodes any of the polypeptides, or fragments or variants thereof. Table 3: Illustrative TNF alpha sequences

Figure imgf000051_0001

Figure imgf000052_0001

[000104] In some embodiments, TAV polynucleotides are designed to provide to a cell, tissue or subject: (1) a TNF alpha polypeptide; and, optionally, (2) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety.

[000105] In particular embodiments, the encoded TNF alpha polypeptide is immunogenic, e.g., it induces a T cell or B cell immune response when administered to a mammal. Immunogenic TNF alpha polypeptides may be referred to herein as TNF alpha antigens.

[000106] In certain embodiments, the encoded TNF alpha polypeptide comprises a polypeptide sequence endogenous to a particular species of mammal, e.g., a human, and is capable of inducing an immune response to the endogenous TNF alpha polypeptide in the same species of mammal, e.g., a human. In particular embodiments, the encoded TNF alpha polypeptide comprises a polypeptide sequence endogenous to one species of mammal, e.g., a mouse, and is capable of inducing an immune response to the endogenous TNF alpha polypeptide in a different species of mammal, e.g., a human. Optionally, TAVs may further comprise a reversing agent to turn off the TAV.

[000107] In particular embodiments, TAV polynucleotides are used to deliver a TNF alpha antigen and, optionally, either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety to a cell, tissue or subject. In particular embodiments, the TAV polynucleotides deliver a TNF alpha antigen and an immunomodulatory agent or moiety to the cell, tissue or subject. In certain embodiments, TAV polynucleotides comprise a polynucleotide that encodes a TNF alpha antigen described herein and, optionally, also encodes either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety. In particular embodiments, TAV polynucleotides comprise a polynucleotide encoding a TNF alpha antigen and an immunomodulatory agent or moiety. In particular embodiments, the polynucleotide is an mRNA, such as a modified mRNA.

[000108] In certain embodiments, a TAV polynucleotide comprises an mRNA encoding both a TNF alpha polypeptide and, optionally, an immunomodulatory polypeptide, such as an immune enhancing polypeptide (IM), e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8), T cell epitope from tetanus toxin, chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid, flagellin derived immunogen, T cell epitopes from Influenza HA antigen, Universal T helper epitope, or Mannose binding protein.. In particular embodiments, the

immunomodulatory polypeptide enhances an immune response, e.g., to the encoded TNF alpha antigen.

[000109] TNF alpha polynucleotides of the present invention may encode any TNF alpha polypeptide, or fragment or variant thereof, including antigenic epitopes of a TNF alpha polypeptide. In particular embodiments, the encoded TNF alpha polypeptides are capable of eliciting an immune response to a TNF alpha polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as TNF alpha antigens. In particular embodiments, a TNF alpha polynucleotide of the present invention may encode any of the TNF alpha polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof.

[000110] The polypeptide sequences of full length human TNF alpha and full length mouse TNF alpha are provided herein, together with the amino acid sequences of various fragments of human, mouse, and rat TNF alpha. In addition, immunogenic epitopes of TNF alpha are described herein. The present invention contemplates the use of polynucleotides encoding any of these polypeptides, and fragments and variants thereof, as TNF alpha polypeptides or antigens. In addition, the present invention contemplates the use of polynucleotides encoding TNF alpha homologs, including TNF alpha polypeptides from other animals, e.g. rat, as well as variants and fragments thereof. In some embodiments, the TNF alpha polynucleotide encodes a TNF alpha antigen that is a C-terminal catalytic domain fragment of a TNF alpha polypeptide. In some embodiments, a TNF alpha polynucleotide encodes a fragment of a TNF alpha polypeptide; in particular embodiments, the TNF alpha fragment is 1- 60, 60-80 amino acids, 30-70 amino acids, or greater than 60, 70, or 80 amino acids in length.

[000111] Particular embodiments of the present invention include polynucleotides encoding antigenic TNF alpha peptides identified herein and the encoded peptides, which include but are not limited to those described in Table 4; variants thereof having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of these TNF alpha peptides; variants thereof having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitution, deletions, or insertions as compared to any one of these TNF alpha peptides; and fragments thereof having at least 6, at least 8, at least 10, or at least 12 contiguous amino acid residues of any of these TNF alpha peptides. Related embodiments of the present invention include polynucleotides, e.g., mRNAs, encoding any of these TNF alpha peptides or variants or fragments thereof;

pharmaceutical compositions comprising any of these TNF alpha peptides or variants or fragments thereof or any polynucleotides encoding them and a pharmaceutically acceptable diluent, excipient, or carrier; and vaccines comprising any of any of these TNF alpha peptides or variants or fragments thereof or any polynucleotides encoding them and an adjuvant. Related embodiments include methods of stimulating an immune response to a TNF alpha polypeptide comprising contacting a cell, tissue or subject with a TNF alpha polynucleotide encoding any of these TNF alpha polypeptides, pharmaceutical compositions comprising a polynucleotide encoding any of these TNF alpha polypeptides, or vaccines comprising a polynucleotide encoding any of these TNF alpha polypeptides. In particular embodiments, the method is practiced in vitro or in vivo, e.g., in a mammal. In particular embodiments, the present invention includes a method for treating or preventing hypercholesterolemia or atherosclerosis in subject in need thereof, comprising providing to the subject an effective amount of any of these TNF alpha polynucleotides, pharmaceutical compositions comprising them, or vaccines comprising them.

[000112] In certain embodiments, the antigen comprises one or more sequences shown in Table 4. In particular embodiments, the antigen comprises an amino acid sequence of one or more of GQGCPDYVLLTHTVSR; FAISYQEKVSLLSAIK; or GDLLSAEVNLPKY. Table 4: Illustrative anti enic TNF al ha ol e tides

Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
[000113] In particular embodiments, multiple TNF alpha antigens can be encoded by a single TAV polynucleotide. For example, a TAV polynucleotide may encode one or more, two or more, or three or more antigenic epitopes of a TNF alpha polypeptide. These antigenic epitopes may be the same or different. In particular embodiments, they are separated from each other by a linker sequence or a cleavage sequence, such as, e.g., a cathepsin S cleavage site sequence, e.g., VVR.

[000114] The present invention further contemplates the use of polynucleotides encoding variants of any of the TNF alpha polypeptides described herein, including variants having one or more amino acid substitutions, insertions or deletions, as compared to a wild type TNF alpha polypeptide or fragment thereof. In particular embodiments, a variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference wild type TNF alpha polypeptide or fragment thereof, including any of those described herein.

[000115] Certain embodiments are directed to a polynucleotide, mRNA encodes a fusion protein comprising a TNF alpha polypepide or fragment or variant thereof and a C-terminal IM comprising PR8M2x3, HA307-318x3, and MBP. Polynucleotides Encoding IL-17A Polypeptides

[000116] Interleukin-17 (IL-17) is a family of cytokines that include six members, IL- 17A to IL- 17F. Members of the IL-17 family bind to IL-17 receptors, a family presently comprising five members, IL- 17RA to IL- 17RE, which share considerable sequence homology with each other. The members of the IL-17 receptor family are type I transmembrane proteins. Presently it is generally accepted that receptors for IL- 17 are abundantly expressed by all cells of the immune system, and stimulation of various cell types with IL- 17A, IL- 17F and IL- 17D can induce the expression of other cytokines like IL- lβ, TNFα, and IL-6, and the chemokines IL-8 and MIP-Ia. Cytokines of the IL-17 family are mainly produced by the recently discovered Th 17 cell. [000117] The interleukin-17 (IL-17) family consists of a subset of cytokines that participate in both acute and chronic inflammatory responses. Since the discovery of IL-17 A (also called IL-17 or CTLA8) in 1993, five other members of this family IL- 17B, IL-17C, IL-17D, IL- 17E (also called IL-25), and IL-17F have been identified based on amino acid sequence homology. Notably, IL-17A is a pro-inflammatory cytokine produced by Thl7 cells, a CD4+ T helper cell subset that has been shown to regulate tissue inflammatory responses. Recent studies indicate that IL- 17A can also be produced by other cell types during inflammatory responses including CD8+ T cells, and γδ T cells, and innate lymphoid cells. IL-17A is a pro-inflammatory cytokine that plays a critical role in the pathogenesis of autoimmune diseases, metabolic disorders, and cancer.

[000118] IL-17A signals through IL-17 receptor complex (IL-17RA and IL-17RC subunits) to transmit signals into cells. The main function of IL-17A is to coordinate local tissue inflammation via the up-regulation of proinflammatory and neutrophil- mobilizing cytokines and chemokines (including IL-6, G-CSF, TNF alpha, IL-1, CXCL1 (KC), CCL2 (MCP-1), CXCL2 (MIP-2)), as well as matrix metalloproteases to allow activated T cells to penetrate extracellular matrix. IL-17A has also been implicated in smooth muscle function and airway remodeling. Previous studies have suggested a central role for IL-17A in severe asthma and COPD. In addition, IL- 17A levels were also increased in synovial fluids from arthritis patients, serum and brain tissue of multiple sclerosis patients, skin lesions of psoriasis patients, serum and tumor tissues of cancer patients.

[000119] Illustrative wild-type IL-17A polynucleotide and polypeptide sequences are shown in Table 5. In particular embodiments, an IL-17A TAV RNA comprises any of these polynucleotide sequences, or fragments or variants thereof, or encodes any of the polypeptides, or fragments or variants thereof. Table 5: Illustrative wild-type IL-17A sequences

Figure imgf000058_0002
Figure imgf000058_0001

Figure imgf000059_0001
[000120] In some embodiments, TAV polynucleotides are designed to provide to a cell, tissue or subject: (1) an IL-17A polypeptide; and, optionally, (2) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety,. In particular embodiments, the encoded IL-17A polypeptide is immunogenic, e.g., it induces a T cell or B cell immune response when administered to a mammal. Immunogenic IL- 17A polypeptides may be referred to herein as IL-17A antigens.

[000121] In certain embodiments, a TAV polynucleotide comprises a

polynucleotide, e.g., an mRNA encoding both an IL-17A polypeptide and, optionally, an immunomodulatory polypeptide. In particular embodiments, the

immunomodulatory polypeptide enhances an immune response, e.g., to the encoded IL-17A antigen.

[000122] In certain embodiments, the encoded IL-17A polypeptide comprises a polypeptide sequence endogenous to a particular species of mammal, e.g., a human, and is capable of inducing an immune response to the endogenous IL-17A

polypeptide in the same species of mammal, e.g., a human. In particular embodiments, the encoded IL-17A polypeptide comprises a polypeptide sequence endogenous to one species of mammal, e.g., a mouse, and is capable of inducing an immune response to the endogenous IL-17A polypeptide in a different species of mammal, e.g., a human. Optionally, TAVs may further comprise a reversing agent to turn off the TAV.

[000123] In particular embodiments, TAV polynucleotides are used to deliver a IL- 17A antigen and, optionally, either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety to a cell, tissue or subject. In particular embodiments, the TAV polynucleotides deliver a IL-17A antigen and an

immunomodulatory agent or moiety to the cell, tissue or subject. In certain embodiments, TAV polynucleotides comprise a polynucleotide that encodes a IL-17A antigen described herein and, optionally, also encodes either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety. In particular

embodiments, TAV polynucleotides comprise a polynucleotide encoding a IL-17A antigen and an immunomodulatory agent or moiety, such as an IM, e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8), T cell epitope from tetanus toxin, chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid, flagellin derived immunogen, T cell epitopes from Influenza HA antigen, Universal T helper epitope, or Mannose binding protein. In particular embodiments, the polynucleotide is an mRNA, such as a modified mRNA.

[000124] IL-17A polynucleotides of the present invention may encode any IL-17A polypeptide, or fragment or variant thereof, including antigenic epitopes of a IL-17A polypeptide. In particular embodiments, the encoded IL-17A polypeptides are capable of eliciting an immune response to a IL-17A polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as IL-17A antigens. In particular embodiments, a IL-17A polynucleotide of the present invention may encode any of the IL-17A polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof.

[000125] IL-17A polynucleotides of the present invention may encode any IL-17A polypeptide, or fragment or variant thereof, including antigenic epitopes of a IL-17A polypeptide. In particular embodiments, the encoded IL-17A polypeptides are capable of eliciting an immune response to a IL-17A polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as IL-17A antigens. In particular embodiments, a IL-17A polynucleotide of the present invention may encode any of the IL-17A polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof.

[000126] The polypeptide sequences of full length human IL-17A and full length mouse IL-17A are provided herein, together with the amino acid sequences of various fragments of human, mouse, and rat IL-17A. In addition, immunogenic epitopes of IL-17A are described herein. The present invention contemplates the use of polynucleotides encoding any of these polypeptides, and fragments and variants thereof, as IL-17A polypeptides or antigens. In addition, the present invention contemplates the use of polynucleotides encoding IL-17A homologs, including IL- 17A polypeptides from other animals, as well as variants and fragments thereof. In some embodiments, the IL-17A polynucleotide encodes a IL-17A antigen that is a C- terminal catalytic domain fragment of a IL-17A polypeptide. In some embodiments, a IL-17A polynucleotide encodes a fragment of a IL-17A polypeptide; in particular embodiments, the IL-17A fragment is 1-60, 60-80 amino acids, 30-70 amino acids, or greater, in length.

[000127] Particular embodiments of the present invention include polynucleotides encoding antigenic IL-17A peptides identified herein and the encoded peptides, which include but are not limited to those described in Table 6; variants thereof having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of these IL- 17A peptides; variants thereof having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitution, deletions, or insertions as compared to any one of these IL-17A peptides; and fragments thereof having at least 6, at least 8, at least 10, or at least 12 contiguous amino acid residues of any of these IL-17A peptides. Related embodiments of the present invention include polynucleotides, e.g., mRNAs, encoding any of these IL- 17A peptides or variants or fragments thereof; pharmaceutical compositions comprising any of these IL-17A peptides or variants or fragments thereof or any polynucleotides encoding them and a pharmaceutically acceptable diluent, excipient, or carrier; and vaccines comprising any of any of these IL-17A peptides or variants or fragments thereof or any polynucleotides encoding them and an adjuvant. Related embodiments include methods of stimulating an immune response to a IL-17A polypeptide comprising contacting a cell, tissue or subject with a IL-17A

polynucleotide encoding any of these IL-17A polypeptides, pharmaceutical compositions comprising a polynucleotide encoding any of these IL-17A polypeptides, or vaccines comprising a polynucleotide encoding any of these IL-17A polypeptides. In particular embodiments, the method is practiced in vitro or in vivo, e.g., in a mammal. In particular embodiments, the present invention includes a method for treating or preventing hypercholesterolemia or atherosclerosis in subject in need thereof, comprising providing to the subject an effective amount of any of these IL-17A polynucleotides, pharmaceutical compositions comprising them, or vaccines comprising them.

Figure imgf000062_0001

Figure imgf000063_0001

[000128] In certain embodiments, the encoded IL-17A polypeptides are capable of eliciting an immune response to an endogenout IL—17A protein in a mammal, e.g. a human.

[000129] In particular embodiments, multiple IL-17A antigens can be encoded by a single TAV polynucleotide. For example, a TAV polynucleotide may encode one or more, two or more, or three or more antigenic epitopes of a IL-17A polypeptide. These antigenic epitopes may be the same or different. In particular embodiments, they are separated from each other by a linker sequence or a cleavage sequence, such as, e.g., a cathepsin S cleavage site sequence, e.g., VVR.

[000130] The present invention further contemplates the use of polynucleotides encoding variants of any of the IL-17A polypeptides described herein, including variants having one or more amino acid substitutions, insertions or deletions, as compared to a wild type IL-17A polypeptide or fragment thereof. In particular embodiments, a variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference wild type IL-17A polypeptide or fragment thereof, including any of those described herein.

[000131] Particular embodiments are directed to mRNA that encodes a fusion protein comprising the at least one IL-17A polypepide or fragment or variant thereof and a C-terminal IM selected from the group consisting of PR8M2x3, STF2D, HA307-318x3, and MBP.

Polynucleotides Encoding GDF8 Polypeptides [000132] Growth and Differentiation Factor (GDF8), also known as myostatin, is a member of the Transforming Growth Factor-beta (TGF-β) superfamily and is a negative regulator of muscle growth. GDF8 is highly conserved throughout evolution and the sequences of human, chicken, mouse and rat are 100% identical in the mature C-terminal domain. GDF8 is synthesized as a precursor protein that contains a signal sequence, a pro-peptide domain and a C-terminal domain. Secreted, circulating forms of GDF8 include the active mature C-terminal domain and an inactive form comprising the mature C-terminal domain in a latent complex associated with the pro- peptide domain and/or other inhibitory proteins.

[000133] GDF8 binds to the activin type II receptor, resulting in a recruitment of either coreceptor Alk-3 or Alk-4. This coreceptor then initiates a cell

signaling cascade in the muscle, which includes the activation of transcription factors in the SMAD family - SMAD2 and SMAD3. These factors then induce myostatin-specific gene regulation. In muscle stem cells, this activation of the activing type II receptor inhibits differentiation into mature muscle fibers.

[000134] Animals lacking GDF8 or animals treated with substances such as follistatin that block the binding of myostatin to its receptor have significantly larger muscles. For example, GDF8 knockout mice have approximate twice the muscle mass as normal mice.

[000135] There are a number of different diseases, disorders and conditions that are associated with reduced muscle mass, muscle strength, and muscle function. The inhibition of GDF8 has emerged as a potential strategy treatments of the diseases and conditions.

[000136] Illustrative wild-type GDF8 polypeptide and polynucleotide sequences are displayed in table 7. Table 7: Illustrative wild-type GDF8 Sequences

Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0002

Figure imgf000068_0001

[000137] In some embodiments, TAV polynucleotides are designed to provide to a cell, tissue or subject: (1) an GDF8 polypeptide; and, optionally, (2) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety. In particular embodiments, the encoded GDF8 polypeptide is immunogenic, e.g., it induces a T cell or B cell immune response when administered to a mammal. Immunogenic GDF8 polypeptides may be referred to herein as GDF8 antigens.

[000138] In certain embodiments, the encoded GDF8 polypeptide comprises a polypeptide sequence endogenous to a particular species of mammal, e.g., a human, and is capable of inducing an immune response to the endogenous GDF8 polypeptide in the same species of mammal, e.g., a human. In particular embodiments, the encoded GDF8 polypeptide comprises a polypeptide sequence endogenous to one species of mammal, e.g., a mouse, and is capable of inducing an immune response to the endogenous GDF8 polypeptide in a different species of mammal, e.g., a human. Optionally, TAVs may further comprise a reversing agent to turn off the TAV.

[000139] In certain embodiments, the encoded GDF8 polypeptides are capable of eliciting an immune response to an endogenout GDF8 protein in a mammal, e.g. a human.

[000140] In particular embodiments, TAV polynucleotides are used to deliver a GDF8 antigen and, optionally, either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety to a cell, tissue or subject. In particular embodiments, the TAV polynucleotides deliver a GDF8 antigen and an

immunomodulatory agent or moiety to the cell, tissue or subject. In certain embodiments, TAV polynucleotides comprise a polynucleotide that encodes a GDF8 antigen described herein and, optionally, also encodes either a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety. In particular

embodiments, TAV polynucleotides comprise a polynucleotide encoding a GDF8 antigen and an immunomodulatory agent or moiety, such as an IM, e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8), T cell epitope from tetanus toxin, chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid, flagellin derived immunogen, T cell epitopes from Influenza HA antigen, Universal T helper epitope, or Mannose binding protein. In particular embodiments, the polynucleotide is an mRNA, such as a modified mRNA.

[000141] In certain embodiments, a TAV polynucleotides comprises a

polynucleotide, e.g., an mRNA encoding both a GDF8 polypeptide and, optionally, an immunomodulatory polypeptide. In particular embodiments, the immunomodulatory polypeptide enhances an immune response, e.g., to the encoded GDF8 antigen.

[000142] GDF8 polynucleotides of the present invention may encode any GDF8 polypeptide, or fragment or variant thereof, including antigenic epitopes of a GDF8 polypeptide. In particular embodiments, the encoded GDF8 polypeptides are capable of eliciting an immune response to a GDF8 polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as GDF8 antigens. In particular embodiments, a GDF8 polynucleotide of the present invention may encode any of the GDF8 polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof.

[000143] GDF8 polynucleotides of the present invention may encode any GDF8 polypeptide, or fragment or variant thereof, including antigenic epitopes of a GDF8 polypeptide. In particular embodiments, the encoded GDF8 polypeptides are capable of eliciting an immune response to a GDF8 polypeptide, e.g., using any of the mouse assays described herein, and are referred to herein as GDF8 antigens. In particular embodiments, a GDF8 polynucleotide of the present invention may encode any of the GDF8 polypeptides described herein, including but not limited to any of those depicted in the Examples, Tables and Figures herein, as well as variants and fragments thereof.

[000144] The polypeptide sequences of full length human GDF8 and full length mouse GDF8 are provided herein, together with the amino acid sequences of various fragments of human, mouse, and rat GDF8. In addition, immunogenic epitopes of GDF8 are described herein. The present invention contemplates the use of polynucleotides encoding any of these polypeptides, and fragments and variants thereof, as GDF8 polypeptides or antigens. In addition, the present invention contemplates the use of polynucleotides encoding GDF8 homologs, including GDF8 polypeptides from other animals, as well as variants and fragments thereof. In some embodiments, the GDF8 polynucleotide encodes a GDF8 antigen that is a C-terminal catalytic domain fragment of a GDF8 polypeptide. In some embodiments, a GDF8 polynucleotide encodes a fragment of a GDF8 polypeptide; in particular

embodiments, the GDF8 fragment is 1-60, 60-80 amino acids, 30-70 amino acids, or greater, in length.

[000145] Particular embodiments of the present invention include polynucleotides encoding antigenic GDF8 peptides identified herein and the encoded peptides, which include but are not limited to those described in Table 8; variants thereof having at least 80%, at least 90%, at least 95%, or at least 98% identity to any one of these GDF8 peptides; variants thereof having 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitution, deletions, or insertions as compared to any one of these GDF8 peptides; and fragments thereof having at least 6, at least 8, at least 10, or at least 12 contiguous amino acid residues of any of these GDF8 peptides. Related embodiments of the present invention include polynucleotides, e.g., mRNAs, encoding any of these GDF8 peptides or variants or fragments thereof; pharmaceutical compositions comprising any of these GDF8 peptides or variants or fragments thereof or any polynucleotides encoding them and a pharmaceutically acceptable diluent, excipient, or carrier; and vaccines comprising any of any of these GDF8 peptides or variants or fragments thereof or any polynucleotides encoding them and an adjuvant. Related embodiments include methods of stimulating an immune response to a GDF8 polypeptide comprising contacting a cell, tissue or subject with a GDF8 polynucleotide encoding any of these GDF8 polypeptides, pharmaceutical compositions comprising a polynucleotide encoding any of these GDF8 polypeptides, or vaccines comprising a polynucleotide encoding any of these GDF8 polypeptides. In particular embodiments, the method is practiced in vitro or in vivo, e.g., in a mammal. In particular embodiments, the present invention includes a method for treating or preventing hypercholesterolemia or atherosclerosis in subject in need thereof, comprising providing to the subject an effective amount of any of these GDF8 polynucleotides, pharmaceutical compositions comprising them, or vaccines comprising them.

[000146] In particular embodiments, multiple GDF8 antigens can be encoded by a single TAV polynucleotide. For example, a TAV polynucleotide may encode one or more, two or more, or three or more antigenic epitopes of a GDF8 polypeptide. These antigenic epitopes may be the same or different. In particular embodiments, they are separated from each other by a linker sequence or a cleavage sequence, such as, e.g., a cathepsin S cleavage site sequence, e.g., VVR. Table 8: Illustrative GDF8 polypeptides

Figure imgf000071_0001
Figure imgf000072_0001

Figure imgf000073_0001

[000147] The present invention further contemplates the use of polynucleotides encoding variants of any of the GDF8 polypeptides described herein, including variants having one or more amino acid substitutions, insertions or deletions, as compared to a wild type GDF8 polypeptide or fragment thereof. In particular embodiments, a variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference wild type GDF8 polypeptide or fragment thereof, including any of those described herein.

[000148] In certain embodiments, the antigen comprises one or more sequences shown in Table 8. In particular embodiments, the antigen comprises an amino acid sequence of one or more of DFGLDCDEHSTESRCCRYPL;

CRYPLTVDFEAFGWDWIIAP; WIIAPKRYKANYCSGECEFV;

ECEFVFLQKYPHTHLVHQAN; VHQANPRGSAGPCCTPTKMS; PTKMSPINMLYFNGKEQIIY; EQIIYGKIPAMWDRCGCS;

DFGLDCDEHSTESRCCRYPL; CRYPLTVDFEAFGWDWIIAP;

WIIAPKRYKANYCSGECEFV; ECEFVFLQKYPHTHLVHQAN;

VHQANPRGSAGPCCTPTKMS; PTKMSPINMLYFNGKEQIIY;

EQIIYGKIPAMWDRCGCS;

DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCS;

PCCTPTKMSPINMLYFN;

DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFL QKYPHT; LVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKI; DFGLDC; FEAFGWDWIIAPKRY; FVFLQKYPHTHLVHQ; CSGECEFVF; or

WIIAPKRYKANYCSGECEFVFLQKY.

[000149] As used herein,“polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In one embodiment, the polypeptides of interest are antigens encoded by the polynucleotides as described herein.

[000150] In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. 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. They may also comprise single chain or multichain polypeptides such as antibodies or insulin 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 one or more amino acid residues are an artificial chemical analogue of a

corresponding naturally occurring amino acid.

[000151] 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 will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical, at least about 95% identical, or at least about 98% identical (homologous) to a native or reference sequence.

[000152] “Homology” as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology.

Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.

[000153] By“homologs” as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.

[000154] “Analogs” is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

[000155] The present invention contemplates several types of compositions which are polypeptide based including variants and derivatives. These include 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.

[000156] 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 invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C- terminal ends). Sequence tags can be used for peptide 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.

[000157] Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis or a priori

incorporation during chemical synthesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

[000158] According to the present invention, the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation. As used herein a“consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.

[000159] 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 of this invention. 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 about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the invention. In certain embodiments, a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.

[000160] In one embodiment, at least one polypeptide of interest may be an antigen or fragment thereof, or any component of a targeted adaptive vaccine.

[000161] In one embodiment, variant polypeptides have a certain identity with a reference polypeptide sequence. As used herein, a“reference polypeptide sequence” refers to a starting polypeptide sequence. Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence. A“reference polypeptide sequence” may be, e.g., any of the PCSK9 polypeptides, including variants and fragments thereof, described herein.

[000162] Reference molecules (polypeptides or polynucleotides) may share a certain identity with the designed molecules (polypeptides or polynucleotides). The term “identity” as known in the art, refers to a relationship between the sequences of two or more peptides, 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 nucleosides. 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 (i.e.,“algorithms”). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:

Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math.48, 1073 (1988).

[000163] In some embodiments, the encoded polypeptide variant may have the same or a similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 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, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.25:3389-3402.) Other tools are described herein, specifically in the definition of“Identity.”

[000164] Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.

Dendritic cell targeting agent or moiety [000165] To further induce potent immune response, TAV polynucleotides of the present invention may encode or include a dendritic cell targeting agent or moiety. In particular embodiments, antigens of the present invention, e.g., PCSK9 antigens, TNF alpha antigens, IL-17A antigens, and GDF8 antigens, can be encoded by a TAV composition of the invention in combination with a dendritic cell targeting agent or moiety. In certain embodiments, the antigen is fused to a dendritic cell targeting agent or moiety. In particular embodiments, an antigen is fused to a polypeptide(s) encoding an antibody, antibody fragment thereof (ScFc, dAb, VHH, etc), engineered protein scaffold (e.g. fibronectin, transferrin, Kunitz domain etc), or a peptide that target one or more dendritic cell surface marker(s). Such markers include, but are not limited to, DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36. In certain embodiments, a TAV includes a polypeptide encoding an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, a IL-17A antigen, or a GDF8 antigen, and also included a dendritic cell targeting agent.

Immunomodulatory agent or moiety

[000166] The TAVs of the present invention may encode or include one or more immunomodulatory agents or moieties. These molecules may be encoded by a polynucleotide or be present as a polypeptide, or they may be non-proteinaceous. Examples of such immunomodulatory agents and/or moieties include, but are not limited to GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti- PD1, anti-PDL1/2, anti-OX40/OX40L, anti-GITR/GITRL, or anti-TIM3. The immunomodulatory agents and/or moieties may act to alter the immune response of the TAV but preferably induce or enhance the immune response. Immunomodulatory agents and/or moieties can also be incorporated into the TAVs as a single transcript vaccine (along with the encoded antigen) as a poly-cistronic mRNA, 2A self-cleavage mediated polypeptide, or protease-mediated polypeptide (using a furin/PACE cleavage system). In certain embodiments, a TAV includes a polynucleotide encoding an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, a IL-17A antigen, or a GDF8 antigen, and also includes an immunomodulatory agent or moiety. In particular embodiments, the polynucleotide encodes both the antigen and the immunomodulatory agent or moiety.

[000167] In certain embodiments, the immunomodulatory agent or moiety is an immunogenicity enhancing polypeptide also referred to herein as an immunogenicity enhancing motif (IM). Non-limiting examples of immunogenicity enhancing peptides include, e.g., mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, or a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid, and fragments and variants thereof. The polypeptide sequences of these immunogenicity enhancing polypeptides are provided in the accompanying Examples. In particular embodiments, a variant has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference wild type immunogenicity enhancing polypeptide, including any of those described herein. In particular embodiments, a variant includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, insertions, or deletions as compared to a reference wild type immunogenicity enhancing polypeptide, including any of those described herein. In particular embodiments, a TAV composition encodes two or more immunogenicity enhancing polypeptides, which may optionally be separated by a linker sequence or a cleavage site, e.g., a cathepsin cleavage site (VVR). The two or more immunogenicity enhancing polypeptides may be the same or different.

[000168] In another non-limiting example, the immunomodulatory agent or moiety may comprise a bacterial or viral protein or fragment thereof to enhance antigenicity. In some embodiments, the immunomodulatory agent may comprise a viral vaccine or a viral cell surface protein or epitope. In some embodiments, the immunomodulatory agent or moiety may comprise a protein or fragment thereof derived from a virus selected from the group of tetanus, chicken pox, rubella, small pox and mumps.

[000169] In some embodiments, the bacterial or viral protein fragment is encoded on the same polynucleotide as the TAV antigen as a poly-cistronic transcript. In some embodiments, the TAV construct may comprise a non-mammalian lead-in sequence.

[000170] In some embodiments, the immunomodulatory agent maybe be 1, 2, 3-5, or greater than 5 amino acids in length. In another non-limiting example, the immunomodulatory agent may be comprised of 6 amino acids, including but not limited to KKPWQ. In some embodiments, the immunomodulatory agent or moiety is engineered into the sequence of the antigen moiety, e.g., a PCSK9 antigen moiety, a TNF alpha antigen moiety, a IL-17A antigen moiety, or a GDF8 antigen moiety, encoded by the TAV composition, to provide an internal epitope. In other embodiments, the immunomodulatory agent or moiety is positioned at the N and/or C terminus of the antigen moiety.

Reversing agent or composition

[000171] A reversing agent may also be administered once the desired effect of TAV is achieved and TAV is no longer needed, to turn off TAV, for example to prevent the accumulation of excessive amounts of autoantibodies.

[000172] In certain embodiments, the reversing agent is the proteasome inhibitor Bortezomib (VELCADE®, Millennium Pharmaceuticals), a boronic acid dipeptide which reversibly binds to the 26 proteasome, which has been approved by the US Food and Drug Administration (FDA) for the treatment of multiple myeloma (a plasma cell tumor) and is also being investigated for the treatment of autoimmune diseases and as a desensitizing agent in transplant patients with donor specific alloantibodies.

[000173] Bortezomib has been reported to eliminate autoantibody production and to improve the clinical condition of glomulo-nephritis in mice in a model of lupus-like disease. The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat. Medicine 200914 (7):748-755) and in a rat model of autoimmune myaththenia gravis (EMAG), (Gomez AM et al.,

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis. J Immunol.2011;186(4):2503-13). In a mouse model of organ transplantation, Bortezomib reduced donor-specific antigen antibodies and depleted the plasma cells that secreted these antibodies. In transplant patients, including sensitized transplant patients, bortezomib provides effective treatment for antibody mediated rejection (AMR) with minimal toxicity and provides sustained reduction in donor-specific antigen antibodies (Everly MJ et al., Bortezomib provides effective therapy for antibody- and cell-mediated acute rejection.

Transplantation.2008;86 (12):1754-61; Walsh RC et al., Proteasome inhibitor-based therapy for antibody-mediated rejection. Kidney Int.2012 (11):1067-74; Heidt S et al., Bortezomib affects the function of human B cells: possible implications for desensitization protocols. Clin. Transpl.2009:387-92; Morrow, WR et al., Rapid Reduction in Donor-Specific Anti-Human Leukocyte Antigen Antibodies and Reversal of Antibody-Mediated Rejection with Bortezomib in Pediatric Heart Transplant Patients Tansplantation 2012, 93:319-324). [000174] Without wishing to be bound by theory, Bortezomib reduces autoantibody titers in autoimmune disease and alloantibody titers in transplant patients by specifically targeting and depleting both short and long-lived plasma cells though induction of apoptosis in these cells. Because of Bortezomib’s ability to desensitize the immune response, in some embodiments, the drug may be used to reverse or turn off TAV.

[000175] CD20 is a glycosylated phospho-protein expressed on the surface of B- cells which plays a role in the differentiation of B cells into plasma cells, and is also a target under investigation for B cell depletion therapy as a treatment to prevent or reduce autoantibody production. In a mouse model of systemic lupus erythematosus (SLE), weekly dosing with an anti-mouse CD20 antibody caused B cell depletion and delayed the onset or reduced progression of nephritis (Bekar, et al. Prolonged Effects of Short-Term Anti CD20 B Cell Depletion Therapy in Murine Systemic Lupus Erthematosus. Arthritis & Rheumatism 2010 (62): 2443-2457). In a mouse model of Hemophilia A, anti-CD20 therapy through selective partial B cell depletion lead to tolerance to FVIII therapy (Zhang et al., Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice. Blood 2011117: 2223-2226). Montalvao et al. found that the liver may be the major site for B cell depletion, with Kupffer cells playing a key role, and with recirculation accounting for the decrease in B cell numbers observed in secondary lymphoid organs (Montalvao et al., The mechanism of anti-CD20-mediated B cell depletion revealed by intravital imaging J. Clin. Invest.2013123:5098-5103).

[000176] In some embodiments, anti-CD20 antibodies may be used as a reversing agent to turn off TAV. Without wishing to be bound by theory, the reversal may occur through elimination of antibodies generated by TAV via B cell depletion.

[000177] Rituximab, a chimeric monoclonal antibody against CD20, has been used to treat antibody mediated rejection (Genberg H et al., Pharmacodynamics of rituximab in kidney allotransplantation. Am J Transplant.20066(10):2418-28).

Without wishing to be bound by theory, Rituximab inhibits activated B cells by binding to CD20 on the B cell surface, leading to their rapid elimination from the circulation. In another non-limiting example of the current invention, Rituximab may be used as a reversing agent to turn off TAV.

Adjuvants [000178] Adjuvants or immune potentiators, may also be administered with or in combination with one or more TAV compositions of the invention. For example, pharmaceutical compositions and liposomal formuations described herein may comprise both a polynucleotide encoding and antigen and one or more adjuvant. Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccines. These may be co-administered by any route, e.g., intramuscular, subcutaneous, IV or intradermal injections.

[000179] Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.

[000180] Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminum hydroxide and aluminum or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilized oil-in-water emulsion, purified saponin, oil-in-water emulsion, stabilized water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes

(unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and/or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensoactive compounds; (9) carbohydrates; or combinations thereof.

[000181] Adjuvants for nucleic acid vaccines (DNA) have been disclosed in, for example, Kobiyama, et al Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. Any of the adjuvants disclosed by Kobiyama may be used in the TAVs of the present invention.

[000182] Other adjuvants which may be utilized in the TAVs of the present invention include any of those listed on the web-based vaccine adjuvant database, http://www.violinet.org/vaxjo/ and described in for example Sayers, et al., J.

Biomedicine and Biotechnology, volume 2012 (2012), Article ID 831486, 13 pages, the content of which is incorporated herein by reference in its entirety.

Polynucleotides

[000183] The present invention provides nucleic acid molecules, specifically polynucleotides that, in some embodiments, encode one or more peptides or polypeptides of interest, such as a PCSK9, TNF alpha, IL-17A, or GDF8 antigen described herein, or a polypeptide listed in Tables 1-8.14, 15, or 17-20. Such peptides or polypeptides, according to the invention may serve as an antigen or antigenic molecule. The term“nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides.

[000184] Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (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 hybrids or combinations thereof.

[000185] In one embodiment, linear polynucleotides encoding one or more antigen, e.g. PCSK9, TNF alpha, IL-17A, or GDF8 antigen, of the TAVs of the present invention that are made using only in vitro transcription (IVT) enzymatic synthesis methods are referred to as“IVT polynucleotides.” Methods of making IVT polynucleotides are known in the art and are described herein and i

PCT/US2014/069155, the contents of each of which is herein incorporated by reference in their entireties.

[000186] In some embodiments, the polynucleotides of the present invention have portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing, and are known as “chimeric polynucleotides.” A“chimera” according to the present invention is an entity having two or more incongruous or heterogeneous parts or regions. As used herein a“part” or“region” of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide. Examples of representative polynucleotide structures are in figures1-8.

[000187] In yet another embodiment, the polynucleotides of the present invention that are circular are known as“circular polynucleotides” or“circP.” As used herein, “circular polynucleotides” or“circP” means a single stranded circular polynucleotide which acts substantially like, and has the properties of, an RNA. The term“circular” is also meant to encompass any secondary or tertiary configuration of the circP.

Examples are shown in Figures 6-8.

[000188] In some embodiments, the polynucleotide includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[000189] 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.”

[000190] In one embodiment, the polynucleotides of the present invention is a messenger RNA or functions as a messenger RNA. 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. [000191] In one embodiment, the polynucleotides of the present invention may be structurally modified or chemically modified. As used herein, a“structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to“AT-5meC-G”. The same polynucleotide may be structurally modified from“ATCG” to“ATCCCG”. Here, the dinucleotide“CC” has been inserted, resulting in a structural modification to the polynucleotide. Structurally or chemically modified polynucleotides may be referred to herein as“modified polynucleotides.”

[000192] In one embodiment, the polynucleotides of the present invention, such as mRNAs or IVT polynucleotides, may have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In another embodiment, the polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way).

[000193] In one embodiment, the polynucleotides of the present invention may include a sequence encoding a self-cleaving peptide. The self-cleaving peptide may be, but is not limited to, a 2A peptide. As a non-limiting example, the 2A peptide may have the protein sequence: GSGATNFSLLKQAGDVEENPGP, fragments or variants thereof. In one embodiment, the 2A peptide cleaves between the last glycine and last proline. As another non-limiting example, the polynucleotides of the present invention may include a polynucleotide sequence encoding the 2A peptide having the protein sequence GSGATNFSLLKQAGDVEENPGP fragments or variants thereof.

[000194] One such polynucleotide sequence encoding the 2A peptide is

GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGA GGAGAACCCTGGACCT. 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.

[000195] In one embodiment, this sequence may be used to separate the coding region of two or more polypeptides of interest. As a non-limiting example, the sequence encoding the 2A peptide may be between a first coding region A and a second coding region B (A-2Apep-B). The presence of the 2A peptide would result in the cleavage of one long protein into protein A, protein B and the 2A peptide. Protein A and protein B may be the same or different peptides or polypeptides of interest. In another embodiment, the 2A peptide may be used in the polynucleotides of the present invention to produce two, three, four, five, six, seven, eight, nine, ten or more proteins. In certain embodiments, the 2A peptide sequence is located between the coding regions of the antigen, e.g., a PCSK9, TNF alpha, IL-17A, or GDF8 antigen, and the immune enhancing polypeptide.

[000196] In certain embodiments, the polynucleotides of the present invention may include a sequence encoding a cleavage site, such as, e.g., a cathepsin S cleavage site. Similarly, this seuqencce may be used to separate the coding region of two or more polypeptides of interest, such as two or more antigens encoded by the same polynucleotide.

IVT Polynucleotide Architecture

[000197] In some embodiments, the polynucleotides are codon optimized, e.g. for expression in human cells or host cells for recombinant production. In particular embodiments, TAV compositions comprise an mRNA encoding the PCSK9, TNF alpha, IL-17A, or GDF8 antigen and, optionally, an immune enhancing polypeptide and/or dendritic cell targeting agent. In particular embodiments, the mRNA is an IVT polynucleotide. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. The IVT polynucleotides of the present invention may function as mRNA but are distinguished from wild-type mRNA in their functional and/or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics.

[000198] In certain embodiments, the present invention comprises a polynucleotide, e.g., an mRNA or modified mRNA comprising in 5’ to 3’ order: (a) a 5’-UTR; (b) a first coding region comprising a sequence encoding one or more polypeptides or antigens; (c) a second coding region comprising a sequence encoding one or more immunomodulatory polypeptides or dendritic cell targeting polypeptides; and (d) a 3’UTR. In certain embodiments, the present invention comprises a polynucleotide, e.g., an mRNA or modified mRNA comprising in 5’ to 3’ order: (a) a 5’-UTR; (b) a first coding region comprising a sequence encoding one or more immunomodulatory polypeptides or dendritic cell targeting polypeptide; (c) a second coding region comprising a sequence encoding one or more polypeptides or antigens; and (d) a 3’UTR. In some embodiments, the one or more polypeptides or antigens are one or more PCSK9 polypeptides or antigens. In certain embodiments, the one or more polypeptides or antigens are one or more TNF alpha polypeptides or antigens. In particular embodiments, the one or more polypeptides or antigens are one or more IL- 17A polypeptides or antigens. In various embodiments, the one or more polypeptides or antigens are one or more GDF8 polypeptides or antigens. In particular embodiments, the polynucleotide comprises a sequence encoding an

immunomodulatory polypeptide. In certain embodiments, the mRNA comprises a modified nucleobase. The mRNA may also comprise a third region between the first region and the second region, wherein the third region comprises a sequence encoding a linker, spacer or a cleavage site. In certain instances, the linker, spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site, which allows cleavage and separation of the antigen and the immunomodulatory polypeptide following translation in a cell. In particular embodiments, the polynucleotide further comprises a 5’-cap and a polyA tail. The present invention further include polynucleotides, e.g., DNA molecules, cDNAs, or vectors, that are transcribed to produce a TAV-encoding polynucleotide, e.g., mRNA, described herein. In particular embodiments, the cDNAs comprise a sequence set forth herein, e.g., in Table 14, or comprise a complement thereof. In particular embodiments, the polynucleotide is a vector, e.g., a vector that may be used as a template for IVT production of a TAV mRNA described herein.

[000199] Figure 1 shows a primary construct 100 of an IVT polynucleotide of the present invention. As used herein,“primary construct” refers to a polynucleotide of the present invention which encodes one or more polypeptides of interest and which retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated.

[000200] According to FIG.1A and 1B, the primary construct 100 of an IVT polynucleotide here contains a first region of linked nucleotides 102 that is flanked by a first flanking region 104 and a second flaking region 106. The first flanking region 104 may include a sequence of linked nucleosides which function as a 5’ untranslated region (UTR) such as the 5’ UTR of any of the nucleic acids encoding the native 5’UTR of the polypeptide or a non-native 5’UTR such as, but not limited to, a heterologous 5’UTR or a synthetic 5’UTR. The polypeptide of interest may comprise at its 5’ terminus one or more signal sequences encoded by a signal sequence region 103. The flanking region 104 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5′ UTRs sequences which may be completely codon optimized or partially codon optimized. The flanking region 104 may include at least one nucleic acid sequence including, but not limited to, miR sequences, TERZAKTM sequences and translation control sequences. The flanking region 104 may also comprise a 5′ terminal cap 108. The 5′ terminal capping region 108 may include a naturally occurring cap, a synthetic cap or an optimized cap. Non-limiting examples of optimized caps include the caps taught by Rhoads in US Patent No. US7074596 and International Patent Publication No. WO2008157668,

WO2009149253 and WO2013103659, the contents of each of which are herein incorporated by reference in its entirety. The second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3′ UTRs which may encode the native 3’ UTR of the polypeptide or a non-native 3’UTR such as, but not limited to, a heterologous 3’UTR or a synthetic 3’ UTR. The second flanking region 106 may be completely codon optimized or partially codon optimized. The flanking region 106 may include at least one nucleic acid sequence including, but not limited to, miR sequences and translation control sequences. The flanking region 106 may also comprise a 3′ tailing sequence 110. The 3’ tailing sequence 110 may include a synthetic tailing region 112 and/or a chain terminating nucleoside 114. Non-liming examples of a synthetic tailing region include a polyA tail, a polyC tail, a polyA-G quartet and/or a stem loop sequence. Non-limiting examples of chain terminating nucleosides include 2’-O methyl, F and locked nucleic acids (LNA).

[000201] Bridging the 5′ terminus of the first region 102 and the first flanking region 104 is a first operational region 105. Traditionally this operational region comprises a Start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a Start codon. [000202] Bridging the 3′ terminus of the first region 102 and the second flanking region 106 is a second operational region 107. Traditionally this operational region comprises a Stop codon. The operational region may alternatively comprise any translation initiation sequence or signal including a Stop codon. Multiple serial stop codons may also be used in the IVT polynucleotide. In one embodiment, the operation region of the present invention may comprise two stop codons. The first stop codon may be“TGA” or“UGA” and the second stop codon may be selected from the group consisting of“TAA,”“TGA,”“TAG,”“UAA,”“UGA” or“UAG.”

[000203] The shortest length of the first region of the primary construct of the IVT polynucleotide of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids. Examples of dipeptides that the polynucleotide sequences can encode or include, but are not limited to, carnosine and anserine.

[000204] The length of the first region of the primary construct of the IVT polynucleotide encoding the polypeptide of interest of the 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).

[000205] In some embodiments, the IVT polynucleotide includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[000206] According to the present invention, the first and second flanking regions of the IVT polynucleotide may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[000207] According to the present invention, the tailing sequence of the IVT polynucleotide may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.

[000208] According to the present invention, the capping region of the IVT polynucleotide may comprise a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be from 1 to 10, e.g.2-9, 3-8, 4-7, 1-5, 5- 10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.

[000209] According to the present invention, the first and second operational regions of the IVT polynucleotide may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences. [000210] In one embodiment, the IVT polynucleotides of the present invention may be structurally modified or chemically modified. When the IVT polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides may be referred to as“modified IVT polynucleotides.”

[000211] In one embodiment, if the IVT polynucleotides of the present invention are chemically modified they may have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In another embodiment, the IVT polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way).

[000212] In one embodiment, the IVT polynucleotides of the present invention may include a sequence encoding a self-cleaving peptide, described herein, such as but not limited to the 2A peptide. The polynucleotide sequence of the 2A peptide in the IVT polynucleotide may be modified or codon optimized by the methods described herein and/or are known in the art.

[000213] In one embodiment, the IVT polynucleotide may encode at least one peptide or polypeptide of interest. In another embodiment, the IVT polynucleotide may encode two or more peptides or polypeptides of interest, such as a PCSK9, TNF alpha, IL-17A, or GDF8 antigen and an immune enhancing polypeptide.

[000214] IVT polynucleotides (such as, but not limited to, primary constructs), formulations and compositions comprising IVT polynucleotides, and methods of making, using and administering IVT polynucleotides are described in

PCT/US2014/069155, the contents of each of which are herein incorporated by reference in their entireties. Any of the recited polypeptides of the IVT

polynucleotides of the foregoing are considered useful as a polypeptide of interest or antigen of the TAVs of the present invention.

Chimeric Polynucleotide Architecture

[000215] The chimeric polynucleotides or RNA constructs of the present invention maintain a modular organization similar to IVT polynucleotides, but the chimeric polynucleotides comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide. As such, the chimeric polynucleotides which are modified mRNA molecules of the present invention are termed“chimeric modified mRNA” or“chimeric mRNA.”

[000216] It is to be understood that the antigens of the TAVs of the present invention may be encoded by a variety of different polynucleotides, including but not limited to a chimeric polynucleotide, IVT polynucleotide, RNA construct, mRNA, modified mRNA, chimeric modified mRNA or chimeric mRNA.

[000217] Chimeric polynucleotides have portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing.

[000218] A chimeric polynucleotide may function as an mRNA and encodes a polypeptide of interest include, and include untranslated regions (UTRs, such as the 5’ UTR or 3’ UTR), coding regions, cap regions, polyA tail regions, start regions, stop regions, signal sequence regions, and combinations thereof. Figure 2 illustrates certain embodiments of the chimeric polynucleotides of the invention which may be used as mRNA. Figure 3 illustrates a schematic of a series of chimeric

polynucleotides identifying various patterns of positional modifications and showing regions analogous to those regions of an mRNA polynucleotide.

[000219] Pattern chimeras may vary in their chemical modification by degree (such as those described above) or by kind (e.g., different modifications).

[000220] Chimeric polynucleotides, including the parts or regions thereof, of the present invention having at least one region with two or more different chemical modifications of two or more nucleoside members of the same nucleoside type (A, C, G, T, or U) are referred to as“positionally modified” chimeras. Positionally modified chimeras are also referred to herein as“selective placement” chimeras or“selective placement polynucleotides”. As the name implies, selective placement refers to the design of polynucleotides which, unlike polynucleotides in the art where the modification to any A, C, G, T or U is the same by virtue of the method of synthesis, can have different modifications to the individual As, Cs, Gs, Ts or Us in a polynucleotide or region thereof. For example, in a positionally modified chimeric polynucleotide, there may be two or more different chemical modifications to any of the nucleoside types of As, Cs, Gs, Ts, or Us. There may also be combinations of two or more to any two or more of the same nucleoside type. For example, a positionally modified or selective placement chimeric polynucleotide may comprise 3 different modifications to the population of adenines in the molecule and also have 3 different modifications to the population of cytosines in the construct—all of which may have a unique, non-random, placement.

[000221] Chimeric polynucleotides, including the parts or regions thereof, of the present invention having a chemical modification percent are referred to as“percent chimeras.” Percent chimeras may have regions or parts which comprise at least 1%, at least 2%, at least 5%, at least 8%, at least 10%, at least 20%, 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 at least 99% positional, pattern or population of modifications. Alternatively, the percent chimera may be completely modified as to modification position, pattern, or population. The percent of modification of a percent chimera may be split between naturally occurring and non-naturally occurring modifications.

[000222] Chimeric polynucleotides, including the parts or regions thereof, of the present invention having a chemical modification population are referred to as “population chimeras.” A population chimera may comprise a region or part where nucleosides (their base, sugar or backbone linkage, or combination thereof) have a select population of modifications. Such modifications may be selected from functional populations such as modifications which induce, alter or modulate a phenotypic outcome. For example, a functional population may be a population or selection of chemical modifications which increase the level of a cytokine. Other functional populations may individually or collectively function to decrease the level of one or more cytokines. Use of a selection of these like-function modifications in a chimeric polynucleotide would therefore constitute a“functional population chimera.” As used herein, a“functional population chimera” may be one whose unique functional feature is defined by the population of modifications as described above or the term may apply to the overall function of the chimeric polynucleotide itself. For example, as a whole the chimeric polynucleotide may function in a different or superior way as compared to an unmodified or non-chimeric polynucleotide.

[000223] It should be noted that polynucleotides which have a uniform chemical modification of all of any of the same nucleoside type or a population of

modifications produced by mere downward titration of the same starting modification in all of any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine, are not considered chimeric. Likewise, polynucleotides having a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (such as all uridines and all cytosines, etc. are modified in the same way) are not considered chimeric polynucleotides. One example of a polynucleotide which is not chimeric is the canonical pseudouridine/5-methyl cytosine modified polynucleotide of the prior art. These uniform polynucleotides are arrived at entirely via in vitro transcription (IVT) enzymatic synthesis; and due to the limitations of the synthesizing enzymes, they contain only one kind of modification at the occurrence of each of the same nucleoside type, i.e., adenosine (A), thymidine (T), guanosine (G), cytidine (C) or uradine (U), found in the polynucleotide. Such polynucleotides may be characterized as IVT polynucleotides.

[000224] The chimeric polynucleotides of the present invention may be structurally modified or chemically modified. When the chimeric polynucleotides of the present invention are chemically and/or structurally modified the polynucleotides may be referred to as“modified chimeric polynucleotides.”

[000225] In some embodiments of the invention, the chimeric polynucleotides may encode two or more peptides or polypeptides of interest. Such peptides or

polypeptides of interest include the heavy and light chains of antibodies, an enzyme and its substrate, a label and its binding molecule, a second messenger and its enzyme or the components of multimeric proteins or complexes.

[000226] The regions or parts of the chimeric polynucleotides of the present invention may be separated by a linker or spacer moiety. Such linkers or spaces may be nucleic acid based or non-nucleosidic.

[000227] In one embodiment, the chimeric polynucleotides of the present invention may include a sequence encoding a self-cleaving peptide described herein, such as, but not limited to, a 2A peptide. The polynucleotide sequence of the 2A peptide in the chimeric polynucleotide may be modified or codon optimized by the methods described herein and/or are known in the art.

[000228] Notwithstanding the foregoing, the chimeric polynucleotides of the present invention may comprise a region or part which is not positionally modified or not chimeric as defined herein.

[000229] For example, a region or part of a chimeric polynucleotide may be uniformly modified at one or more A, T, C, G, or U but according to the invention, the polynucleotides will not be uniformly modified throughout the entire region or part.

[000230] Regions or parts of chimeric polynucleotides may be from 15-1000 nucleosides in length and a polynucleotide may have from 2-100 different regions or patterns of regions as described herein.

[000231] In one embodiment, chimeric polynucleotides encode one or more polypeptides of interest. In another embodiment, the chimeric polynucleotides are substantially non-coding. In another embodiment, the chimeric polynucleotides have both coding and non-coding regions and parts.

[000232] Figure 2 illustrates the design of certain chimeric polynucleotides of the present invention when based on the scaffold of the polynucleotide of Figure 1.

Shown in the figure are the regions or parts of the chimeric polynucleotides where patterned regions represent those regions which are positionally modified and open regions illustrate regions which may or may not be modified but which are, when modified, uniformly modified. Chimeric polynucleotides of the present invention may be completely positionally modified or partially positionally modified. They may also have subregions which may be of any pattern or design. Shown in Figure 2 are a chimeric subregion and a hemimer subregion.

[000233] According to the present invention, chimeric polynucleotides which function as an mRNA may have a capping region. The capping region may comprise a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be from 1 to 10, e.g.2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.

[000234] The present invention contemplates chimeric polynucleotides which are circular or cyclic. As the name implies circular polynucleotides are circular in nature meaning that the termini are joined in some fashion, whether by ligation, covalent bond, common association with the same protein or other molecule or complex or by hybridization. Any of the circular polynucleotides as taught in for example PCT Application Publication No. WO2015034928, the contents of which are incorporated herein by reference in their entirety, may be made chimeric according to the present invention.

[000235] Chimeric polynucleotides, formulations and compositions comprising chimeric polynucleotides, and methods of making, using and administering chimeric polynucleotides are also described in PCT Application Publication No.

WO2015034928, which is incorporated by reference in its entirety.

Circular Polynculeotide Architecture

[000236] The present invention contemplates polynucleotides which are circular or cyclic. As the name implies circular polynucleotides are circular in nature meaning that the termini are joined in some fashion, whether by ligation, covalent bond, common association with the same protein or other molecule or complex or by hybridization. Any of the circular polynucleotides as taught in for example PCT Application Publication No. WO2015034928, the contents of which are incorporated herein by reference in their entirety.

[000237] Circular polynucleotides of the present invention may be designed according to the circular RNA construct scaffolds shown in Figures 6-8. Such polynucleotides are circular polynucleotides or circular constructs.

[000238] The circular polynucleotides or circPs of the present invention, which encode at least one peptide or polypeptide of interest, are known as circular RNAs or circRNA. The antigens of the TAVs of the present invention may be encoded by one or more circular RNAs or circRNAs.

[000239] Figure 6 shows a representative circular construct 200 of the circular polynucleotides of the present invention. As used herein, the term“circular construct” refers to a circular polynucleotide transcript which may act substantially similar to and have properties of a RNA molecule. In one embodiment the circular construct acts as an mRNA. If the circular construct encodes one or more peptides or polypeptides of interest (e.g., a PCSK9, TNF alpha, IL-17A, or GDF8 antigen) then the polynucleotide transcript retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated. Circular constructs may be polynucleotides of the invention. When structurally or chemically modified, the construct may be referred to as a modified circP, modified circSP, modified circRNA or modified circRNA-SP.

[000240] Returning to FIG.6, the circular construct 200 here contains a first region of linked nucleotides 202 that is flanked by a first flanking region 204 and a second flanking region 206. As used herein, the“first region” may be referred to as a “coding region,” a“non-coding region” or“region encoding” or simply the“first region.” In one embodiment, this first region may comprise nucleotides such as, but is not limited to, encoding at least one peptide or polypeptide of interest and/or nucleotides encoding a sensor region. The peptide or polypeptide of interest may comprise at its 5’ terminus one or more signal peptide sequences encoded by a signal peptide sequence region 203. The first flanking region 204 may comprise a region of linked nucleosides or portion thereof which may act similarly to an untranslated region (UTR) in an mRNA and/or DNA sequence. The first flanking region may also comprise a region of polarity 208. The region of polarity 208 may include an IRES sequence or portion thereof. As a non-limiting example, when linearlized this region may be split to have a first portion be on the 5’ terminus of the first region 202 and second portion be on the 3’ terminus of the first region 202. The second flanking region 206 may comprise a tailing sequence region 210 and may comprise a region of linked nucleotides or portion thereof 212 which may act similarly to a UTR in an mRNA and/or DNA.

[000241] Bridging the 5′ terminus of the first region 202 and the first flanking region 104 is a first operational region 205. In one embodiment, this operational region may comprise a start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a start codon.

[000242] Bridging the 3′ terminus of the first region 202 and the second flanking region 106 is a second operational region 207. Traditionally this operational region comprises a stop codon. The operational region may alternatively comprise any translation initiation sequence or signal including a stop codon. According to the present invention, multiple serial stop codons may also be used. In one embodiment, the operation region of the present invention may comprise two stop codons. The first stop codon may be“TGA” or“UGA” and the second stop codon may be selected from the group consisting of“TAA,”“TGA,”“TAG,”“UAA,”“UGA” or“UAG.”

[000243] Turning to Figure 7, at least one non-nucleic acid moiety 201 may be used to prepare a circular construct 200 where the non-nucleic acid moiety 201 is used to bring the first flanking region 204 near the second flanking region 206. Non-limiting examples of non-nucleic acid moieties which may be used in the present invention are described herein. The circular construct 200 may comprise more than one non- nucleic acid moiety wherein the additional non-nucleic acid moieties may be heterologous or homologous to the first non-nucleic acid moiety.

[000244] Turning to Figure 8, the first region of linked nucleosides 202 may comprise a spacer region 214. This spacer region 214 may be used to separate the first region of linked nucleosides 202 so that the circular construct can include more than one open reading frame, non-coding region or an open reading frame and a non- coding region.

[000245] Circular polynucleotides, formulations and compositions comprising circular polynucleotides, and methods of making, using and administering circular polynucleotides are also described in PCT Application Publication No.

WO2015034928, which is incorporated by reference in its entirety.

Conjugates and Combinations of Polynucleotides

[000246] In order to further enhance protein production, polynucleotides of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, 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, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.

[000247] In a preferred embodiment, the polynucleotides of the present invention which encode an antigen are conjugated to one or more dendritic cell markers.

[000248] Conjugation may result in increased stability and/or half-life and may be particularly useful in targeting the polynucleotides to specific sites in the cell, tissue or organism.

[000249] According to the present invention, the polynucleotides may be administered with, conjugated to or further encode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.

Polynucleotide Regions

[000250] In some embodiments, polynucleotides may be designed to comprise regions, subregions or parts which function in a similar manner as known regions or parts of other nucleic acid based molecules. Such regions include those mRNA regions discussed herein as well as noncoding regions. Noncoding regions may be at the level of a single nucleoside such as the case when the region is or incorporates one or more cytotoxic nucleosides. In particular embodiments, polynucleotides of the present invention, including mRNAs comprise one or more of the following regions, e.g., untranslated regions.

Polynucleotides having Untranslated Regions (UTRs)

[000251] The polynucleotides of the present invention may comprise one or more regions or parts which act or function as an untranslated region. Where

polynucleotides are designed to encode at least one polypeptide of interest, the polynucleotides may comprise one or more of these untranslated regions.

[000252] By definition, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. In mRNA, the 5′UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3′UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present invention to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5’UTR and 3’UTR sequences are known and available in the art. 5′ UTR and Translation Initiation

[000253] Natural 5′UTRs bear features which play roles in translation initiation. They harbor signatures like Kozak sequences which 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′UTR also have been known to form secondary structures which are involved in elongation factor binding.

[000254] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides of the invention. 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 Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a 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 (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D). [consider referencing the PCT application]

[000255] Other non-UTR sequences may also be used as regions or subregions within the polynucleotides. For example, introns or portions of introns sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucletoide levels.

[000256] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5’UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5’UTRs described in US Patent Application Publication No.20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety.

[000257] It should be understood that any UTR from any gene may be incorporated into the regions of the polynucleotide. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term“altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ or 5′ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR. [000258] In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′ UTR may be used. As used herein, a“double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.

[000259] It is also within the scope of the present invention to have patterned UTRs. As used herein“patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[000260] In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a“family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[000261] The untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art.

3′ UTR and the AU Rich Elements

[000262] Natural or wild type 3′ UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well- studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[000263] Introduction, removal or modification of 3′ UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the invention. When engineering specific polynucleotides, one or more copies of an ARE can be introduced to make polynucleotides of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using polynucleotides of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

microRNA Binding Sites

[000264] microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bind to the 3′UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. The polynucleotides of the invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US Publication

US2005/0261218 and US Publication US2005/0059005, the contents of which are incorporated herein by reference in their entirety.

[000265] A microRNA sequence comprises a“seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson- Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. In some embodiments, a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. See for example, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6;27(1):91-105; each of which is herein incorporated by reference in their entirety. The bases of the microRNA seed have complete complementarity with the target sequence. By engineering microRNA target sequences into the polynucleotides (e.g., in a 3’UTR like region or other region) of the invention one can target the molecule for degradation or reduced translation, provided the microRNA in question is available. This process will reduce the hazard of off target effects upon nucleic acid molecule delivery. Identification of microRNA, microRNA target regions, and their expression patterns and role in biology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; 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 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; each of which is herein incorporated by reference in its entirety).

[000266] For example, if the nucleic acid molecule is an mRNA and is not intended to be delivered to the liver but ends up there, then miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3′ UTR region of the polynucleotides. Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of polynucleotides.

[000267] As used herein, the term“microRNA site” refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that“binding” may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.

[000268] Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they occur, e.g., in order to increase protein expression in specific tissues. For example, miR-122 binding sites may be removed to improve protein expression in the liver. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA binding sites. [000269] Examples of tissues where microRNA 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). MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176; herein incorporated by reference in its entirety).

[000270] Expression profiles, microRNA and cell lines useful in the present invention include those taught in for example, U.S. Provisional Application Nos 61/857,436 (Attorney Docket Number M39) and 61/857,304 (Attorney Docket Number M37) each filed July 23, 2013, the contents of which are incorporated by reference in their entirety.

[000271] In the polynucleotides of the present invention, binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the polynucleotides expression to biologically relevant cell types or to the context of relevant biological processes. A listing of microRNA, miR sequences and miR binding sites is listed in Table 9 of U.S.

Provisional Application No.61/753,661 filed January 17, 2013, in Table 9 of U.S. Provisional Application No.61/754,159 filed January 18, 2013, and in Table 7 of U.S. Provisional Application No.61/758,921 filed January 31, 2013, each of which are herein incorporated by reference in their entireties.

[000272] Examples of use of microRNA to drive tissue or disease-specific gene expression are listed (Getner and Naldini, Tissue Antigens.2012, 80:393-403; herein incorporated by reference in its entirety). In addition, microRNA seed sites can be incorporated into mRNA to decrease expression in certain cells which results in a biological improvement. An example of this is incorporation of miR-142 sites into a UGT1A1-expressing lentiviral vector. The presence of miR-142 seed sites reduced expression in hematopoietic cells, and as a consequence reduced expression in antigen-presenting cells, leading to the absence of an immune response against the virally expressed UGT1A1 (Schmitt et al., Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al. Gastroenterology 2010, 139:726-729; both herein incorporated by reference in its entirety) . Incorporation of miR-142 sites into modified mRNA could not only reduce expression of the encoded protein in hematopoietic cells, but could also reduce or abolish immune responses to the mRNA-encoded protein. Incorporation of miR-142 seed sites (one or multiple) into mRNA would be important in the case of treatment of patients with complete protein deficiencies (UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.) .

[000273] Lastly, through an understanding of the expression patterns of microRNA in different cell types, polynucleotides can be engineered for more targeted expression in specific cell types or only under specific biological conditions. Through introduction of tissue-specific microRNA binding sites, polynucleotides could be designed that would be optimal for protein expression in a tissue or in the context of a biological condition.

[000274] Transfection experiments can be conducted in relevant cell lines, using engineered polynucleotides and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different microRNA binding site-engineering polynucleotides and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7 days post-transfection. In vivo experiments can also be conducted using microRNA-binding site-engineered molecules to examine changes in tissue- specific expression of formulated polynucleotides.

Regions having a 5′ Cap

[000275] 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 removal during mRNA splicing.

[000276] Endogenous mRNA molecules may 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 may 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 may optionally also be 2′-O-methylated. 5′-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[000277] In some embodiments, polynucleotides may be designed to incorporate a cap moiety. Modifications to the polynucleotides of the present invention may generate 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 may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may 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 may be used such as α-methyl- phosphonate and seleno-phosphate nucleotides.

[000278] 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 which functions as an mRNA molecule.

[000279] 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 may be chemically (i.e. non- enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.

[000280] 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 may 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.

[000281] 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). [000282] In one embodiment, the cap is a dinucleotide cap analog. As a non- limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in US Patent No. US 8,519,110, the contents of which are herein incorporated by reference in its entirety.

[000283] 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)-m3’-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 invention is a 4-chloro/bromophenoxyethyl analog.

[000284] 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, may lead to reduced translational competency and reduced cellular stability.

[000285] Polynucleotides of the invention may 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 invention are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5′ endonucleases and/or reduced 5′decapping, 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).

[000286] As a non-limiting example, capping chimeric polynucleotides post- manufacture may be more efficient as nearly 100% of the chimeric polynucleotides may 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.

[000287] According to the present invention, 5′ terminal caps may include endogenous caps or cap analogs. According to the present invention, a 5′ terminal cap may 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.

Viral Sequences

[000288] Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus (See e.g., International Pub. No. WO2012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the polynucleotides of the invention and can stimulate the translation of the construct in vitro and in vivo. Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.

IRES Sequences

[000289] Further, provided are polynucleotides which may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5′ cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).

Poly-A tails

[000290] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3' end of the transcript may 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.

[000291] PolyA tails may also be added after the construct is exported from the nucleus.

[000292] According to the present invention, terminal groups on the poly A tail may be incorporated for stabilization. Polynucleotides of the present invention may include des-3' hydroxyl tails. They may 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).

[000293] The polynucleotides of the present invention may 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.

[000294] Unique poly-A tail lengths provide certain advantages to the

polynucleotides of the present invention.

[000295] 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).

[000296] In one embodiment, 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 may 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.

[000297] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail may 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 may enhance expression.

[000298] Additionally, multiple distinct polynucleotides may 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, 72 hr and day 7 post-transfection.

[000299] In one embodiment, the polynucleotides of the present invention 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

[000300] In some embodiments, the polynucleotides of the present invention may have regions that are analogous to or function like a start codon region.

[000301] In one embodiment, the translation of a polynucleotide may initiate on a codon which is not the start codon AUG. Translation of the polynucleotide may 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, 2010 5:11; 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.

[000302] 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 may be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.

[000303] In one embodiment, a masking agent may 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).

[000304] In another embodiment, a masking agent may 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.

[000305] In one embodiment, a masking agent may 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.

[000306] In one embodiment, a start codon or alternative start codon may be located within a perfect complement for a miR binding site. The perfect complement of a miR binding site may 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 may be located in the middle of a perfect complement for a miR-122 binding site. The start codon or alternative start codon may 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.

[000307] In another embodiment, the start codon of a polynucleotide may be removed from the polynucleotide sequence in order to have the translation of the polynucleotide begin on a codon which is not the start codon. Translation of the polynucleotide may 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 may 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

[000308] In one embodiment, the polynucleotides of the present invention may include at least two stop codons before the 3’ untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In one embodiment, the polynucleotides of the present invention include the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA. In another embodiment, the polynucleotides of the present invention include three stop codons.

Signal Sequences

[000309] The polynucleotides may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a“signal sequence” or“signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5′ (or N-terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.

[000310] Additional signal sequences which may be utilized in the present invention include those taught in, for example, databases such as those found at

http://www.signalpeptide.de/ or http://proline.bic.nus.edu.sg/spdb/. Those described in US Patents 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the invention and the contents of each are incorporated herein by reference in their entirety.

Target

[000311] According to the present invention, the polynucleotides encode at least one polypeptide of interest, e.g., an antigen. In particular embodiments, the antigen is a PCSK9 polypeptide or fragment or variant thereof, including any of the PCSK9 antigens described herein, and variants or fragments thereof. Indications that may be treated using a TAV comprising or encoding a PCSK9 antigen include but are not limited to hypercholesterolemia and atherosclerosis.

[000312] In certain embodiments, the antigen is a TNF alpha polypeptide or fragment or variant thereof, including any of the TNF alpha antigens described herein, and variants or fragments thereof. Indications that may be treated using a TAV comprising or encoding a PCSK9 antigen include but are not limited to inflammatory disease, autoimmune disease, and cancer.

[000313] In particular embodiments, the antigen is an IL-17A polypeptide or fragment or variant thereof, including any of the IL-17A antigens described herein, and variants or fragments thereof. Indications that may be treated using a TAV comprising or encoding a IL-17A antigen include but are not limited to inflammatory disease, autoimmune disease, and cancer.

[000314] In some embodiments, the antigen is an GDF8 polypeptide or fragment or variant thereof, including any of the GDF8 antigens described herein, and variants or fragments thereof. Indications that may be treated using a TAV comprising or encoding a GDF8 antigen include but are not limited to muscle weakness (for example due to advanced age), degenerative muscle diseases such as muscular dystrophy, and muscle injury.

[000315]

Protein Cleavage Signals and Sites

[000316] In one embodiment, the polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C- termini such as, but not limited to, half-way between the N- and C-termini, between the N-terminus and the half way point, between the half way point and the C-terminus, and combinations thereof.

[000317] The polypeptides of the present invention may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin or Factor Xa protein cleavage signal. Proprotein convertases are a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that cleave at non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).

[000318] The polypeptides of the present invention may include, but are not limited to, members of the TNF super family. The TNF superfamily are a group of cytokines that are characterized by their ability to cuase cell death. The TNF superfamily includes TNF alpha (also known as TNF), lymphotoxin alpha, lymphotoxin beta, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[000319] The polypeptides of the present invention may include, but are not limited to, members of the IL-17 family. The IL-17 family includes cytokines that are proinflammatory. Members of the IL-17 family include IL-17A, IL-17B, IL-17C, IL- 17D, IL-17E, IL-17F, and IL-25.

[000320] The polypeptides of the present invention may include, but are not limited to, members of the Growth/differentiation factors (GDF) family, which is a subfamily of proteins belonging to the transforming growth factor beta superfamily that have functions predominantly in development. Members of the GDF family include GDF1, GDF2, GDF3, GDF4, GDF5, GDF6, GDF8 (myostatin), GDF9, GDF10, GDF11, and GDF15.

[000321]

[000322] In one embodiment, the polynucleotides of the present invention may be engineered such that the polynucleotide contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may be located in any region including but not limited to before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof.

[000323] In one embodiment, the polynucleotides of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal.

[000324] As a non-limiting example, U.S. Pat. No.7,374,930 and U.S. Pub. No. 20090227660, herein incorporated by reference in their entireties, use a furin cleavage site to cleave the N-terminal methionine of GLP-1 in the expression product from the Golgi apparatus of the cells. In one embodiment, the polypeptides of the present invention include at least one protein cleavage signal and/or site with the proviso that the polypeptide is not GLP-1.

Insertions and Substitutions

[000325] In one embodiment, the 5’UTR of the polynucleotide may be replaced by the insertion of at least one region and/or string of nucleosides of the same base. The region and/or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides may be natural and/or unnatural. As a non-limiting example, the group of nucleotides may include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.

[000326] In one embodiment, the 5’UTR of the polynucleotide may be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof. For example, the 5’UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5’UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.

[000327] In one embodiment, the polynucleotide may include at least one substitution and/or insertion downstream of the transcription start site which may be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and/or insertion may occur downstream the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site may affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its entirety). The modification, substitution and/or insertion of at least one nucleoside may cause a silent mutation of the sequence or may cause a mutation in the amino acid sequence.

[000328] In one embodiment, the polynucleotide may include the substitution of 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 or at least 13 guanine bases downstream of the transcription start site.

[000329] In one embodiment, the polynucleotide may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site. As a non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides. In another non- limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.

[000330] In one embodiment, the polynucleotide may include at least one substitution and/or insertion upstream of the start codon. For the purpose of clarity, one of skill in the art would appreciate that the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins. The polynucleotide may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases. The nucleotide bases may be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon. The nucleotides inserted and/or substituted may be the same base (e.g., all A or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases. As a non-limiting example, the guanine base upstream of the coding region in the polynucleotide may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example the substitution of guanine bases in the polynucleotide may be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et al. Nature (2011) 472(7344):499-503; the contents of which is herein incorporated by reference in its entirety). As a non-limiting example, at least 5 nucleotides may be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides may be the same base type. Incorporating Post Transcriptional Control Modulators [000331] In one embodiment, the polynucleotides of the present invention may include at least one post transcriptional control modulator. These post transcriptional control modulators may be, but are not limited to, small molecules, compounds and regulatory sequences. As a non-limiting example, post transcriptional control may be achieved using small molecules identified by PTC Therapeutics Inc. (South Plainfield, NJ) using their GEMSTM (Gene Expression Modulation by Small-Moleclues) screening technology.

[000332] The post transcriptional control modulator may be a gene expression modulator which is screened by the method detailed in or a gene expression modulator described in International Publication No. WO2006022712, herein incorporated by reference in its entirety. Methods identifying RNA regulatory sequences involved in translational control are described in International Publication No. WO2004067728, herein incorporated by reference in its entirety; methods identifying compounds that modulate untranslated region dependent expression of a gene are described in International Publication No. WO2004065561, herein incorporated by reference in its entirety.

[000333] In one embodiment, the polynucleotides of the present invention may include at least one post transcriptional control modulator is located in the 5’ and/or the 3’ untranslated region of the polynucleotides of the present invention.

[000334] In another embodiment, the polynucleotides of the present invention may include at least one post transcription control modulator to modulate premature translation termination. The post transcription control modulators may be compounds described in or a compound found by methods outlined in International Publication Nos. WO2004010106, WO2006044456, WO2006044682, WO2006044503 and WO2006044505, each of which is herein incorporated by reference in its entirety. As a non-limiting example, the compound may bind to a region of the 28S ribosomal RNA in order to modulate premature translation termination (See e.g.,

WO2004010106, herein incorporated by reference in its entirety).

[000335] In one embodiment, polynucleotides of the present invention may include at least one post transcription control modulator to alter protein expression. As a non- limiting example, the expression of VEGF may be regulated using the compounds described in or a compound found by the methods described in International

Publication Nos. WO2005118857, WO2006065480, WO2006065479 and

WO2006058088, each of which is herein incorporated by reference in its entirety. [000336] The polynucleotides of the present invention may include at least one post transcription control modulator to control translation. In one embodiment, the post transcription control modulator may be a RNA regulatory sequence. As a non- limiting example, the RNA regulatory sequence may be identified by the methods described in International Publication No. WO2006071903, herein incorporated by reference in its entirety. II. Design, Synthesis and Quantitation of Polynucleotides

Codon Optimization

[000337] The polynucleotides contained in the TAVs of the invention, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon 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. In one embodiment, the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.

Table 9. Codon Options

Figure imgf000119_0001
Figure imgf000120_0001

[000338] Features, which may be considered beneficial in some embodiments of the present invention, may be encoded by regions of the polynucleotide and such regions may be upstream (5’) or downstream (3’) to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon optimization of the protein encoding region or open reading frame (ORF). It is not required that a polynucleotide contain both a 5′ and 3′ flanking region. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags and may include multiple cloning sites which may have XbaI recognition.

[000339] In some embodiments, a 5′ UTR and/or a 3′ UTR region may be provided as flanking regions. Multiple 5′ or 3′ UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization.

[000340] After optimization (if desired), the polynucleotides components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, the optimized polynucleotide may 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.

[000341] Synthetic polynucleotides and their nucleic acid analogs play an important role in the research and studies of biochemical processes. Various enzyme-assisted and chemical-based methods have been developed to synthesize polynucleotides and nucleic acids.

Enzymatic Methods

In Vitro Transcription-enzymatic synthesis

[000342] cDNA encoding the polynucleotides described herein may 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 may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural

(modified) NTPs. The polymerase may 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 (e.g., modified nucleic acids). Any number of RNA polymerases or variants may be used in the synthesis of the polynucleotides of the present invention. In one embodiment, a Syn5 promoter may be used in the synthesis of the polynucleotides. As a non-limiting example, the Syn5 promoter may be 5’-ATTGGGCACCCGTAAGGG-3’ as described by Zhu et al. (Nucleic Acids Research 2013, the contents of which is herein incorporated by reference in its entirety). In one embodiment, a Syn5 RNA polymerase may 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 described in Zhu et al. Nucleic Acids Research 2013, the contents of which is herein incorporated by reference in its entirety). Assembling polynucleotides or nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond.

Solid-phase chemical synthesis [000343] Chimeric polynucleotides or circular polynucleotides of the present invention may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the polynucleotide or nucleic acid sequences.

Liquid Phase Chemical Synthesis

The synthesis of chimeric polynucleotides or circular polynucleotides of the present invention by the sequential addition of monomer building blocks may be carried out in a liquid phase.

Combination of Synthetic Methods

[000344] The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present invention. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain

polynucleotides that cannot be obtained by chemical synthesis alone.

Ligation of Polynucleotide Regions or Subregions

[000345] Polynucleotides such as chimeric polynucleotides and/or circular polynucleotides may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase. Modified and Conjugated Polynucleotides

[000346] Non-natural modified nucleotides may be introduced to polynucleotides or nucleic acids during synthesis or post-synthesis of the chains to achieve desired functions or properties. The modifications may be on internucleotide lineage, the purine or pyrimidine bases, or sugar. The modification may be introduced at the terminal of a chain or anywhere else in the chain; with chemical synthesis or with a polymerase enzyme. For example, hexitol nucleic acids (HNAs) are nuclease resistant and provide strong hybridization to RNA. Short messenger RNAs (mRNAs) with hexitol residues in two codons have been constructed (Lavrik et al.,

Biochemistry, 40, 11777-11784 (2001), the contents of which are incorporated herein by reference in their entirety). The antisense effects of a chimeric HNA gapmer oligonucleotide comprising a phosphorothioate central sequence flanked by 5’ and 3’ HNA sequences have also been studied (See e.g., Kang et al., Nucleic Acids Research, vol.32(4), 4411-4419 (2004), the contents of which are incorporated herein by reference in their entirety). The preparation and uses of modified nucleotides comprising 6-member rings in RNA interference, antisense therapy or other applications are disclosed in US Pat. Application No.2008/0261905, US Pat.

Application No.2010/0009865, and PCT Application No. WO97/30064 to Herdewijn et al.; the contents of each of which are herein incorporated by reference in their entireties). Modified nucleic acids and their synthesis are disclosed in copending PCT applications No. PCT/US2012/058519 (Attorney Docket Number M09), the contents of which are incorporated herein by reference for their entirety. The synthesis and strategy of modified polynucleotides is reviewed by Verma and Eckstein in Annual Review of Biochemistry, vol.76, 99-134 (1998), the contents of which are

incorporated herein by reference in their entirety.

[000347] Either enzymatic or chemical ligation methods can be used to conjugate polynucleotides or their regions with different functional blocks, such as fluorescent labels, liquids, nanoparticles, delivery agents, etc. The conjugates of polynucleotides and modified polynucleotides are reviewed by Goodchild in Bioconjugate Chemistry, vol.1(3), 165-187 (1990), the contents of which are incorporated herein by reference in their entirety. US Pat. No.6,835,827 and US Pat. No.6,525,183 to Vinayak et al. (the contents of each of which are herein incorporated by reference in their entireties) teach synthesis of labeled oligonucleotides using a labeled solid support.

Quantification

[000348] In one embodiment, the polynucleotides of the present invention may 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 may 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.

[000349] Assays may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using

immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

[000350] 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 invention differ from the endogenous forms due to the structural or chemical modifications.

[000351] In one embodiment, the polynucleotide may 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 may be analyzed in order to determine if the polynucleotide may be of proper size, check that no degradation of the polynucleotide has occurred. Degradation of the polynucleotide may 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).

Purification

[000352] Purification of the polynucleotides described herein may include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to,

AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM 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 which 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.

[000353] A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[000354] In another embodiment, the polynucleotides may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. III. Modifications

[000355] Polynucleotides of the present invention may comprise one or more modification, including any of those described herein. As used herein in a polynucleotide (such as a modified mRNA, chimeric polynucleotide, IVT

polynucleotide or a circular polynucleotide), the terms“chemical modification” or, as appropriate,“chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or

deoxyribnucleosides in one or more of their position, pattern, percent or population. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5′-terminal mRNA cap moieties.

[000356] The modifications may be various distinct modifications. In some embodiments, the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified polynucleotide, introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide.

[000357] Modifications of the polynucleotides of the TAVs which are useful in the present invention include, but are not limited to those in Table 2. Noted in the table are the symbol of the modification, the nucleobase type and whether the modification is naturally occurring or not.

Table 10. Modifications

Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001

Figure imgf000147_0001

[000358] Other modifications which may be useful in the polynucleotides of the TAVs of the present invention are listed in Table 3.

Figure imgf000147_0002
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001

Figure imgf000151_0001
[000359] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo- cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio- cytidine (s2C), 2-thio-5-methyl-cytidine.

[000360] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza- adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A).

[000361] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza- guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7- methyl-8-oxo-guanosine.

[000362]

[000363] The polynucleotides of the TAVs can include any useful linker between the nucleosides. Such linkers, including backbone modifications are given in Table 4.

Table 12. Linker modifications

Figure imgf000152_0001

Figure imgf000153_0001

[000364] The polynucleotides can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

[000365] In some embodiments, the polynucleotides of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced. Features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.

[000366] In certain embodiments, it may desirable to intracellularly degrade a polynucleotide introduced into the cell. For example, degradation of a polynucleotide may be preferable if precise timing of protein production is desired. Thus, in some embodiments, the invention provides a polynucleotide containing a degradation domain, which is capable of being acted on in a directed manner within a cell.

[000367] Any of the regions of the polynucleotides may be chemically modified as taught herein or as taught in International Application Number PCT/2012/058519 filed October 3, 2012 (Attorney Docket Number M9) and U.S. Provisional

Application Number 61/837297 filed June 20, 2013 (Attorney Docket Number M36) the contents of each of which are incorporated herein by reference in its entirety. Modified Polynucleotide Molecules

[000368] The present invention also includes building blocks, e.g., modified ribonucleosides, and modified ribonucleotides, of polynucleotide molecules. For example, these building blocks can be useful for preparing the polynucleotides of the invention. Such building blocks are taught in International Application Number PCT/2012/058519 filed October 3, 2012 (Attorney Docket Number M9) and U.S. Provisional Application Number 61/837297 filed June 20, 2013 (Attorney Docket Number M36) the contents of each of which are incorporated herein by reference in its entirety.

Modifications on the Sugar

[000369] The modified nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide (e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2′-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy;

optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy, optionally substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), - O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20);“locked” nucleic acids (LNA) in which the 2′-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4’-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein; and amino acid, as defined herein

[000370] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a

phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and“unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with α-L-threofuranosyl-(3′→2′)) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides containing, e.g., arabinose, as the sugar. Such sugar modifications are taught International Application Number PCT/2012/058519 filed October 3, 2012 (Attorney Docket Number M9) and U.S. Provisional Application Number 61/837297 filed June 20, 2013 (Attorney Docket Number M36) the contents of each of which are incorporated herein by reference in its entirety.

Modifications on the Nucleobase

[000371] The present disclosure provides for modified nucleosides and nucleotides. As described herein“nucleoside” is defined as a compound containing a sugar molecule (e.g., a pentose 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”). As described herein,“nucleotide” is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). The polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphoester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[000372] The 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. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.

[000373] The modified nucleosides and nucleotides can include a modified nucleobase. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. Such modified nucleobases (including the distinctions between naturally occurring and non-naturally occurring) are taught in International Application Number

PCT/2012/058519 filed October 3, 2012 (Attorney Docket Number M9) and U.S. Provisional Application Number 61/837297 filed June 20, 2013 (Attorney Docket Number M36) the contents of each of which are incoroporated herein by reference in its entirety.

Modified mRNAs

[000374] In some embodiments, an mRNA of the invention comprises one or more modified nucleobases, nucleosides, or nucleotides (termed“modified mRNAs” or “mmRNAs”). In some embodiments, modified mRNAs may have useful properties, including enhanced stability, intracellular retention, enhanced translation, and/or the lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, as compared to a reference unmodified mRNA. Therefore, use of modified mRNAs may enhance the efficiency of protein production, intracellular retention of nucleic acids, as well as possess reduced immunogenicity.

[000375] In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3 or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, an mRNA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified mRNA may have reduced degradation in a cell into which the mRNA is introduced, relative to a corresponding unmodified mRNA.

[000376] In some embodiments, the modified nucleobase is a modified uracil.

Exemplary nucleobases and nucleosides having a modified uracil include

pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio- uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5- carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio- pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1- methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl- uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2- thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl- uridine (m3Um), and 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1- thio-uridine, deoxythymidine, 2’‐F‐ara‐uridine, 2’‐F‐uridine, 2’‐OH‐ara‐uridine, 5‐(2‐ carbomethoxyvinyl) uridine, and 5‐[3‐(1‐E‐propenylamino)]uridine.

[000377] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl- cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl- cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1- deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m5Cm), N4- acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine (m4Cm), 5-formyl- 2′-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m4

2Cm), 1-thio- cytidine, 2’‐F‐ara‐cytidine, 2’‐F‐cytidine, and 2’‐OH‐ara‐cytidine.

[000378] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6- halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2- methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2- methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl- adenosine (m6

2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio- N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7- methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O- methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl- adenosine (m6

2Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’‐F‐ara‐adenosine, 2’‐F‐adenosine, 2’‐OH‐ara‐adenosine, and N6‐(19‐amino‐ pentaoxanonadecyl)-adenosine.

[000379] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7- deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl- guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy- guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m2

2G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl- guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio- guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m2

2Gm), 1-methyl-2′-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m1Im), 2′-O-ribosylguanosine (phosphate) (Gr(p)) , 1-thio- guanosine, O6-methyl-guanosine, 2’‐F‐ara‐guanosine, and 2’‐F‐guanosine.

[000380] In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[000381] In some embodiments, the modified nucleobase is pseudouridine (ψ), N1- methylpseudouridine (m1ψ), 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- methoxyuridine, or 2’-O-methyl uridine. In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

[000382] In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α- thio-guanosine, or α-thio-adenosine. In some embodiments, an mRNA of the invention includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.) [000383] In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl- cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl- cytidine (m5C). In some embodiments, the mRNA comprises 2’-O-methyl uridine. In some embodiments, the mRNA comprises 2’-O-methyl uridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[000384] In some embodiments, an mRNA of the invention may be modified in a coding region (e.g., an open reading frame encoding a polypeptide). In other embodiments, an mRNA may be modified in regions besides a coding region. For example, in some embodiments, a 5′-UTR and/or a 3′-UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the coding region.

[000385] Examples of nucleoside modifications and combinations thereof that may be present in mmRNAs of the present invention include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813. Combinations of Modified Sugars, Nucleobases, and Internucleoside Linkages

[000386] The polynucleotides of the invention can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.

[000387] Examples of modified nucleotides and modified nucleotide combinations are provided below in Tables 8 and 9. These combinations of modified nucleotides can be used to form the polynucleotides of the invention. Unless otherwise noted, the modified nucleotides may be completely substituted for the natural nucleotides of the polynucleotides of the invention. As a non-limiting example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleotide uridine may be partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed herein. Any combination of base/sugar or linker may be incorporated into the polynucleotides of the invention and such modifications are taught in International Application Number PCT/2012/058519 filed October 3, 2012 (Attorney Docket Number M9) and International Application Number

PCT/US2013/075177 filed December 13, 2013 (Attorney Docket Number M36), the contents of each of which are incoroporated herein by reference in its entirety.

Table 13. Combinations

Figure imgf000161_0001

Figure imgf000162_0001

Figure imgf000162_0002
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001

Figure imgf000168_0001
[000388] According to the invention, polynucleotides of the invention may be synthesized to comprise the combinations or single modifications of Tables 5 or 6.

[000389] Where a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of that A, U, G or C nucleotide or nucleoside having been modified. Where percentages are listed, these represent the percentage of that particular A, U, G or C nucleobase triphosphate of the total amount of A, U, G, or C triphosphate present.

[000390] For example, the combination: 25 % 5-Aminoallyl-CTP + 75 % CTP/ 25 % 5-Methoxy-UTP + 75 % UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of the uracils are UTP. Where no modified UTP is listed then the naturally occurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of those nucleotides found in the polynucleotide. In this example all of the GTP and ATP nucleotides are left unmodified. IV. Pharmaceutical Compositions

Formulation, Administration, Delivery and Dosing

[000391] The present invention provides TAVs and TAV pharmaceutical compositions and complexes optionally in combination with one or more

pharmaceutically acceptable excipients, carriers or diluents. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances. Pharmaceutical compositions of the present invention may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[000392] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase“active ingredient” generally refers to the TAVs or the polynucleotides contained therein to be delivered as described herein.

[000393] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the

compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys. [000394] Formulations of the pharmaceutical 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 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.

[000395] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending 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 .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

Formulations

[000396] The TAVs of the invention 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 (antigen) 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 of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with TAVs (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[000397] Accordingly, the formulations of the invention can include one or more excipients, each in an amount that may increases the stability of the TAV, increases cell transfection by the TAV, increases the expression of polynucleotides encoded protein, and/or alters the release profile of polynucleotide encoded proteins. Further, the polynucleotides of the present invention may be formulated using self-assembled nucleic acid nanoparticles.

[000398] Formulations of the pharmaceutical 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 associating the active ingredient with an excipient and/or one or more other accessory ingredients.

[000399] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a“unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[000400] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

[000401] In some embodiments, the formulations described herein may contain at least one polynucleotide. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 polynucleotides.

[000402] In one embodiment, the formulations described herein may comprise more than one type of polynucleotide. In one embodiment, the formulation may comprise a chimeric polynucleotide in linear and circular form. In another embodiment, the formulation may comprise a circular polynucleotide and an IVT polynucleotide. In yet another embodiment, the formulation may comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.

[000403] Pharmaceutical formulations may additionally comprise a

pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, 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, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams Ħ Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[000404] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.

Lipidoids

[000405] The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem.2010 21:1448-1454; 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).

[000406] While these lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol.200826:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U S A.2008105:11915-11920; Akinc et al., Mol Ther.200917:872-879; Love et al., Proc Natl Acad Sci U S A.2010107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety), the present disclosure describes their formulation and use in delivering TAVs or polynucleotides contained therein.

[000407] Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in 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.

[000408] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther.2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA–5LAP; aka 98N12-5, see Murugaiah et al., Analytical

Biochemistry, 401:61 (2010); herein incorporated by reference in its entirety), C12- 200 (including derivatives and variants), and MD1, can be tested for in vivo activity.

[000409] The lipidoid referred to herein as“98N12-5” is disclosed by Akinc et al., Mol Ther.200917:872-879 and is incorporated by reference in its entirety.

[000410] The lipidoid referred to herein as“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; both of which are herein incorporated by reference in their entirety. The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotides. As an example, formulations with certain lipidoids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length). As another example, formulations with certain lipidoids, include, but are not limited to, C12-200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

[000411] In one embodiment, a polynucleotide formulated with a lipidoid for systemic intravenous administration can target the liver. For example, a final optimized intravenous formulation using polynucleotides, and comprising a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid to polynucleotides, and a C14 alkyl chain length on the PEG lipid, with a mean particle size of roughly 50–60 nm, can result in the distribution of the formulation to be greater than 90% to the liver.(see, Akinc et al., Mol Ther.200917:872-879; herein incorporated by reference in its entirety). In another example, an intravenous formulation using a C12-200 (see US provisional application 61/175,770 and published international application WO2010129709, each of which is herein incorporated by reference in their entirety) lipidoid may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl

choline/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipid to polynucleotides, and a mean particle size of 80 nm may be effective to deliver polynucleotides to hepatocytes (see, Love et al., Proc Natl Acad Sci U S A.2010 107:1864-1869 herein incorporated by reference in its entirety). In another embodiment, an MD1 lipidoid-containing formulation may be used to effectively deliver polynucleotides to hepatocytes in vivo.

[000412] The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther.200917:872-879 herein incorporated by reference in its entirety), use of a lipidoid-formulated TAVs to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited.

[000413] Use of lipidoid formulations to deliver siRNA in vivo to other non- hepatocyte cells such as myeloid cells and endothelium has been reported (see Akinc et al., Nat Biotechnol.200826:561-569; Leuschner et al., Nat Biotechnol.2011 29:1005-1010; Cho et al. Adv. Funct. Mater.200919:3112-3118; 8th International Judah Folkman Conference, Cambridge, MA October 8-9, 2010; each of which is herein incorporated by reference in its entirety). Effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the TAVs for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% C12-200, 10%

disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see

Leuschner et al., Nat Biotechnol 201129:1005-1010; herein incorporated by reference in its entirety). The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the TAV. [000414] Combinations of different lipidoids may be used to improve the efficacy of polynucleotides directed protein production as the lipidoids may be able to increase cell transfection by the TAV; and/or increase the translation of encoded protein (see Whitehead et al., Mol. Ther.2011, 19:1688-1694, herein incorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

[000415] The TAVs of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of TAVs include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[000416] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[000417] As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375,

US20130183373 and US20130183372, the contents of each of which are herein incorporated by reference in its entirety. [000418] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[000419] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy.1999 6:271-281; Zhang et al. Gene Therapy.19996:1438-1447; Jeffs et al. Pharm Res. 200522:362-372; Morrissey et al., Nat Biotechnol.20052:1002-1007; Zimmermann et al., Nature.2006441:111-114; Heyes et al. J Contr Rel. 2005107:276-287;

Semple et al. Nature Biotech.201028:172-176; Judge et al. J Clin Invest.2009 119:661-673; deFougerolles Hum Gene Ther.200819:125-132; U.S. Patent

Publication No US20130122104; all of which are incorporated herein in their entireties). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2-distearloxy- N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy- 3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[000420] In some embodiments, liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and/or from about 48.5% cholesterol to about 60% cholesterol. In a preferred embodiment, formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.

[000421] In one embodiment, pharmaceutical compositions may include liposomes which may be formed to deliver polynucleotides which may encode at least one immunogen (antigen) or any other polypeptide of interest. The TAV may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos.

WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684; the contents of each of which are herein incorporated by reference in their entirety).

[000422] In another embodiment, liposomes may be formulated for targeted delivery. As a non-limiting example, the liposome may be formulated for targeted delivery to the liver. The liposome used for targeted delivery may include, but is not limited to, the liposomes described in and methods of making liposomes described in US Patent Publication No. US20130195967, the contents of which are herein incorporated by reference in its entirety.

[000423] In another embodiment, the polynucleotide which may encode an immunogen (antigen) may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the polynucleotide anchoring the molecule to the emulsion particle (see International Pub. No. WO2012006380; herein incorporated by reference in its entirety).

[000424] In one embodiment, the TAVs may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed. As a non-limiting example, the emulsion may be made by the methods described in International Publication No. WO201087791, herein incorporated by reference in its entirety.

[000425] In another embodiment, the lipid formulation may include at least cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No.

WO2011076807 and U.S. Pub. No.20110200582; the contents of each of which is herein incorporated by reference in their entirety). In another embodiment, the polynucleotides encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No.

20120177724, the contents of which is herein incorporated by reference in its entirety).

[000426] In one embodiment, the polynucleotides may be formulated in a liposome as described in International Patent Publication No. WO2013086526, herein incorporated by reference in its entirety. The TAVs may be encapsulated in a liposome using reverse pH gradients and/or optimized internal buffer compositions as described in International Patent Publication No. WO2013086526, herein

incorporated by reference in its entirety.

[000427] In one embodiment, the TAV pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006

5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan- coated liposomes (Quiet Therapeutics, Israel).

[000428] In one embodiment, the cationic lipid may be a low molecular weight cationic lipid such as those described in US Patent Application No.20130090372, the contents of which are herein incorporated by reference in its entirety.

[000429] In one embodiment, the TAVs may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.

[000430] In one embodiment, the TAVs may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phosphates in the RNA (N:P ratio) of between 1:1 and 20:1 as described in International Publication No. WO2013006825, herein incorporated by reference in its entirety. In another embodiment, the liposome may have a N:P ratio of greater than 20:1 or less than 1:1.

[000431] In one embodiment, the TAVs may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No.20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326 or US Patent Pub. No. US20130142818; each of which is herein incorporated by reference in its entirety. In another embodiment, the TAVs may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[000432] In one embodiment, the TAVs may be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in the present invention may be prepared by the methods described in U.S. Patent No.8,450,298, herein incorporated by reference in its entirety.

[000433] The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Semple et al. Nature Biotech.201028:172- 176; herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther.201119:2186-2200; herein incorporated by reference in its entirety). In some embodiments, liposome formulations may comprise from about 35 to about 45% cationic lipid, from about 40% to about 50% cationic lipid, from about 50% to about 60% cationic lipid and/or from about 55% to about 65% cationic lipid. In some embodiments, the ratio of lipid to mRNA in liposomes may be from about 5:1 to about 20:1, from about 10:1 to about 25:1, from about 15:1 to about 30:1 and/or at least 30:1.

[000434] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations. As a non-limiting example, LNP formulations may contain from about 0.5% to about 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about 3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG (1,2-Dipalmitoyl-sn- glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

[000435] In one embodiment, the TAVs may be formulated in a lipid nanoparticle such as those described in International Publication No. WO2012170930, herein incorporated by reference in its entirety.

[000436] In one embodiment, the TAV formulation comprising the polynucleotide is a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3- DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and/or made by the methods described in US Patent Publication No. US20130150625, herein incorporated by reference in its entirety. As a non- limiting example, the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien- 1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en- 1-yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[000437] In one embodiment, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos.

WO2012040184, WO2011153120, WO2011149733, WO2011090965,

WO2011043913, WO2011022460, WO2012061259, WO2012054365,

WO2012044638, WO2010080724, WO201021865, WO2008103276,

WO2013086373 and WO2013086354, US Patent Nos.7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115,

US20120202871, US20130064894, US20130129785, US20130150625,

US20130178541 and US20130225836; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733,

WO2011090965, WO2011043913, WO2011022460, WO2012061259,

WO2012054365, WO2012044638 and WO2013116126 or US Patent Publication No. US20130178541 and US20130225836; the contents of each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of US Patent No.7,893,302, formula CLI-CLXXXXII of US Patent No.7,404,969 and formula I-VI of US Patent Publication No. US20100036115, formula I of US Patent Publication No

US20130123338; each of which is herein incorporated by reference in their entirety. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N- dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20- dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-l 6, 19-dien-8-amine, (13Z,16Z)- N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15- dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N- dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21- dien-10-amine, (15Ζ,18Ζ)-Ν,Ν-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)- N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22- dien-9-amine, (18Z,21 Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8–amine, (17Z,20Z)- N,N-dimethylhexacosa- 17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa- 16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z ,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimetylheptacos-18- en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N- dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine,

(20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-l0-amine, 1-[(11Z,14Z)-l- nonylicosa-11,14-dien-l-yl] pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-l 0- amine, (15Z)-N,N-dimethyl eptacos-15-en-l 0-amine, (14Z)-N,N-dimethylnonacos- 14-en-l0-amine, (17Z)-N,N-dimethylnonacos-17-en-l0-amine, (24Z)-N,N- dimethyltritriacont-24-en-l0-amine, (20Z)-N,N-dimethylnonacos-20-en-l 0-amine, (22Z)-N,N-dimethylhentriacont-22-en-l0-amine, (16Z)-N,N-dimethylpentacos-16-en- 8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1–amine, (13Z,16Z)- N,N-dimethyl-3-nonyldocosa-l3,16-dien-l–amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl] eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N- dimethylnonadecan-10-amine, Ν,Ν-dimethyl-1-[(1S ,2R)-2- octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(lS,2R)-2- octylcyclopropyl]henicosan-l0-amine,Ν,Ν-dimethyl-1-[(1S,2S)-2-{[(lR,2R)-2- pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,Ν,Ν-dimethyl-1- [(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, Ν,Ν-dimethyl-[(lR,2S)-2- undecyIcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2- octylcyclopropyl]heptyl} dodecan-1–amine, 1-[(1R,2S)-2-hepty lcyclopropyl]-Ν,Ν- dimethyloctadecan-9–amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N- dimethylpentadecan-6-amine, N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12- dien-1-yloxy]-3-(octyloxy)propan-2-amine, S-N,N-dimethyl-1-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien- 1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1- (hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2- amine, Ν,Ν-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan- 2-amine, Ν,Ν-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3- (octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N- dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14- dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1- yloxy]-Ν,Ν-dimethy1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-l3,16-dien- l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa- 13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)- docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos- 13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1- yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-metoylo ctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7- dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2- amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2- pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1- {[8-(2-oc1ylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (1lE,20Z,23Z)- N,N-dimethylnonacosa-l1,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[000438] In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.

[000439] In another embodiment, the lipid may be a cationic lipid such as, but not limited to, Formula (I) of U.S. Patent Application No. US20130064894, the contents of which are herein incorporated by reference in its entirety.

[000440] In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos.

WO2012040184, WO2011153120, WO2011149733, WO2011090965,

WO2011043913, WO2011022460, WO2012061259, WO2012054365,

WO2012044638, WO2010080724, WO201021865, WO2013086373 and

WO2013086354; the contents of each of which are herein incorporated by reference in their entirety.

[000441] In another embodiment, the cationic lipid may be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids and methods of making and using the trialkyl cationic lipids are described in International Patent Publication No.

WO2013126803, the contents of which are herein incorporated by reference in its entirety.

[000442] In one embodiment, the LNP formulations of the TAVs may contain PEG- c-DOMG at 3% lipid molar ratio. In another embodiment, the LNP formulations TAVs may contain PEG-c-DOMG at 1.5% lipid molar ratio.

[000443] In one embodiment, the pharmaceutical compositions of the TAVs may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, herein incorporated by reference.

[000444] In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000). In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID:

22908294; herein incorporated by reference in its entirety).

[000445] In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or

WO2008103276, the contents of each of which is herein incorporated by reference in their entirety. As a non-limiting example, the TAVs described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or

WO2008103276; each of which is herein incorporated by reference in their entirety.

[000446] In one embodiment, the TAVs described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No. US20120207845; the contents of which are herein incorporated by reference in its entirety.

[000447] In one embodiment, the TAVs may be formulated in a lipid nanoparticle made by the methods described in US Patent Publication No US20130156845 or International Publication No WO2013093648 or WO2012024526, each of which is herein incorporated by reference in its entirety.

[000448] The lipid nanoparticles described herein may be made in a sterile environment by the system and/or methods described in US Patent Publication No. US20130164400, herein incorporated by reference in its entirety.

[000449] In one embodiment, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in US Patent No.

8,492,359, the contents of which are herein incorporated by reference in its entirety. As a non-limiting example, the lipid particle may comprise one or more active agents or therapeutic agents; one or more cationic lipids comprising from about 50 mol % to about 85 mol % of the total lipid present in the particle; one or more non-cationic lipids comprising from about 13 mol % to about 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from about 0.5 mol % to about 2 mol % of the total lipid present in the particle. The nucleic acid in the nanoparticle may be the polynucleotides described herein and/or are known in the art.

[000450] In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or

WO2008103276, the contents of each of which are herein incorporated by reference in their entirety. As a non-limiting example, modified RNA described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or

WO2008103276; the contents of each of which are herein incorporated by reference in their entirety.

[000451] In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety. In another embodiment, the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA described herein in vivo and/or in vitro.

[000452] In one embodiment, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.

[000453] In one embodiment, the TAV pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006

5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan- coated liposomes (Quiet Therapeutics, Israel).

[000454] In one embodiment, the TAVs may be formulated in a lyophilized gel- phase liposomal composition as described in US Publication No. US2012060293, herein incorporated by reference in its entirety.

[000455] The nanoparticle formulations may comprise a phosphate conjugate. The phosphate conjugate may increase in vivo circulation times and/or increase the targeted delivery of the nanoparticle. Phosphate conjugates for use with the present invention may be made by the methods described in International Application No. WO2013033438 or US Patent Publication No. US20130196948, the contents of each of which are herein incorporated by reference in its entirety. As a non-limiting example, the phosphate conjugates may include a compound of any one of the formulas described in International Application No. WO2013033438, herein incorporated by reference in its entirety. [000456] The nanoparticle formulation may comprise a polymer conjugate. The polymer conjugate may be a water soluble conjugate. The polymer conjugate may have a structure as described in U.S. Patent Application No.20130059360, the contents of which are herein incorporated by reference in its entirety. In one aspect, polymer conjugates with the polynucleotides of the present invention may be made using the methods and/or segmented polymeric reagents described in U.S. Patent Application No.20130072709, herein incorporated by reference in its entirety. In another aspect, the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in US Patent Publication No. US20130196948, the contents of which is herein incorporated by reference in its entirety.

[000457] The nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present invention in a subject. Further, the conjugate may inhibit phagocytic clearance of the nanoparticles in a subject. In one aspect, the conjugate may be a“self” peptide designed from the human membrane protein CD47 (e.g., the“self” particles described by Rodriguez et al (Science 2013339, 971-975), herein incorporated by reference in its entirety). As shown by Rodriguez et al. the self-peptides delayed macrophage-mediated clearance of nanoparticles which enhanced delivery of the nanoparticles. In another aspect, the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al . Science 2013339, 971-975, herein incorporated by reference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in a subject as compared to scrambled peptides and PEG coated nanoparticles.

[000458] In one embodiment, the TAVs of the present invention are formulated in nanoparticles which comprise a conjugate to enhance the delivery of the nanoparticles of the present invention in a subject. The conjugate may be the CD47 membrane or the conjugate may be derived from the CD47 membrane protein, such as the“self” peptide described previously. In another aspect the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof. In yet another aspect, the nanoparticle may comprise both the“self” peptide described above and the membrane protein CD47.

[000459] In another aspect, a“self” peptide and/or CD47 protein may be conjugated to a virus-like particle or pseudovirion, as described herein for delivery of the TAVs of the present invention. [000460] In another embodiment, TAV pharmaceutical compositions comprising the polynucleotides of the present invention and a conjugate which may have a degradable linkage. Non-limiting examples of conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer. As a non-limiting example, pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in US Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in its entirety.

[000461] The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a TAV. As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121; the contents of which are herein incorporated by reference in its entirety).

[000462] Nanoparticle formulations of the present invention may be coated with a surfactant or polymer in order to improve the delivery of the particle. In one embodiment, the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and/or coatings that have a neutral surface charge. The hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, TAVs within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in its entirety.

[000463] In one embodiment, the lipid nanoparticles of the present invention may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in US Patent Publication No. US20130210991, the contents of which are herein

incorporated by reference in its entirety.

[000464] In another embodiment, the lipid nanoparticles of the present invention may be hydrophobic polymer particles.

[000465] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.

[000466] In one embodiment, the internal ester linkage may be located on either side of the saturated carbon.

[000467] In one embodiment, an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen. (U.S. Publication No.20120189700 and International Publication No.

WO2012099805; each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and/or a polynucleotide described herein. In one embodiment, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.

[000468] Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007104(5):1482-487; Lai et al. Adv Drug Deliv Rev.200961(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No.8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.

[000469] The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are herein incorporated by reference in its entirety. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (See e.g., International App. No. WO201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co- caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co- PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),

poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),

poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co- caprolactone), PEG-PLGA-PEG and trimethylene carbonate,

polyvinylpyrrolidone.The lipid nanoparticle may be coated or associated with a co- polymer such as, but not limited to, a block co-polymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed.201150:2597- 2600; the contents of which are herein incorporated by reference in its entirety). A non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (See e.g., J Control Release 2013, 170(2):279-86; the contents of which are herein incorporated by reference in its entirety).

[000470] The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains). [000471] The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.. The surface altering agent may be embedded or enmeshed in the particle’s surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle. (see e.g., US Publication 20100215580 and US Publication 20080166414 and US20130164343; each of which is herein incorporated by reference in their entirety).

[000472] In one embodiment, the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein. The polynucleotide may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the paricle. The polynucleotide may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.

[000473] In another embodiment, the mucus penetrating lipid nanoparticles may be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation may be hypotonice for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations may be found in International Patent Publication No. WO2013110028, the contents of which are herein incorporated by reference in its entirety.

[000474] In one embodiment, in order to enhance the delivery through the mucosal barrier the TAV formulation may comprise or be a hypotonic solution. Hypotonic solutions were found to increase the rate at which mucoinert particles such as, but not limited to, mucus-penetrating particles, were able to reach the vaginal epithelial surface (See e.g., Ensign et al. Biomaterials 201334(28):6922-9; the contents of which is herein incorporated by reference in its entirety).

[000475] In one embodiment, the TAV is formulated as a lipoplex, such as, without limitation, the ATUPLEXTM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTM from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (Aleku et al. Cancer Res.200868:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.201023:334-344; Kaufmann et al. Microvasc Res 201080:286-293Weide et al. J Immunother.200932:498-507; Weide et al. J Immunother.200831:180-188; Pascolo Expert Opin. Biol. Ther.

4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother.34:1-15; Song et al., Nature Biotechnol.2005, 23:709-717; Peer et al., Proc Natl Acad Sci U S A.2007

6;104:4095-4100; deFougerolles Hum Gene Ther.200819:125-132; all of which are incorporated herein by reference in its entirety).

[000476] In one embodiment such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes. One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther.201018:1357-1364; herein incorporated by reference in its entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol.20118:197-206; Musacchio and Torchilin, Front Biosci.201116:1388-1412; Yu et al., Mol Membr Biol.2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.200825:1-61; Benoit et al., Biomacromolecules.201112:2708-2714; Zhao et al., Expert Opin Drug Deliv.2008 5:309-319; Akinc et al., Mol Ther.201018:1357-1364; Srinivasan et al., Methods Mol Biol.2012820:105-116; Ben-Arie et al., Methods Mol Biol.2012757:497-507; Peer 2010 J Control Release.20:63-68; Peer et al., Proc Natl Acad Sci U S A.2007 104:4095-4100; Kim et al., Methods Mol Biol.2011721:339-353; Subramanya et al., Mol Ther.201018:2028-2037; Song et al., Nat Biotechnol.200523:709-717; Peer et al., Science.2008319:627-630; Peer and Lieberman, Gene Ther.201118:1127-1133; all of which are incorporated herein by reference in its entirety).

[000477] In one embodiment, the TAV is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696–1702; the contents of which are herein incorporated by reference in its entirety). As a non-limiting example, the SLN may be the SLN described in International Patent Publication No. WO2013105101, the contents of which are herein incorporated by reference in its entirety. As another non-limiting example, the SLN may be made by the methods or processes described in International Patent Publication No. WO2013105101, the contents of which are herein incorporated by reference in its entirety.

[000478] Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the TAV; and/or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 200715:713-720; herein incorporated by reference in its entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide.

[000479] In another embodiment, the TAV controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, herein incorporated by reference in its entirety.

[000480] In one embodiment, the TAVs of the present invention may be encapsulated in a therapeutic nanoparticle. Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286,

US20120288541, US20130123351 and US20130230567 and US Pat No.8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.

[000481] In one embodiment, the TAV therapeutic nanoparticles may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518; herein incorporated by reference in its entirety). In one embodiment, the therapeutic nanoparticles may be formulated to be cancer specific. As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725,

WO2011084521 and US Pub No. US20100069426, US20120004293 and

US20100104655, each of which is herein incorporated by reference in their entirety.

[000482] In one embodiment, the therapeutic nanoparticle comprising at least one TAV may be formulated using the methods described by Podobinski et al in US Patent No.8,404,799, the contents of which are herein incorporated by reference in its entirety.

[000483] In one embodiment, the TAV compositions may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255,

WO2012149259, WO2012149265, WO2012149268, WO2012149282,

WO2012149301, WO2012149393, WO2012149405, WO2012149411,

WO2012149454 and WO2013019669, and US Pub. Nos. US20110262491,

US20100104645, US20100087337 and US20120244222, each of which is herein incorporated by reference in their entirety.

[000484] In one embodiment, the TAV compositions may be encapsulated in, linked to and/or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in US Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in its entirety. In one aspect, the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein. [000485] In one embodiment, the TAV compositions may be formulated in colloid nanocarriers as described in US Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in its entirety.

[000486] In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No.

20120282343; herein incorporated by reference in its entirety.

[000487] In some embodiments, LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No.2012/0295832 expressly incorporated herein by reference in its entirety). Activity and/or safety (as measured by examining one or more of ALT/AST, white blood cell count and cytokine induction) of LNP administration may be improved by incorporation of such lipids. LNPs comprising KL52 may be administered intravenously and/or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and/or protein expression as compared to LNPs comprising MC3.

[000488] In some embodiments, TAV compositions may be delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1 um up to 100 nm such as, but not limited to, less than 0.1 um, less than 1.0 um, less than 5 um, less than 10 um, less than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35 um, less than 40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um, less than 70 um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less than 95 um, less than 100 um, less than 125 um, less than 150 um, less than 175 um, less than 200 um, less than 225 um, less than 250 um, less than 275 um, less than 300 um, less than 325 um, less than 350 um, less than 375 um, less than 400 um, less than 425 um, less than 450 um, less than 475 um, less than 500 um, less than 525 um, less than 550 um, less than 575 um, less than 600 um, less than 625 um, less than 650 um, less than 675 um, less than 700 um, less than 725 um, less than 750 um, less than 775 um, less than 800 um, less than 825 um, less than 850 um, less than 875 um, less than 900 um, less than 925 um, less than 950 um, less than 975 um,

[000489] In another embodiment, TAV s may be delivered using smaller LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from about 70 to about 90 nm.

[000490] In one embodiment, the TAV compositions of the invention may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No.8,440,614, each of which is herein incorporated by reference in its entirety.

[000491] In one embodiment, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[000492] In one embodiment, the TAV compositions described herein may be formulated in polymeric nanoparticles as described in or made by the methods described in US Patent No.8,420,123 and 8,518,963 and European Patent No.

EP2073848B1, the contents of each of which are herein incorporated by reference in their entirety.

[000493] In one embodiment, the TAV compositions of the present invention may be formulated in a swellable nanoparticle. The swellable nanoparticle may be, but is not limited to, those described in U.S. Patent No.8,440,231, the contents of which is herein incorporated by reference in its entirety. [000494] The TAV compositions of the present invention may be formulated in polyanhydride nanoparticles such as, but not limited to, those described in U.S. Patent No.8,449,916, the contents of which is herein incorporated by reference in its entirety.

[000495] In one embodiment, the nanoparticles of the present invention may be water soluble nanoparticles such as, but not limited to, those described in International Publication No. WO2013090601, the contents of which is herein incorporated by reference in its entirety. The nanoparticles may be inorganic nanoparticles which have a compact and zwitterionic ligand in order to exhibit good water solubility. The nanoparticles may also have small hydrodynamic diameters (HD), stability with respect to time, pH, and salinity and a low level of non-specific protein binding.

[000496] In one embodiment the nanoparticles of the present invention may be developed by the methods described in US Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in its entirety.

[000497] In one embodiment, the nanoparticles of the present invention are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in US Patent Publication No. US20130172406; the contents of which is herein incorporated by reference in its entirety.

[000498] In one embodiment, the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high density nucleic acid layer. As a non-limiting example, the nanoparticle-nucleic acid hybrid structure may made by the methods described in US Patent Publication No. US20130171646, the contents of which are herein incorporated by reference in its entirety. The nanoparticle may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and/or known in the art.

[000499] The TAV compositions of the invention may be formulated with or in a polymeric compound. The polymer may include at least one polymer such as, 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, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, 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, polyacrylates, polymethacrylates,

polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof .

[000500] As a non-limiting example, the TAV compositions of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No.6,177,274; herein incorporated by reference in its entirety. The formulation may be used for transfecting cells in vitro or for in vivo delivery of polynucleotide. In another example, the polynucleotide may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos.

20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.

[000501] As another non-limiting example the TAV compositions of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No.

US20120004293 and US Pat No.8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No.6,004,573, herein incorporated by reference in its entirety). As a non-limiting example, the TAVs of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).

[000502] A polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No.20100260817 (now U.S. Patent No.8,460,696) the contents of each of which is herein incorporated by reference in its entirety). As a non-limiting example, a pharmaceutical composition may include the TAV composition and the polyamine derivative described in U.S. Pub. No.20100260817 (now U.S. Patent No.8,460,696; the contents of which are incorporated herein by reference in its entirety. As a non- limiting example the TAV compositions of the present invention may be delivered using a polyaminde polymer such as, but not limited to, a polymer comprising a 1,3- dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No.8,236,280; herein incorporated by reference in its entirety).

[000503] The TAV compositions of the invention may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),

polycyanoacrylates and combinations thereof.

[000504] In one embodiment, the TAV compositions of the present invention may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos. WO2011115862, WO2012082574 and

WO2012068187 and U.S. Pub. No.20120283427, each of which are herein incorporated by reference in their entireties.

[000505] In another embodiment, the TAV compositions of the present invention may be formulated with a polymer of formula Z as described in WO2011115862, herein incorporated by reference in its entirety. In yet another embodiment, the TAV compositions may be formulated with a polymer of formula Z, Z’ or Z’’ as described in International Pub. Nos. WO2012082574 or WO2012068187 and U.S. Pub. No. 2012028342, each of which are herein incorporated by reference in their entireties. The polymers formulated with the modified RNA of the present invention may be synthesized by the methods described in International Pub. Nos. WO2012082574 or WO2012068187, each of which are herein incorporated by reference in their entireties.

[000506] The TAV compositions of the invention may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),

polycyanoacrylates and combinations thereof.

[000507] Formulations of TAV compositions of the invention may include 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. As a non-limiting example, the poly(amine-co-esters) may be the polymers described in and/or made by the methods described in International Publication No

WO2013082529, the contents of which are herein incorporated by reference in its entirety.

[000508] For example, the TAV compositions of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi- block copolymer or combinations thereof. The biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Pat. No.

6,696,038, U.S. App. Nos.20030073619 and 20040142474 each of which is herein incorporated by reference in their entireties. The poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No.20100004315, herein incorporated by reference in its entirety. The biodegradabale polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos.6,517,869 and 6,267,987, the contents of which are each incorporated herein by reference in their entirety. The linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No.6,217,912 herein incorporated by reference in its entirety. The PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co- glycolides). The biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No.8,057,821, 8,444,992 or U.S. Pub. No.2012009145 each of which are herein incorporated by reference in their entireties. For example, the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines. Further, the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos.6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.

[000509] The TAV compositions of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[000510] The TAV compositions of the invention may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No.20120269761, the contents of which is herein incorporated by reference in its entirety.

[000511] The TAV compositions of the invention may be formulated in or with at least one cyclodextrin polymer. Cyclodextrin polymers and methods of making cyclodextrin polymers include those known in the art and described in US Pub. No. 20130184453, the contents of which are herein incorporated by reference in its entirety.

[000512] In one embodiment, the TAV compositions of the invention may be formulated in or with at least one crosslinked cation-binding polymers. Crosslinked cation-binding polymers and methods of making crosslinked cation-binding polymers include those known in the art and described in International Patent Publication No. WO2013106072, WO2013106073 and WO2013106086, the contents of each of which are herein incorporated by reference in its entirety.

[000513] In one embodiment, the TAV compositions of the invention may be formulated in or with at least one branched polymer. Branched polymers and methods of making branched polymers include those known in the art and described in International Patent Publication No. WO2013113071, the contents of each of which are herein incorporated by reference in its entirety.

[000514] In one embodiment, the TAV compositions of the invention may be formulated in or with at least PEGylated albumin polymer. PEGylated albumin polymer and methods of making PEGylated albumin polymer include those known in the art and described in US Patent Publication No. US20130231287, the contents of each of which are herein incorporated by reference in its entirety. [000515] In one embodiment, the polymers described herein may be conjugated to a lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present invention are described in International Publication No. WO2008103276, herein incorporated by reference in its entirety. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No.8,273,363, herein incorporated by reference in its entirety.

[000516] In one embodiment, the TAV compositions disclosed herein may be mixed with the PEGs or the sodium phosphate/sodium carbonate solution prior to administration.

[000517] In another embodiment, a polynucleotides encoding a protein of interest may be mixed with the PEGs and also mixed with the sodium phosphate/sodium carbonate solution.

[000518] In yet another embodiment, polynucleotides encoding a protein of interest may be mixed with the PEGs and a polynucleotides encoding a second protein of interest may be mixed with the sodium phosphate/sodium carbonate solution.

[000519] In one embodiment, the TAV compositions described herein may be conjugated with another compound. Non-limiting examples of conjugates are described in US Patent Nos.7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. In another embodiment, modified RNA of the present invention may be conjugated with conjugates of formula 1-122 as described in US Patent Nos.7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. The TAV compositions described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc.2009131(6): 2072-2073; herein incorporated by reference in its entirety). In another embodiment, the TAV compositions described herein may be conjugated and/or encapsulated in gold-nanoparticles. (International Pub. No. WO201216269 and U.S. Pub. No.20120302940 and US20130177523; the contents of each of which is herein incorporated by reference in its entirety).

[000520] As described in U.S. Pub. No.20100004313, herein incorporated by reference in its entirety, a gene delivery composition may include a nucleotide sequence and a poloxamer. For example, the TAVs of the present invention may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. 20100004313.

[000521] In one embodiment, the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No.20090042829 herein incorporated by reference in its entirety. The cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoyl- 3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol

Hydrochloride (DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop- 3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl- N,N-dimethylammonium chloride DODAC) and combinations thereof. As a non- limiting example, the TAV compositions may be formulated with a cationic lipopolymer such as those described in U.S. Patent Application No.20130065942, herein incorporated by reference in its entirety.

[000522] The TAV compositions of the invention may be formulated in a polyplex of one or more polymers (See e.g., U.S. Pat. No.8,501,478, U.S. Pub. No.

20120237565 and 20120270927 and 20130149783 and International Patent Pub. No. WO2013090861; the contents of each of which is herein incorporated by reference in its entirety). As a non-limiting example, the polyplex may be formed using the noval alpha-aminoamidine polymers described in International Publication No.

WO2013090861, the contents of which are herein incorporated by reference in its entirety. As another non-limiting example, the polyplex may be formed using the click polymers described in US Patent No.8,501,478, the contents of which is herein incorporated by reference in its entirety. [000523] In one embodiment, the polyplex comprises two or more cationic polymers. The catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI. In another embodiment, the polyplex comprises p(TETA/CBA) its PEGylated analog p(TETA/CBA)-g-PEG2k and mixtures thereof (see e.g., US Patent Publication No. US20130149783, the contents of which are herein incorporated by reference in its entirety.

[000524] The TAV compositions of the invention can also 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 may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the TAV compositions, may be enhanced (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 its entirety). As a non-limiting example, the nanoparticle may 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 (International Pub. No. WO20120225129; the contents of which is herein incorporated by reference in its entirety).

Peptides and Proteins

[000525] The TAV compositions of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide. In one embodiment, peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations. A non-limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther.3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and

Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci.62(16):1839- 49 (2005), all of which are incorporated herein by reference in their entirety). The compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space. TAV compositions of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologics (Cambridge, MA) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol.20105:747-752;

McNaughton et al., Proc. Natl. Acad. Sci. USA 2009106:6111-6116; Sawyer, Chem Biol Drug Des.200973:3-6; Verdine and Hilinski, Methods Enzymol.2012;503:3- 33; all of which are herein incorporated by reference in its entirety).

[000526] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein,“protein-binding partner” includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein- binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the polynucleotide may be introduced.

[000527] Formulations of the including peptides or proteins may be used to increase cell transfection by the TAV compositions, alter the biodistribution of the

polynucleotide (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein. (See e.g., International Pub. No. WO2012110636 and WO2013123298; the contents of which are herein incorporated by reference in its entirety).

[000528] In one embodiment, the cell penetrating peptide may be, but is not limited to, those described in US Patent Publication No US20130129726, US20130137644 and US20130164219, each of which is herein incorporated by reference in its entirety. Introduction Into Cells

[000529] A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques and any of these may be used to introduce the TAVs of the present invention. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Conjugates

[000530] The TAV compositions of the invention include conjugates, such as a polynucleotide 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). In one embodiment, the conjugate of the present invention may function as a carrier for the TAVs of the present invention.

[000531] The conjugates of the invention 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 may 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.

[000532] 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-gulucosamine 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.

[000533] 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 may 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-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.

[000534] 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.

[000535] In one embodiment, the TAV compositions disclosed herein may be formulated as self-assembled nanoparticles. As a non-limiting example, nucleic acids may be used to make nanoparticles which may be used in a delivery system for the TAVs of the present invention (See e.g., International Pub. No. WO2012125987; herein incorporated by reference in its entirety).

Suspension formulations

[000536] In some embodiments, suspension formulations are provided comprising TAV compositions, water immiscible oil depots, surfactants and/or co-surfactants and/or co-solvents. Combinations of oils and surfactants may enable suspension formulation with TAVs. Delivery of TAVs in a water immiscible depot may be used to improve bioavailability through sustained release of mRNA from the depot to the surrounding physiologic environment and prevent polynucleotides degradation by nucleases.

[000537] In some embodiments, suspension formulations of TAV compositions may be prepared using combinations of polynucleotides, oil-based solutions and surfactants. Such formulations may be prepared as a two-part system comprising an aqueous phase comprising polynucleotides and an oil-based phase comprising oil and surfactants. Exemplary oils for suspension formulations may include, but are not limited to sesame oil and Miglyol (comprising esters of saturated coconut and palmkernel oil-derived caprylic and capric fatty acids and glycerin or propylene glycol), corn oil, soybean oil, peanut oil, beeswax and/or palm seed oil. Exemplary surfactants may include, but are not limited to Cremophor, polysorbate 20, polysorbate 80, polyethylene glycol, transcutol, Capmul®, labrasol, isopropyl myristate, and/or Span 80. In some embodiments, suspensions may comprise co- solvents including, but not limited to ethanol, glycerol and/or propylene glycol.

Microparticles

[000538] In one embodiment, formulations comprising TAV compositions may comprise microparticles. The microparticles may comprise a polymer described herein and/or known in the art such as, but not limited to, poly(α-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester and a polyanhydride. The microparticle may have adsorbent surfaces to adsorb biologically active molecules such as TAV compositions. As a non-limiting example microparticles for use with the present invention and methods of making microparticles are described in US Patent Publication No. US2013195923 and US20130195898 and US Patent No. 8,309,139 and 8,206,749, the contents of each of which are herein incorporated by reference in its entirety.

[000539] In another embodiment, the formulation may be a microemulsion comprising microparticles and TAV compositions. As a non-limiting example, microemulsions comprising microparticles are described in US Patent Publication No. US2013195923 and US20130195898 and US Patent No.8,309,139 and 8,206,749, the contents of each of which are herein incorporated by reference in its entirety.

Amino Acid Lipids

[000540] In one embodiment, the TAV compositions may be formulated in amino acid lipids. 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 US Patent No.8,501,824, the contents of which are herein incorporated by reference in its entirety.

Excipients

[000541] Pharmaceutical formulations may additionally comprise a

pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, 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, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[000542] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.

[000543] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.

[000544] In some embodiments, the pH of pharmaceutical compositions is maintained between pH 5 and pH 8, e.g., to improve stability. Exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not preferred due to complex formation and/or mRNA degradation.

[000545] Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used. [000546] In some embodiments, pharmaceutical compositions may comprise cyroprotectants. As used herein, there term“cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some embodiments, cryoprotectants are combined with TAV compositions in order to stabilize them during freezing. Frozen storage of mRNA between -20°C and -80°C may be advantageous for long term (e.g.36 months) stability of polynucleotide. In some embodiments, cryoprotectants are included in TAV formulations to stabilize polynucleotide through freeze/thaw cycles and under frozen storage conditions. Cryoprotectants of the present invention may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and/or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

Delivery

[000547] The present disclosure encompasses the delivery of TAV compositions for any of therapeutic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.

Naked Delivery

[000548] The TAV compositions of the present invention may be delivered to a cell naked. As used herein in,“naked” refers to delivering TAV compositions free from agents which promote transfection. For example, the TAV compositions delivered to the cell may contain no modifications. The naked TAV compositions may be delivered to the cell using routes of administration known in the art and described herein.

Formulated Delivery

[000549] The TAV compositions of the present invention may be formulated, using the methods described herein. The formulations may contain polynucleotides which may be modified and/or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained- release delivery depot. The formulated TAV compositions may be delivered to the cell using routes of administration known in the art and described herein. [000550] The compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.

Administration

The TAV compositions of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal

administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura),

intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier. In one embodiment, a formulation for a route of administration may include at least one inactive ingredient.

[000551] Non-limiting examples of routes of administration for the TAV compositions of the present invention are described below.

Parenteral and Injectable Administration

[000552] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration,

compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or

combinations thereof.

[000553] A pharmaceutical composition for parenteral administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by the US Food and Drug Administration (FDA). A non- exhaustive list of inactive ingredients for use in pharmaceutical compositions for parenteral administration includes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodium chloride and sodium hydroxide.

[000554] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. The sterile formulation may also comprise adjuvants such as local anesthetics, preservatives and buffering agents.

[000555] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Topical or Transdermal Administration

[000556] As described herein, compositions containing the TAV compositions of the invention may be formulated for administration topically and/or transdermally. The skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.

[000557] The site of cutaneous expression of the delivered compositions will depend on the route of nucleic acid delivery. Three routes are commonly considered to deliver TAV compositions to the skin: (i) topical application (e.g. for local/regional treatment and/or cosmetic applications); (ii) intradermal injection (e.g. for local/regional treatment and/or cosmetic applications); and (iii) systemic delivery (e.g. for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions). TAV compositions can be delivered to the skin by several different approaches known in the art. Most topical delivery approaches have been shown to work for delivery of DNA, such as but not limited to, topical application of non-cationic liposome–DNA complex, cationic liposome–DNA complex, particle-mediated (gene gun), puncture- mediated gene transfections, and viral delivery approaches. After delivery of the nucleic acid, gene products have been detected in a number of different skin cell types, including, but not limited to, basal keratinocytes, sebaceous gland cells, dermal fibroblasts and dermal macrophages.

[000558] Ointments, creams and gels for topical administration, can, for example, can be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents.

[000559] Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.

[000560] Topically-administrable formulations may, for example, comprise from about 0.1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Combinations [000561] The TAV compositions of the present invention may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By“in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

[000562] TAV compositions of the present invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.

[000563] The combinations referred to above can conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

[000564] The individual compounds of such combinations can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. In one embodiment, the individual compounds will be administered simultaneously in a combined pharmaceutical formulation.

[000565] It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In one embodiment, the combinations, each or together may be administered according to the split dosing regimens described herein.

Dosing

[000566] The present invention provides methods comprising administering TAV compositions and in accordance with the invention 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.

Compositions in accordance with the invention 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 the compositions of the present invention 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.

[000567] In certain embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect (see e.g., the range of unit doses described in International Publication No WO2013078199, 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, or every four weeks. 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.

[000568] According to the present invention, TAV compositions may be administered in split-dose regimens. As used herein, a“split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a“single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a“total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the TAV compositions of the present invention are administered to a subject in split doses. The TAV compositions may be formulated in buffer only or in a formulation described herein.

Dosage Forms

[000569] A TAV pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

Injectable

[000570] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[000571] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[000572] In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the TAV compositions then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered TAV compositions may be accomplished by dissolving or suspending the TAV compositions in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the TAV compositions in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of TAV compositions to polymer and the nature of the particular polymer employed, the rate of polynucleotides release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the TAV compositions in liposomes or microemulsions which are compatible with body tissues.

Multi-dose and repeat-dose administration

[000573] In some embodiments, TAV compositions of the present invention may be administered in two or more doses (referred to herein as“multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and/or volume of components or an altered mass and/or volume of components in comparison to a previous dose. In some embodiments, multi-dose administration may comprise repeat- dose administration. As used herein, the term“repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising substantially the same components provided at substantially the same mass and/or volume. In some embodiments, subjects may display a repeat-dose response. As used herein, the term“repeat-dose response” refers to a response in a subject to a repeat-dose that differs from that of another dose administered within a repeat-dose administration regimen. In some embodiments, such a response may be the expression of a protein in response to a repeat-dose comprising TAV. In such embodiments, protein expression may be elevated in comparison to another dose administered within a repeat-dose administration regimen or protein expression may be reduced in comparison to another dose administered within a repeat-dose administration regimen. Alteration of protein expression may be from about 1% to about 20%, from about 5% to about 50% from about 10% to about 60%, from about 25% to about 75%, from about 40% to about 100% and/or at least 100%. A reduction in expression of mRNA administered as part of a repeat-dose regimen, wherein the level of protein translated from the administered RNA is reduced by more than 40% in comparison to another dose within the repeat-dose regimen is referred to herein as “repeat-dose resistance.” V. Uses of polynucleotides of the Invention

Therapeutics

Therapeutic Agents

[000574] The TAV compositions of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, a TAV composition described herein can be administered to a subject, wherein the polynucleotide is translated in vivo to produce a therapeutic or prophylactic polypeptide in the subject. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include TAV compositions, cells containing TAV compositions or polypeptides translated from the

polynucleotides contained in said TAV compositions.

[000575] Provided herein are methods of inducing translation of a polypeptide (antigen) in a cell, tissue or organism using the polynucleotides of the TAV compositions described herein. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing a TAV which contains a polynucletotide that has at least one a translatable region encoding the polypeptide of interest (antigen).

[000576] An“effective amount” of the TAV composition 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 TAV, and other determinants. In general, an effective amount of the TAV composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen. Increased antigen production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the TAV compositions), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered innate immune response of the host cell.

[000577] Aspects of the invention are directed to methods of inducing in vivo translation of a polypeptide antigen (e.g., a polypeptide PCSK9, TNF alpha, IL-17A, or GDF8 antigen) in a mammalian subject in need thereof. Therein, an effective amount of a TAV composition containing a polynucleotide that has at least one structural or chemical modification and a translatable region encoding the polypeptide (antigen) is administered to the subject using the delivery methods described herein. The polynucleotide is provided in an amount and under other conditions such that the polynucleotide is localized into a cell of the subject and the polypeptide is translated in the cell from the polynucleotide. The cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of TAV composition administration.

In some embodiments, modified polynucleotides of the TAV compositions and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions associated with PCSK9, including but not limited to hypercholesterolemia and atherosclerosis. They may also be used to reduce cholesterol levels. In certain embodiments, modified polynucleotides of the TAV compositions and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions associated with TNF alpha, including but not limited to inflammatory disease, autoimmune disease, and/or cancer. In certain embodiments, modified polynucleotides of the TAV compositions and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions associated with IL-17A, including but not limited to inflammatory disease, autoimmune disease, and/or cancer. In certain embodiments, modified polynucleotides of the TAV compositions and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions associated with muscle injury, degeneration, or weakness, including but not limited to muscular dystrophy.

Modulation of the Immune Response

Activation of the immune response: Vaccines

[000578] According to the present invention, the TAV compositions disclosed herein encode an antigenic polypeptide and act as a vaccine when provided to a subject. As used herein, a“vaccine” is a biological preparation that improves immunity to a particular disease or pathological agent. A vaccine introduces an antigen into the tissues or cells of a subject and elicits an immune response, thereby protecting the subject from a particular disease or pathogenic agent. The polynucleotides of the present invention may encode a PCSK9, TNF alpha, IL-17A, or GDF8 antigen and when the polynucleotides are expressed in cells, a desired immune response is achieved.

[000579] The use of RNA in or as a vaccine overcomes the disadvantages of conventional genetic vaccination involving incorporating DNA into cells in terms of safeness, feasibility, applicability, and effectiveness to generate immune responses. RNA molecules are considered to be significantly safer than DNA vaccines, as RNAs are more easily degraded. They are cleared quickly out of the organism and cannot integrate into the genome and influence the cell’s gene expression in an

uncontrollable manner. It is also less likely for RNA vaccines to cause severe side effects like the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol.2010, article ID623687). Transfection with RNA requires only insertion into the cell’s cytoplasm, which is easier to achieve than into the nucleus. However, RNA is susceptible to RNase degradation and other natural decomposition in the cytoplasm of cells.

[000580] Various attempts to increase the stability and shelf life of RNA vaccines. US 2005/0032730 to Von Der Mulbe et al. discloses improving the stability of mRNA vaccine compositions by increasing G(guanosine)/C(cytosine) content of the mRNA molecules. US 5580859 to Felgner et al. teaches incorporating polynucleotide sequences coding for regulatory proteins that binds to and regulates the stabilities of mRNA. While not wishing to be bound by theory, it is believed that the

polynucleotides vaccines (TAVs) of the invention will result in improved stability and therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.

[000581] Additionally, certain modified nucleosides, or combinations thereof, when introduced into the TAV polynucleotides of the invention will activate the innate immune response. Such activating molecules are useful as adjuvants when combined with polypeptides and/or other vaccines. In certain embodiments, the activating molecules contain a translatable region which encodes for a polypeptide sequence useful as a vaccine, thus providing the ability to be a self-adjuvant.

[000582] In one embodiment, the TAV compositions of the invention may encode an immunogen. The delivery of the polynucleotides encoding an immunogen may activate the immune response. As a non-limiting example, the polynucleotides encoding an immunogen may be delivered to cells to trigger multiple innate response pathways (see International Pub. No. WO2012006377 and US Patent Publication No. US20130177639; herein incorporated by reference in its entirety). As another non- limiting example, TAV polynucleotides of the present invention encoding an immunogen may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see International Pub. No. WO2012006372 and WO2012006369 and US Publication No. US20130149375 and US20130177640; the contents of each of which are herein incorporated by reference in their entirety).

[000583] The TAV polynucleotides of the invention may encode a polypeptide sequence for a vaccine and may further comprise an inhibitor. The inhibitor may impair antigen presentation and/or inhibit various pathways known in the art. As a non-limiting example, the polynucleotides of the invention may be used for a vaccine in combination with an inhibitor which can impair antigen presentation (see

International Pub. No. WO2012089225 and WO2012089338; each of which is herein incorporated by reference in their entirety).

[000584] In one embodiment, a formulation of the TAV polynucleotides of the invention may further comprise an amphipathic and/or immunogenic amphipathic peptide. As a non-limiting example, the polynucleotides comprising an amphipathic and/or immunogenic amphipathic peptide may be formulated as described in US. Pub. No. US20110250237 and International Pub. Nos. WO2010009277 and

WO2010009065; each of which is herein incorporated by reference in their entirety.

[000585] In one embodiment, the TAV polynucleotides of the invention may be immunostimultory. In another non-limiting example, the immunostimultory polynucleotides of the present invention may be formulated with an excipient for administration as described herein and/or known in the art (see International Pub No. WO2012068295 and US Pub No. US20120213812, each of which is herein incorporated by reference in their entirety). The polynucleotides may further comprise a sequence region encoding a cytokine that promotes the immune response, such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, GM-CFS, LT-α, or growth factors such as hGH.

[000586] In one embodiment, the response of the vaccine formulated by the methods described herein may be enhanced by the addition of various compounds to induce the therapeutic effect. As a non-limiting example, the vaccine formulation may encode a MHC II binding peptide or a peptide having a similar sequence to a MHC II binding peptide (see International Pub Nos. WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965, each of which is herein incorporated by reference in their entirety).

[000587] In one embodiment, the effective amount of the polynucleotides of the TAVs of the invention provided to a cell, a tissue or a subject may be enough for immune prophylaxis.

[000588] In one embodiment, the TAV polynucleotides of the invention may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a 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.

[000589] In one embodiment, the TAV polynucleotides of the invention may be administered intranasally similar to the administration of live vaccines. In another aspect the polynucleotide may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.

[000590] As a non-limiting example, the polynucleotide encode at least one antigen, at least one dendritic cell targeting agent or moiety and at least one

immunomodulatory agent or moiety. [000591] In one embodiment, the TAV may be used as a vaccine and may further comprise an adjuvant which may enable the vaccine to elicit a higher immune response. As a non-limiting example, the adjuvant could be a sub-micron oil-in-water emulsion which can elicit a higher immune response in human pediatric populations (see e.g., the adjuvanted vaccines described in US Patent Publication No.

US20120027813 and US Patent No. US8506966, the contents of each of which are herein incorporated by reference in its entirety).

[000592] In another embodiment, the TAV may be used to as a vaccine where the TAV polynucleotides may also comprise 5’ cap analogs to improve the stability and increase the expression of the vaccine. Non-limiting examples of 5’cap analogs are described in US Patent Publication No. US20120195917, the contents of which are herein incorporated by reference in its entirety.

[000593] In one embodiment, the present invention includes a method of enhancing an immune response to an endogenous PCSK9 polypeptide, comprising providing to a cell, tissue or subject a TAV polynucleotide encoding a PCSK9 antigen. In particular embodiments, an mRNA encoding a PCSK9 antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided.

[000594] In one embodiment, the present invention includes a method of treating or preventing a disease or disorder associated with PCSK9 overexpression or a PCSK9 mutation in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a TAV compositions of the present invention. In particular embodiments, the pharmaceutical composition comprises an mRNA encoding a PCSK9 antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide. In particular embodiments, the dendritic cell targeting agent or immune enhancing polypeptide is also encoded by the mRNA. In certain embodiments, the disease or disorder is hypercholesterolemia or atherosclerosis.

[000595] Certain embodiments are directed to a method of enhancing an immune response to a TNF alpha polypeptide, e.g., an endogenous TNF alpha polypeptide, comprising providing to a cell, tissue or subject a TAV polynucleotide encoding a TNF alpha antigen. In particular embodiments, an mRNA encoding a TNF alpha antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided. [000596] In one embodiment, the present invention includes a method of treating or preventing a TNF alpha-associated disease in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a TAV compositions of the present invention. A TNF alpha associated disease, as used herein, refers to a disease in which the presence of TNF alpha is thought to contribute to at least one feature or symptom of the disease, and/or a disease that may be treated through inhibition of TNF alpha. TNF alpha associated diseases include, but are not limited to, inflammatory diseases (e.g. diabetic complication such as retinopathy, nephrosis, neuropathy, great vessel disorder and the like; arthritis such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma,

osteoarthritis, rheumatoid myelitis, gouty arthritis, osteomyelitis and the like;

lumbago; gout; inflammation after operation and trauma; remission of swelling; neuralgia; pharyngitis; cystitis; pneumonia; atopic dermatitis; inflammatory bowel disease such as Crohn's disease, ulcerative colitis, and the like; meningitis;

inflammatory ocular disease; inflammatory pulmonary disease such as pneumonia, silicosis, pulmonary sarcoidosis, pulmonary tuberculosis and the like), circulatory system diseases (e.g. angina pectoris, myocardial infarction, congestive heart failure, disseminated intravascular coagulation and the like), diabetic nephropathy, asthma, allergic disease, chronic obstructive pulmonary disease, systemic lupus

erythematosus, Crohn's disease, autoimmune hemolytic anemia, psoriasis, nervous degenerative diseases (e.g., Alzheimer disease, Parkinson's disease, amyotrophic lateral sclerosis, AIDS encephalopathy and the like), central nervous disorder (for example, cerebrovascular disorders such as cerebral hemorrhage and cerebral infarction, head trauma, spinal damage, cerebral edema, multiple scleroma and the like), toxemia (e.g., sepsis, septic shock, endotoxin shock, gram negative sepsis, toxic shock syndrome and the like), Addison's disease, Creutzfeldt-Jakob disease, virus infective disease (e.g., virus infective disease such as cytomegalovirus, influenza virus, herpesvirus and the like), rejection response upon transplantation and dialytic hypotension.

[000597]

[000598] Certain embodiments are directed to a method of enhancing an immune response to an IL-17A polypeptide, e.g., an endogenous IL-17A polypeptide, comprising providing to a cell, tissue or subject a TAV polynucleotide encoding a IL- 17A antigen. In particular embodiments, an mRNA encoding a IL-17A antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided.

[000599] In one embodiment, the present invention includes a method of treating or preventing a IL-17A-mediated disease in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a TAV compositions of the present invention. IL-17A-mediated disease, as defined herein, is a disease in which IL-17A plays a significant role in the pathology of the disease, and include, but are not limited to inflammatory diseases such as atopic and contact dermatitis, multiple sclerosis, colitis, endotoxemia, arthritis, rheumatoid arthritis, psoriatic arthritis, mucosal immune diseases, adult respiratory disease (ARD), septic shock, multiple organ failure, inflammatory lung injury such as asthma, chronic obstructive pulmonary disease (COPD), airway hyper- responsiveness, chronic bronchitis, allergic asthma, psoriasis, eczema, IBS and inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's

disease, Helicobacter pylori infection, intraabdominal adhesions and/or abscesses as results of peritoneal inflammation (i.e. from infection, injury, etc.), systemic lupus erythematosus (SLE), multiple sclerosis, systemic sclerosis, nephrotic syndrome, organ allograft rejection, graft vs. host disease (GVHD), kidney, lung, heart, etc. transplant rejection, streptococcal cell wall (SCW)-induced arthritis, osteoarthritis, gingivitis/periodontitis, herpetic stromal keratitis, restenosis, and Kawasaki disease.

[000600] Particular embodiments are directed to methods of treating or preventing cancer a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising TAV compositions encoding TNF alpha or IL-17A antigens. In some embodiments, pharmaceutical compositions comprising TAV compositions encoding TNF alpha or IL-17A antigens are administered for the treatment of a cancer, including but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non- Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic

Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T- Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gautier’s Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational

Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer,

Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma,

Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male

Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplasia Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative

Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma,

Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System

Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal

Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous

Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer,

Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer,

Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

[000601] In certain embodiments, a pharmaceutical composition comprising a TAV composition that encodes IL-17A antigen is administered to a subject for the treatment or prevention of breast cancer, colon cancer, gastric cancer, glioma, hepatocellular carcinoma, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer. In particular embodiments, a pharmaceutical composition comprising a TAV composition that encodes a TNF alpha antigen is administered to a subject for the treatment or prevention of breast cancer, colon cancer, gastric cancer, glioma, hepatocellular carcinoma, kidney cancer, leukemia, lung cancer, lymphoma, melanoma, multiple myeloma, ovarian cancer, pancreatic cancer, prostate cancer.

[000602] Certain embodiments are directed to a method of enhancing an immune response to a GDF8 polypeptide, e.g., an endogenous GDF8 polypeptide, comprising providing to a cell, tissue or subject a TAV polynucleotide encoding a GDF8 antigen. In particular embodiments, an mRNA encoding a GDF8 antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided. [000603] Particular embodiments contemplate reducing the amount or activity of GDF8 in a subject will increase or sustain muscle mass, or prevent muscle loss.

Therefore, in one embodiment, the present invention includes a method of treating or preventing a disease associated with muscle loss in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition comprising a TAV composition encoding GDF8 antigen. Diseases associated with muscle loss include, but are not limited to, inflammatory muscle disease, e.g. polymyositis, dermatomyositis, and inclusion-body myositis, muscular dystrophy, e.g. Duchenne muscular dystrophy, Becker muscular dystrophy, Emery- Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy,

facioscapulohumeral muscular dystrophy, Limb-Girdle muscular dystrophies, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy, Myotonic dystrophy (Steinert's disease) and congenital muscular dystrophy, cachexia, muscular attenuation, muscular atrophy, sarcopenia, intensive care unit (ICU) induced weakness, and surgery-induced weakness. VI. Kits and Devices

Kits

[000604] The invention provides a variety of kits for conveniently and/or effectively carrying out methods of the present invention. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.

[000605] In one aspect, the present invention provides kits comprising the TAV compositions of the invention. In one embodiment, the kit comprises one or more functional antibodies or function fragments thereof.

[000606] Said kits can be for protein production, comprising a first polynucleotide encoding an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen. The kit may comprise a second polynucleotide encoding an immune enhancing polypeptide. The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, a lipidoid or any delivery agent disclosed herein.

[000607] In one aspect, the present invention provides kits for protein production, comprising a polynucleotide comprising a translatable region, wherein the polynucleotide exhibits reduced degradation by a cellular nuclease, and packaging and instructions. In certain embodiments, the translatable region encodes an antigen, e.g., a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen, and an immune enhancing polypeptide.

[000608] In one aspect, the present invention provides kits for protein production, comprising a polynucleotide comprising a translatable region, wherein the polynucleotide exhibits reduced degradation by a cellular nuclease, and a mammalian cell suitable for translation of the translatable region of the first nucleic acid. VII. Definitions

[000609] At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges

[000610] About: As used herein, the term“about” means +/- 10% of the recited value.

[000611] Administered in combination: As used herein, the term“administered in combination” or“combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

[000612] Adjuvant: As used herein, the term“adjuvant” means a substance that enhances a subject’s immune response to an antigen. The TAVs of the present invention may optionally comprise one or more adjuvants.

[000613] Animal: As used herein, the term“animal” refers to any member of the animal kingdom. In some embodiments,“animal” refers to humans at any stage of development. In some embodiments,“animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone. [000614] Antigen: As used herein, the term“antigen” refers to a substance or molecule that induces, elicits or triggers an immune response in a cell, tissue or organism. An antigen may originate either from the body, such as cancer antigen, or from the external environment, for instance, from infectious agents. Antigens may be, in whole or part, endogenous or exogenous peptides, proteins or polypeptides of interest or fragments thereof.

[000615] Antigens of interest or desired antigens: As used herein, the terms “antigens of interest” or“desired antigens” include those proteins and other biomolecules provided herein that are components of or encoded by polynucleotides which are components of one or more TAVs.

[000616] Approximately: As used herein, the term“approximately” or“about,” as applied to one or more values of interest, refers 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).

[000617] Associated with: As used herein, the terms“associated with,”

“conjugated,”“linked,”“attached,” and“tethered,” when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An“association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the“associated” entities remain physically associated.

[000618] Biocompatible: As used herein, the term“biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system.

[000619] Biologically active: As used herein, the phrase“biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, a polynucleotide of the present invention may be considered biologically active if even a portion of the polynucleotides is biologically active or mimics an activity considered biologically relevant.

[000620] Chimera: As used herein,“chimera” is an entity having two or more incongruous or heterogeneous parts or regions.

[000621] Chimeric polynucleotide: As used herein,“chimeric polynucleotides” are those nucleic acid polymers having portions or regions which differ in size and/or chemical modification pattern, chemical modification position, chemical modification percent or chemical modification population and combinations of the foregoing.

[000622] Compound: As used herein, the term“compound,” is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[000623] Conserved: As used herein, the term“conserved” refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.

[000624] In some embodiments, two or more sequences are said to be“completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be“highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be“highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be“conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of an polynucleotide or polypeptide or may apply to a portion, region or feature thereof. [000625] Controlled Release: As used herein, the term“controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.

[000626] Cyclic or Cyclized: As used herein, the term“cyclic” refers to the presence of a continuous loop. Cyclic molecules need not be circular, only joined to form an unbroken chain of subunits. Cyclic molecules such as the engineered RNA or mRNA of the present invention may be single units or multimers or comprise one or more components of a complex or higher order structure.

[000627] Delivery: As used herein,“delivery” refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[000628] Delivery Agent: As used herein,“delivery agent” refers to any substance which facilitates, at least in part, the in vivo delivery of a polynucleotide to targeted cells.

[000629] Detectable label: As used herein,“detectable label” refers to one or more markers, signals, or moieties which are attached, incorporated or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, or located at the N- or C- termini.

[000630] Dosing regimen: As used herein, a“dosing regimen” is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.

[000631] Dose splitting factor (DSF)-ratio of PUD of dose split treatment divided by PUD of total daily dose or single unit dose. The value is derived from comparison of dosing regimens groups.

[000632] Encapsulate: As used herein, the term“encapsulate” means to enclose, surround or encase.

[000633] Encoded protein cleavage signal: As used herein,“encoded protein cleavage signal” refers to the nucleotide sequence which encodes a protein cleavage signal. [000634] Engineered: As used herein, embodiments of the invention are “engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

[000635] Effective Amount: As used herein, the term“effective amount” of an agent is that amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an“effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that treats cancer, an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of cancer, as compared to the response obtained without administration of the agent.

[000636] Exosome: As used herein,“exosome” is a vesicle secreted by mammalian cells or a complex involved in RNA degradation.

[000637] Expression: As used herein,“expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[000638] Feature: As used herein, a“feature” refers to a characteristic, a property, or a distinctive element.

[000639] Formulation: As used herein, a“formulation” includes at least a polynucleotide of a TAV and a delivery agent.

[000640] Fragment: A“fragment,” as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.

[000641] Functional: As used herein, a“functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[000642] Homology: As used herein, the term“homology” refers to the overall relatedness between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 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). In accordance with the invention, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. In accordance with the invention, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.

[000643] Identity: As used herein, the term“identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 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 nucleotide 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 nucleotide 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 nucleotide 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)).

[000644] In vitro: As used herein, the term“in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[000645] In vivo: As used herein, the term“in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[000646] Isolated: As used herein, the term“isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is“pure” if it is substantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure. Substantial separation can include compositions containing 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 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.

[000647] IVT Polynucleotide: As used herein, an“IVT polynucleotide” is a linear polynucleotide which may be made using only in vitro transcription (IVT) enzymatic synthesis methods.

[000648] Linker: As used herein, a“linker” refers to a group of atoms, e.g., 10- 1,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached to a modified nucleoside or nucleotide on the nucleobase or sugar moiety at a first end, and to a payload, e.g., a detectable or therapeutic agent, at a second end. The linker may be of sufficient length as to not interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as to form polynucleotide multimers (e.g., through linkage of two or more chimeric polynucleotides molecules or IVT polynucleoties) or polynucleotides conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomeric units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and derivatives thereof., Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bond include an amido bond can be cleaved for example by the use of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond can be cleaved for example by acidic or basic hydrolysis.

[000649] MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA) binding site represents a nucleotide location or region of a nucleic acid transcript to which at least the“seed” region of a miRNA binds.

[000650] Modified: As used herein“modified” refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the polynucleotide molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Noncanonical nucleotides such as the cap structures are not considered “modified” although they differ from the chemical structure of the A, C, G, U ribonucleotides.

[000651] Naturally occurring: As used herein,“naturally occurring” means existing in nature without artificial aid.

[000652] Open reading frame: As used herein,“open reading frame” or“ORF” refers to a sequence which does not contain a stop codon in a given reading frame.

[000653] Operably linked: As used herein, the phrase“operably linked” refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

[000654] Optionally substituted: Herein a phrase of the form“optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to“X, wherein X is optionally substituted” (e.g.,“alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature“X” (e.g. alkyl) per se is optional.

[000655] Part: As used herein, a“part” or“region” of a polynucleotide is defined as any portion of the polynucleotide which is less than the entire length of the polynucleotide.

[000656] Peptide: As used herein,“peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[000657] Pharmaceutically acceptable: The phrase“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[000658] Pharmaceutically acceptable excipients: The phrase“pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non- inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben,

microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[000659] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. [000660] Pharmaceutically acceptable solvate: The term“pharmaceutically acceptable solvate,” as used herein, means a compound of the invention wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N- methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N’-dimethylformamide (DMF), N,N’-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a“hydrate.”

[000661] Pharmacokinetic: As used herein,“pharmacokinetic” refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.

[000662] Physicochemical: As used herein,“physicochemical” means of or relating to a physical and/or chemical property.

[000663] Polypeptide per unit drug (PUD): As used herein, a PUD or product per unit drug, is defined as a subdivided portion of total daily dose, usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) as measured in body fluid or tissue, usually defined in concentration such as pmol/mL, mmol/mL, etc divided by the measure in the body fluid.

[000664] Preventing: As used herein, the term“preventing” refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.

[000665] Prophylactic: As used herein,“prophylactic” refers to a therapeutic or course of action used to prevent the spread of disease.

[000666] Prophylaxis: As used herein, a“prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease. An“immune prophylaxis” refers to a measure to produce active or passive immunity to prevent the spread of disease.

[000667] Protein cleavage site: As used herein,“protein cleavage site” refers to a site where controlled cleavage of the amino acid chain can be accomplished by chemical, enzymatic or photochemical means.

[000668] Protein cleavage signal: As used herein“protein cleavage signal” refers to at least one amino acid that flags or marks a polypeptide for cleavage.

[000669] Protein of interest: As used herein, the terms“proteins of interest” or “desired proteins” include those provided herein and fragments, mutants, variants, and alterations thereof.

[000670] Proximal: As used herein, the term“proximal” means situated nearer to the center or to a point or region of interest.

[000671] Pseudouridine: As used herein, pseudouridine refers to the C-glycoside isomer of the nucleoside uridine. A“pseudouridine analog” is any modification, variant, isoform or derivative of pseudouridine. For example, pseudouridine analogs include but are not limited to 1-carboxymethyl-pseudouridine, 1-propynyl- pseudouridine, 1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m1ψ), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1- methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza- pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2- thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3 ψ), and 2′-O-methyl-pseudouridine (ψm). [000672] Purified: As used herein,“purify,”“purified,”“purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.

[000673] Repeated transfection: As used herein, the term“repeated transfection” refers to transfection of the same cell culture with a polynucleotide a plurality of times. The cell culture can be transfected 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, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times at least 18 times, at least 19 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times or more.

[000674] Sample: As used herein, the term“sample” or“biological sample” refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.

[000675] Signal Sequences: As used herein, the phrase“signal sequences” refers to a sequence which can direct the transport or localization of a protein.

[000676] Single unit dose: As used herein, a“single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.

[000677] Similarity: As used herein, the term“similarity” refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art. [000678] Split dose: As used herein, a“split dose” is the division of single unit dose or total daily dose into two or more doses.

[000679] Stable: As used herein“stable” refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

[000680] Stabilized: As used herein, the term“stabilize”,“stabilized,”“stabilized region” means to make or become stable.

[000681] Stereoisomer: As used herein, the term“stereoisomer” refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.

[000682] Subject: As used herein, the term“subject” or“patient” refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

[000683] Substantially: As used herein, the term“substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a

characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[000684] Substantially equal: As used herein as it relates to time differences between doses, the term means plus/minus 2%.

[000685] Substantially simultaneously: As used herein and as it relates to plurality of doses, the term means within 2 seconds.

[000686] Suffering from: An individual who is“suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition. [000687] Susceptible to: An individual who is“susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

[000688] Sustained release: As used herein, the term“sustained release” refers to a pharmaceutical composition or compound release profile that conforms to a release rate over a specific period of time.

[000689] Synthetic: The term“synthetic” means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.

[000690] Targeted Adaptive Vaccine: As used herein, a“targeted adaptive vaccine” or“TAV” is a vaccine which comprises at least one antigenic polypeptide or a polynucleotide encoding an antigenic polypeptide, optionally further comprising a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety.

[000691] Targeted Cells: As used herein,“targeted cells” refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

[000692] Therapeutic Agent: The term“therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. [000693] Therapeutically effective amount: As used herein, the term

“therapeutically effective amount” means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[000694] Therapeutically effective outcome: As used herein, the term

“therapeutically effective outcome” means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.

[000695] Total daily dose: As used herein, a“total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.

[000696] Transcription: As used herein, the term“transcription” refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures.

[000697] Treating: As used herein, the term“treating” refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition. For example,“treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.

[000698] Unmodified: As used herein,“unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification. [000699] Vaccine: As used herein, the phrase“vaccine” refers to a biological preparation that improves immunity in the context of a particular disease, disorder or condition.

[000700] Viral protein: As used herein, the phrase“viral protein” means any protein originating from a virus. Equivalents and Scope

[000701] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[000702] In the claims, articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context.

Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes

embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[000703] It is also noted that the term“comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term“comprising” is used herein, the term“consisting of” is thus also encompassed and disclosed.

[000704] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[000705] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[000706] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[000707] Section and table headings are not intended to be limiting.

EXAMPLES

Example 1

MANUFACTURE AND CHARACTERIZATION OF CHIMERIC POLYNUCLEOTIDES

[000708] According to the present invention, the manufacture of chimeric polynucleotides and or parts or regions thereof may be accomplished utilizing the methods taught in USSN 61/800,049 filed March 15, 2013 entitled“Manufacturing Methods for Production of RNA Transcripts” (Attorney Docket number M500), the contents of which is incorporated herein by reference in its entirety.

[000709] Purification methods may include those taught in USSN 61/799,872 filed March 15, 2013 entitled“Methods of removing DNA fragments in mRNA

production” (Attorney Docket number M501); USSN 61/794,842 filed March 15, 2013, entitled“Ribonucleic acid purification” (Attorney Docket number M502), each of which is incorporated herein by reference in its entirety.

[000710] Characterization of the chimeric polynucleotides of the invention 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, USSN 61/798,945 filed March 15, 2013 entitled“Characterization of mRNA molecules” (Attorney Docket number M505); USSN 61/799,905 filed March 15, 2013 entitled “Analysis of mRNA Heterogeneity and Stability” (Attorney Docket number M506) and USSN 61/800,110 filed March 15, 2013 entitled“Ion Exchange Purification of mRNA” (Attorney Docket number M507) the contents of each of which is incorporated herein by reference in its entirety.

Chimeric polynucleotide synthesis: triphosphate route

Introduction

[000711] According to the present invention, two regions or parts of a chimeric polynucleotide may be joined or ligated using triphosphate chemistry.

[000712] 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. [000713] 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.

[000714] Monophosphate protecting groups may be selected from any of those known in the art.

[000715] 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.

[000716] It is noted that for ligation methods, ligation with DNA T4 ligase, followed by treatment with DNAse should readily avoid concatenation.

[000717] 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.

[000718] Ligation is then performed using any known click chemistry, orthoclick chemistry, solulink, or other bioconjugate chemistries known to those in the art.

Synthetic route

[000719] The chimeric polynucleotide is made using a series of starting segments. Such segments include:

[000720] (a) Capped and protected 5’ segment comprising a normal 3’OH (SEG.1)

[000721] (b) 5’ triphosphate segment which may include the coding region of a polypeptide and comprising a normal 3’OH (SEG.2)

[000722] (c) 5’ monophosphate segment for the 3’ end of the chimeric

polynucleotide (e.g., the tail) comprising cordycepin or no 3’OH (SEG.3)

[000723] After synthesis (chemical or IVT), segment 3 (SEG.3) is treated with cordycepin and then with pyrophosphatase to create the 5’monophosphate.

[000724] 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. [000725] 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).

[000726] The yields of each step may be as much as 90-95%.

PCR for cDNA Production

[000727] 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.

[000728] The reverse primer of the instant invention incorporates a poly-T120 for a poly-A120 in the mRNA. Other reverse primers with longer or shorter poly(T) tracts can be used to adjust the length of the poly(A) tail in the polynucleotide mRNA.

[000729] 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 NANODROPTM 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.

In vitro Transcription (IVT)

A. Preparation of DNA Template

Restriction Digest of Plasmid DNA

[000730] DNA plasmid is digested by incubation at 37°C for 2 hr in a 50 µL reaction containing DNA plasmid (50 ng/µL), BSA (1X), 1X NEBuffer 4 (50mM potassium acetate, 20mM Tris-acetate, 10mM magnesium acetate, 1mM DTT, pH 7.9), and XbaI (400 U/mL) (New England Biolabs). The restriction digest is analyzed by 1% agarose gel and used directly for PCR.

DNA Template Amplification

The desired DNA template is amplified by PCR in 100 uL reactions using linearized plasmid (20 ng), dNTPs (0.2 µM each), forward primer (0.2 µM), reverse primer (0.2 µM), 1X Q5 reaction buffer, and Q5 high-fidelity DNA polymerase (20 U/mL) (New England Biolabs). All components are kept on ice until added to the thermocycler. The reaction conditions are at 95° C for 4 min. and 30 cycles of 98° C for 15 sec, then 72° C for 45 sec, then 72° C for 20 sec per kb, then 72° C for 5 min. then 4° C to termination. The PCR product is analyzed by capillary electrophoresis (CE) (Agilent 2100 Bioanalyzer) and desalted by ultrafiltration (Amicon).

B. IVT Reaction

[000731] In vitro transcription (IVT) reactions are performed in 50 uL containing template DNA (25 ng/µL), NTPs (7.6 mM each), 1X T7 IVT buffer, RNase Inhibitor (1 U/µL), Pyrophosphatase (1 U/µL), and T7 RNA polymerase (7 U/µL) (NEB). In general, 2450uL reactions per construct are used. Modified mRNA may be generated using 5-methyl-CTP and 1-methyl-pseudoUTP or any chosen modified triphosphate. IVT reactions are incubated at 37 °C for 4 hr, after which 2.5 µL of DNase I (2000 U/mL) (NEB) is added and the reaction allowed to incubated for another 45 min. The reactions are combined and purified using MEGAclear spin columns (Ambion) and eluted in 250 µL water. The IVT product is analyzed by CE (Agilent 2100

Bioanalyzer).

Enzymatic Capping

[000732] 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.

[000733] 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.

[000734] 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.

C. PolyA Tailing Reaction [000735] 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.

[000736] 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.

D. Natural 5′ Caps and 5′ Cap Analogues

[000737] 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.

[000738] 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.

Characterization of Capped Chimeric Polynucleotides

A. Protein Expression Assay [000739] Chimeric 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 chimeric polynucleotides that secrete higher levels of protein into the medium would correspond to a synthetic chimeric polynucleotide with a higher translationally-competent Cap structure.

B. Purity Analysis Synthesis

[000740] Chimeric 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. Chimeric polynucleotides with a single, consolidated band by electrophoresis correspond to the higher purity product compared to chimeric polynucleotides with multiple bands or streaking bands.

Synthetic chimeric 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 chimeric polynucleotide population.

C. Cytokine Analysis

[000741] Chimeric 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. Chimeric polynucleotides resulting in the secretion of higher levels of pro- inflammatory cytokines into the medium would correspond to a chimeric polynucleotides containing an immune-activating cap structure.

D. Capping Reaction Efficiency

[000742] Chimeric 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 chimeric 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 chimeric 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.

Agarose Gel Electrophoresis of Modified RNA or RT PCR Products [000743] Individual chimeric 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.

Nanodrop Modified RNA Quantification and UV Spectral Data

[000744] Modified chimeric polynucleotides in TE buffer (1 µl) are used for Nanodrop UV absorbance readings to quantitate the yield of each chimeric polynucleotide from an chemical synthesis or in vitro transcription reaction.

Formulation of Modified mRNA Using Lipidoids

[000745] Chimeric polynucleotides are formulated for in vitro experiments by mixing the chimeric 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 be used as a starting point. After formation of the particle, chimeric polynucleotide is added and allowed to integrate with the complex. The

encapsulation efficiency is determined using a standard dye exclusion assays.

Method of Screening for Protein Expression

A. Electrospray Ionization

[000746] A biological sample which may contain proteins encoded by a chimeric polynucleotide administered to the subject is prepared and analyzed according to the manufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or 4 mass analyzers. A biologic sample may also be analyzed using a tandem ESI mass spectrometry system.

[000747] Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

[000748] A biological sample which may contain proteins encoded by one or more chimeric polynucleotides administered to the subject is prepared and analyzed according to the manufacturer protocol for matrix-assisted laser desorption/ionization (MALDI).

Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass spectrometry-Mass spectrometry [000749] A biological sample, which may contain proteins encoded by one or more chimeric polynucleotides, may be treated with a trypsin enzyme to digest the proteins contained within. The resulting peptides are analyzed by liquid chromatography-mass spectrometry-mass spectrometry (LC/MS/MS). The peptides are fragmented in the mass spectrometer to yield diagnostic patterns that can be matched to protein sequence databases via computer algorithms. The digested sample may be diluted to achieve 1 ng or less starting material for a given protein. Biological samples containing a simple buffer background (e.g. water or volatile salts) are amenable to direct in-solution digest; more complex backgrounds (e.g. detergent, non-volatile salts, glycerol) require an additional clean-up step to facilitate the sample analysis.

[000750] Patterns of protein fragments, or whole proteins, are compared to known controls for a given protein and identity is determined by comparison.

D. Cyclization and/or concatemerization

[000751] According to the present invention, a chimeric polynucleotide may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5′-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5′- /3′-linkage may be intramolecular or intermolecular.

[000752] In the first route, the 5′-end and the 3′-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5′-end and the 3′-end of the molecule. The 5′-end may contain an NHS- ester reactive group and the 3′-end may contain a 3′-amino-terminated nucleotide such that in an organic solvent the 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNA molecule will undergo a nucleophilic attack on the 5′-NHS-ester moiety forming a new 5′-/3′-amide bond.

[000753] In the second route, T4 RNA ligase may be used to enzymatically link a 5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In an example reaction, 1µg of a nucleic acid molecule is incubated at 37ºC for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer’s protocol. The ligation reaction may occur in the presence of a split polynucleotide capable of base- pairing with both the 5′- and 3′- region in juxtaposition to assist the enzymatic ligation reaction. [000754] In the third route, either the 5′-or 3′-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5′-end of a nucleic acid molecule to the 3′-end of a nucleic acid molecule. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37ºC. Example 2

INDUCTION OF AUTO-ANTIBODY AGAINST PCSK9 TO TREAT AUTOSOMAL DOMINANT

HYPERCHOLESTEROLEMIA CAUSED BY PCSK9 MUTATION

[000755] A polynucletotide-based vaccine or targeted adaptive vaccine (TAV) is prepared using a polynucleotide encoding the antigen which is either the full length or fragment of the PCSK9 protein (e.g. human or murine PSCK9). In certain embodiments, the polynucelodte encoding the antigen is fused in-frame to a polynucleotide encoding an immunomodulatory IM, either directly or through a linker. The sequences of the human and murine forms are provided in the Table 12. Table 12. PCSK9 Forms and Sequences

Figure imgf000256_0001
Figure imgf000257_0001

Figure imgf000258_0001

In vivo induction of antibody against PCSK9 in mice

[000756] The polynucleotide encoding the PCSK9 antigen as described above and being chemically modified (e.g. as decribed herein) is formulated for administration to the mammalian subject. The formulation is either in saline or any of the formulations taught herein.

[000757] The TAV containing the PCSK9 polynucleotide optionally contains one or more dendritic targeting agent or moieties.

[000758] The TAV comprising the polynucleotide encoding the antigen is injected via a suitable route, either intradermal, subcutaneous, intramuscular, or intravenous route at Day 0.

[000759] A polynucleotide encoding an immunostimulatory agent or moiety can be co-administered with the polynucleotide encoding the PCSK9 antigen to stimulate immune response. In certain embodiments, the two polynucleotides are present within the same polynucleotide.

[000760] Additional challenges of the TAV containing the polynucleotide encoding the PCSK9 antigen are given on a weekly, bi-weekly, every three week, or every four week basis until detection of anti-PCSK9 antibody and/or lowering of serum PCSK9 concentration is detected.

[000761] LDL is monitored for a period to determine the long term efficacy of induced auto-antibody against endogenous PCSK9.

[000762] In the event of diminishing of auto-antibody against PCSK9, additional TAV challenges are administered to boost the production of auto-antibody. [000763] Where auto-immunity mediated side effects occur, tolerizing agents and/or compositions are co-administered with the PCSK9 targeted adaptive vaccine to induce antigen specific tolerance.

In vivo induction of PCSK9 antibodies in mice

[000764] Table 13 lists the sequences of PSCK9 antigens that are administered to measure and evaluate their ability to generate antibodies directed against PCSK9. Table 13. Table of Murine PCSK9 fragments

Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001

[000765] The polynucleotides encoding PCSK9 antigen as described in Table 13 are chemically modified. The polynucleotides may further comprise a sequence encoding an IM. The modified polynucleotides are then formulated for administration to the mammalian subject with the objective to measure and compare the immunogenicity of (1) intracellular PCSK9 (SEQ ID NO: 44), (2) secreted PCSK9 (SEQ ID NO: 43), (3) secreted PCSK9 that includes a dentritic cell targeting moiety (SEQ ID NO: 42) and (4) secreted PCSK9 formulated and administered in combination with

immunopotentiation factors selected from the group of GMCSF, IL-15, IL-21, etc. A control administration of the formulation without polynucleotide is performed in parallel. The formulation is either in saline or any of the formulations taught herein.

[000766] The TAV comprising the polynucleotide encoding the PCSK9 antigen is injected via a suitable route, either intradermal, subcutaneous, intramuscular, or intravenous route at Day 0. Additional dosages of the TAV containing the

polynucleotide encoding the PCSK9 antigen are given on a weekly, bi-weekly, every three week, or every four week basis. During the time course, anti-PCSK9 antibody and total IgG concentrations, and serum PCSK9 protein and LDL levels are measured. The time course is continued until anti-PCSK9 antibody and/or lowering of serum PCSK9 concentration is detected.

[000767] LDL levels are further monitored for a period to determine the long term efficacy of induced antibody against endogenous PCSK9. In the event of diminishing of antibody against PCSK9, additional TAV challenges are administered to boost the production of antibody.

[000768] A polycistronic construct which contains a C-terminal fragment of PCSK9 flanked on its C- and N-termini by an immunomodulatory agent or moiety comprising a fragment of a viral or bacterial protein to increase antigenicity is administered in parallel according to the same formulation, delivery method and time course as the polynucleotides described above. Another polycistronic construct, in which the immunomodulatory moiety is located within the sequences of the PCSK9 fragment, is also administered in parallel. The immunomodulatory agent or moiety comprises a protein or fragment thereof derived from a virus selected from the group of tetanus, chicken pox, rubella, small pox and mumps, which is 1, 2, 3-5, or greater than 5 amino acids in length. During the time course, anti-PCSK9 antibody and total IgG concentrations, and serum PCSK9 protein and LDL levels are measured in parallel with the measurements described above. The time course is continued until anti- PCSK9 antibody and/or lowering of serum PCSK9 concentration is detected.

Treatment with reversing agents to desensitize mice and turn off TAV

[000769] In one embodiment, the polynucleotides comprising the TAV are administered in combination with a reversing agent, which functions to turn off the TAV after sufficient immune response is mounted to avoid excess PCSK9 antibodies. In non-limiting examples, the reversing agent may be Bortezomib (VELCADE®) or Rituximab (RITUXAN®) to deplete the mice of the antibody-producing plasma cells.

[000770] Mice previously treated with TAV to produce PCSK9 antibodies as described above are either treated with saline alone or are treated with 0.75 mg/kg of Bortezomib of Rituximab twice a week for 4 weeks. The reversing agents are injected via a suitable route, either intradermal, subcutetanous, intramuscular, or intravenous route. During the course of the treatment, the anti-PCSK9 antibody titer, total serum IgG, serum PCSK9 protein, and total cholesterol levels are measured. A reduction in anti-PCSK9 antibodies over constant total serum IgG is observed in the Bortezomib and Rituximab-treated group over the saline-treated control group during the time course of the treatment. Serum PCSK9 protein and serum LDL levels increase over the time course of the treatment in the Bortezomib and Rituximab-treated group over the saline treated control group.

[000771] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

Example 3

PCSK9 TAV INDUCES ENDOGENOUS ANTIBODY RESPONSE

[000772] To demonstrate that PCSK9 TAVs could induce an endogenous antibody response against mouse PCSK9, mice were immunized with an mRNA encoding a either murine PCSK9 fragment containing the catalytic domain (mouse C-terminal domain deletion; see Table 13) or the corresponding human PCSK9 fragment, and levels of anti-human PCSK9 antibodies and anti-mouse PCDK9 antibodies were determined. mRNAs were prepared by IVS as described in Example 1.

[000773] Briefly, mice were immunized with mRNA encoding human PCSK9 fragment, mRNA encoding the murine PCSK9 fragment, or a PBS control at a first time point, and then two weeks later and four weeks later after the first immunization. The mRNAs were encapsulated in a lipid nanoparticle. Blood samples were obtained from the animals about one week before the first immunization, about the time of each of the three immunizations, and 10 or 11 weeks following the first

immunization, and levels of anti-human PCSK9 antibody and anti-mouse PCSK9 antibodies were determined for each blood sample (mcg/mL).

[000774] As shown in Figure 11A (left panel), mice immunized with the murine PCSK9 TAV construct or PBS showed no production of anti-human PCSK9 antibodies, whereas mice immunized with the human PCSK9 TAV construct showed increased levels of anti-human PCSK9 antibodies, which peaked at about 40-80 days following the first immunization. Interestingly, mice immunized with the human PCSK9 TAV construct showed increased levels of both anti-human PCSK9 antibodies and anti-mouse PCSK9 antibodies, both of which peaked at about 40-50 days following the first immunization (Figure 11B, right panel).

[000775] To ascertain the functional results of this increase in anti-mouse PCSK9 antibodies, levels of mouse pCSK9 protein (ng/mL) in the serum samples were also measured. As shown in Figure 12, immunization with the human PCSK9 TAV resulted in a rapid and sustained decrease in endogenous mouse PCSK9 levels, wherein immunization with the murine PCSK9 TAV or PBS had little or no sustained effect on levels of endogenous mouse PCSK9.

[000776] In addition, total serum lipid (LDL/VLDL/HDL) levels were determined over a time course following the first immunization, using a moving average (3 period) time-course plot to control for variability due to: (1) food being available ad libitum throughout the study with no fasting levels; and (2) to control for any variability due to the use of a colorometric assay. As shown in Figures 13A and 13B, mice immunized with the human PCSK9 TAV construct showed a substantially reduced level of total serum lipid level.

[000777] These studies demonstrate that a human PCSK9 TAV can be used to break tolerance and induce functional antibodies against endogenous mouse PCSK9, which resulted in reduced levels of endogenous mouse PCSK9 and lowered serum lipid levels. Example 4

IDENTIFICATION OF CROSS-REACTIVE PCSK9 EPITOPES

[000778] Studies were performed to identify B cell epitopes or immunodominant regions of human PCSK9 and mouse PCSK9, including regions of cross-reactivity. Briefly, overlapping peptides derived from human PCSK9 or mouse PCSK9 were displayed on peptide microarrays and incubated with purified polyclonal or monoclonal antibodies or plasma of interest, to identify peptides bound by the antibodies or plasma. These experiments were performed using the ProArray Ultra peptidic microarray (ProImmune Inc., Sarasota, FL).

[000779] An overlapping peptide library of 15-mer synthetic peptides, overlapping by 12 amino acids, representing human PCSK9.delC (SEQ ID NOS 1-136) and mouse PCSK.delC (SEQ IN NOS: 137-272) was synthesized. The sequences of these peptides are shown in Figure 14.

[000780] The peptides were then bound to the ProArray Ultra slide surface, blocked using a blocking buffer to reduce non-specific binding, and then incubated with various concentrations/dilutions of different test samples (e.g., antibodies or serum), followed by incubation with a fluorescent-labeled secondary antibody specific for each of the test samples. After several washing steps, the arrays were dried and scanned using a high resolution fluorescence microarray scanning system, and scanner images were recorded and evaluated using image analysis software, which enabled the quantification of the levels of fluorescence intensities associated with each peptide. Test samples included anti-human PCSK9 antibodies and anti-mouse PCSK9 antibodies.

[000781] When the peptide library was screened using serum from mice previously immunized with PCSK9, several human and mouse PCSK9 peptides were identified, which mapped to three regions of overlapping peptides: peptides 10 to 15 and peptides 129-133 from the human PCSK9.delC sequence; and peptides 265-268 from the mouse PCSK9.delC sequence. Peptides 129-133 from human PCSK9 and peptides 265-268 from mouse PCSK9 displayed some sequence homology and were identified as a cross-reactive peptide candidate for use in PCSK9 TAVs.

[000782] Peptide 10: HGTTATFHRCAKDPW [000783] Peptide 11: TATFHRCAKDPWRLP

[000784] Peptide 12: FHRCAKDPWRLPGTY

[000785] Peptide 13: CAKDPWRLPGTYVVV

[000786] Peptide 14: DPWRLPGTYVVVLKE

[000787] Peptide 15: RLPGTYVVVLKEETH

[000788] Peptide 129: LIHFSAKDVINEAWF

[000789] Peptide 130: FSAKDVINEAWFPED

[000790] Peptide 131: KDVINEAWFPEDQRV

[000791] Peptide 132: INEAWFPEDQRVLTP

[000792] Peptide 133: AWFPEDQRVLTPNLV

[000793] Peptide 265: IHFSTKDVINMAWFP

[000794] Peptide 266: STKDVINMAWFPEDQ

[000795] Peptide 267: DVINMAWFPEDQQVL

[000796] Peptide 268: NMAWFPEDQQVLTPN

[000797] The cross-reactive peptide candidates are shown below with alignment between the human and mouse peptides shown in gray:

LIHFSAKDVINEAWFPEDQRVLTPNLV (Peptide 129-133 (human PCSK9)) IHFSTKDVINMAWFPEDQQVLTPN (Peptide 265-268 (mouse PCSK9)).

[000798] Studies were next performed to identify human PCSK9 or mouse PCSK9 MCH Class II T cell epitopes using the ProImmune Reveal Immunogenicity System, which measures MHC-peptide binding, and the ProImumne CFSE-based T cell proliferation assays. The same overlapping peptide library was assayed for binding against mouse allele H-2 IAd. Briefly, the peptides were assembled with the allele H- 2 IAd and analyzed using the ProImmune REVEAL MHC-peptide binding assay to determine their level of incorporation into MHC molecules. Binding was compared to that of a known T-cell epitope with very strong binding properties used as a positive control (100%).

[000799] Peptides considered to be immunologically significant or considered good binders were those peptides with scores >15% of the positive control. Several clusters of overlapping peptides with scores >15% of the positive control were identified, included clusters of peptides 10-15, 21-22, 24-28, 33-36, 62-63, 70-74, 85-92, 94, 106-109, 126, 144-152, 157-164, 174-175, 177, 197-198, 207-209, 220, 222-224, 226, 230-236, and 243-249. Several of these clusters included peptides having activity >60% of the positive control, including peptides 11, 22, 25, 33, 71, 94, 107, 147-150, 158-163. Of note, peptides 10-15 were immunogenic in this MHC study, as well as the B cell epitope study. Example 5

TAVS COMPRISING PCSK9 EPITOPES AND IMMUNOGENICITY ENHANCING

POLYPEPTIDES INDUCE ENDOGENOUS IMMUNE RESPONSE

[000800] TAVs compositions encoding mouse PCSK9 polypeptide with a C- terminal deletion and various immunogenicity enhancing polypeptides (IMs) were constructed and tested for their ability to generate an immune response in mice. The specific PCSK9 sequence (muPCSK9.delC) and immunogenicity enhancing polypeptide and polynucleotide sequences tested are included in Tables 17, 18, and 19. The PCSK9 TAV compositions included immunogenicity enhancing

polypeptides either N-terminal or C-terminal to the muPCSK9.delC sequences. Table 17 includes the sequences of particular peptides, immune enhancing polypeptides, and additional polypeptide elements present in certain PCSK9 TAV compositions, as well as certain illustrative TAV polynucleotide constructs.

[000801] For each PCSK9 TAV polynucleotide construct tested, five BALB/c mice were immunized by delivery of an LNP-encapsulated mRNA encoding the PCSK9 TAV composition or PBS as a negative control. The LNP comprised the MC3 lipid. Five mice were also immunized with recombinant full length mouse PCSK9 polypeptide as a positive control. Mice were provided with three weekly doses of the PCSK9 TAV composition at LNP doses of 0.5 mg/kg or recombinant muPCSK9.delC protein dosed at 2.5 mg/kg, in combination with Complete Freund’s Adjuvant on day 0 and Incomplete Freund’s Adjuvant on days 3, 7 and 14. Serum was collected from the mice pre-dosing on day 0 and also on day 7, day 14 and day 28 following the first dosing, and serum levels of anti-PCSK9 antibodies were determined.

[000802] Immunization with muPCSK9.delC protein resulted in the production of antibodies against PCSK9 by the mice, as compared to the PBS control (FIG.15).

[000803] The ability of the various immunogenicity enhancing polypeptides (IMs) to enhance the antibody response to a PCSK9 polypeptide encoded by a TAV composition was determined using modified mRNA constructs encoding the muPCSK9.delC polypeptide and an immunogenicity enhancing peptide (or multiple copies thereof) located either N- or C-terminal to the muPCSK9.delC polypeptide. TAV compositions encoding an immunogenicity enhancing polypeptide N-terminal to the muPCSK9.delC polypeptide included: nTetanus, nPR8M2, nMBP,

nUnivThEpitopeX3, nTpD; and TAV compositions encoding an immunogenicity enhancing polypeptide C-terminal to the muPCSK9.delC polypeptide included:

cTetanus, cPR8M2, cMBP, cUnivThEpitopeX3, cTpD, cSTF2d and cHA307-318x3, as described in Table 17. Table 17 also provides polypeptide and/or polynucleotide seuqnces for certain TAV composition sequences, including PCSK9 epitopes, immune enhancing agents, and non-coding sequences, as well as sequences of illustrative TAV polynucleotide constructs of the present invention.

[000804] The presence of certain immunogenicity peptides in the PCSK9 TAVs led to higher antibody titers at either day 14 or day 48 following first immunization, as compared to the antibody titer levels achieved with the positive control. For example, C-terminal PR8M2 and C-terminal MBP both resulted in antibody titers at 14 days following first immunization that were at least twice as high as those observed for the positive control at day 14 (about 65 ug/mL as compared to about 28 ug/mL). N- terminal PR8M2 and N-terminal MBP both resulted in antibody titers at 14 days following first immunization that were almost twice as high as those observed for the positive control at day 14 (about 52 ug/mL or about 45 ug/mL, respectively, as compared to about 28 ug/mL). At 28 days, the antibody titers were comparable for the positive control, C- or N-terminal PR8M2, and C- or N-terminal MBP. nTetanus, nUnivThEpitopeX3, nTpD, cTetanus, cUnivThEpitopeX3, cTpD, cSTF2d, and cHA307-318x3 did not enhance antibody titer. In general, C-terminally located immunogenicity enhancing peptides showed a greater enhancement of antibody production. C-terminal PR8M2 also showed a trend towards lowering serum levels of PCSK9 in mice over time, with reduced levels being observed at day 14 and day 28 following initial dosing as compared to day 0.

Table 17. Illustrative PCSK9 Antigen, Epitope, Immune Enhancing Agents

Figure imgf000267_0001
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Figure imgf000316_0001
Figure imgf000317_0001

Figure imgf000318_0001

Poynuc eot e sequences correspon to t e mRNA sequence w ere T may alternatively be U)

**Lower case one letter amino acid abbreviations indicate a linker, spacer or cleavage site

Example 6

SCREENING OF C-TERMINAL AND N-TERMINAL IMMUNOPOTENTIATION MOTIFS (IMS) [000805] A screen was performed to evaluate the immunogenic potential of fusion polypeptide TAVs fused with immunopotentiation motifs (IMs). mRNA encoding fusion polypeptides containing mouse TAVs with C-terminal or N-terminal IMs were tested in mice. mRNAs encoding fusion proteins combining mouse PCSK9, IL-17A, TNA alpha, or GDF8 proteins as TAVs and UnivThEpitope, PR8M2, TpD, HA307- 318, Tetanus epitope, and MBP, as C-terminal or N-terminal IMs were constructed and tested for their ability to generate an immune response in mice. For controls, mice were administered phosphate buffered saline (PBS), or recombinant PCSK9, IL- 17A, TNA alpha, or GDF8 polypeptides.

[000806] For each mRNA encoding a TAV and IM fusion polypeptide tested, five BALB/c mice were immunized by delivery of an LNP-encapsulated mRNA encoding the TAV or PBS control. The LNP comprised the MC3 lipid. Mice were provided with three weekly doses of the PCSK9 TAV at LNP doses of 0.5 mg/kg, with recombinant protein dosed at 2.5 mg/kg using CFA on day 0 and IFA on days 3, 7 and 14.

[000807] Polynucleotides encoding GDF8 TAVs were administered either once or three times on day 0, day 7, and day 14. [000808] Serum was collected from the mice pre-dosing on day 0 and also on day 7, day 14 and day 28 following the first dosing, and serum levels and relative strength of anti-PCSK9, anti-IL-17A, anti-TNF alpha, and anti-GDF8 antibodies were determined.

[000809] The relative strength of the antibody response was ascertained by determining the lowest dilution of serum whereby antibodies present in the serum would bind to the antigens (i.e., PCSK9, IL-17A, TNF alpha, or GDF8), that would result in a detectable signal following binding by fluorescent labeled anti-mouse secondary antibody. The antigens were bound to the ProArray Ultra slide surface, blocked using a blocking buffer to reduce non-specific binding, and then incubated with various concentrations/dilutions of different test samples (i.e., serum), followed by incubation with a fluorescent-labeled secondary antibody specific for each of the test samples. After several washing steps, the arrays were dried and scanned using a high resolution fluorescence microarray scanning system, and scanner images were recorded and evaluated using image analysis software, which enabled the

quantification of the levels of fluorescent intensities associated with each peptide.

[000810] The specific PCSK9, TNF alpha, IL-17A, and GDF8 and immunogenicity enhancing polypeptide and polynucleotide fusion sequences tested are included in Tables 17, 18, and 19. The TAV compositions included immunogenicity enhancing polypeptides either N-terminal or C-terminal. Tables 17, 18, and 19 also include the sequences of particular peptides, immune enhancing polypeptides, and additional polypeptide elements present in certain TAV compositions, as well as certain illustrative TAV polynucleotide constructs.

[000811] Table 15 shows the relative strengths of antigen specific antibody responses as measured by the dilution factor in serum collected at day 0 and day 28. The dilution factors listed in Table 15 are the highest dilution factor of sample serum that resulted in a detection of antigen-specific antibody binding.“PBS” refers to a negative PBS control where PBS was administered in place of a polynucleotide. “Recombinant protein” refers to experimental groups that were injected with recombinant mouse wild-type TNF alpha, IL-17A, PCSK9, or GDF8 polypeptides in place of a polynucleotide vaccine.“WT RNA” refers to experimental groups that were administered with mRNA comprising TAVs encoding the respective full length wild-type protein (i.e., TNF alpha, IL-17A, PCSK9, or GDF8) without IMs.

Polynucleotides encoding fusion polypeptides of TAVs with C-terminal IMs included “cPR8M2x3” (C-terminal T cell epitope of M2 protein of H1N1 Puerto Rico/8), “cTetanus” (C-terminal T cell epitope from tetanus toxin), cTpDx3 (three copies of chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid at the C-terminal), cSTF2d (C-terminal flagellin derived immunogen), cHA307-318x3 (three copies of T cell epitopes from Influenza HA antigen at the C- terminal), cUnivThEpitopeX3 (three copies of universal T helper epitope at the C- terminal), and cMBP (C-terminal mannose binding protein). Polynucleotides encoding fusion polypeptides of TAVs with N-terminal IMs included“nPR8M2x3” (N-terminal T cell epitope of M2 protein of H1N1 Puerto Rico/8),“nTetanus” (N- terminal T cell epitope from tetanus toxin), nTpDx3 (three copies of chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid at the N- terminal), nSTF2d (N-terminal flagellin derived immunogen), nHA307-318x3 (three copies of T cell epitopes from Influenza HA antigen at the N-terminal),

nUnivThEpitopeX3 (three copies of universal T helper epitope at the N-terminal), and nMBP (N-terminal mannose binding protein). In addition, polynucleotides encoding fusion polypeptides of PCSK9 with C-terminal or N-terminal single copies of TpD (cTpD and nTpD, respectively) and PR8M2 ( cPR8M2 and nPR8M2, respectively) were also tested. A dilution of“<500” indicates that only background signal was detected (i.e., no antigen-specific antibody binding was detected at a serum dilution of 1:500.) For all groups, no antigen specific antibody binding was detected at day 0. Table 15: Summary of relative strength of antigen specific antibody response

Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001

Figure imgf000323_0002
[000812] Tables 14, and 17-20 include polynucleotide sequences of the mRNAs tested in the experiments described in Examples 5 and 6, and the amino acid sequences of the encoded polypeptides. Table 18: Illustrative Antigen, Epitope, Immune Enhancing Agents (IMs), and TAV Construct Sequences

Figure imgf000323_0001

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Figure imgf000331_0001

Figure imgf000332_0001

Figure imgf000333_0001

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Figure imgf000335_0001

Figure imgf000336_0001

Figure imgf000337_0001

Figure imgf000338_0001

Figure imgf000339_0001

Figure imgf000340_0001
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Figure imgf000342_0001

Figure imgf000343_0001
Figure imgf000344_0001

Figure imgf000345_0001

Figure imgf000346_0001

Figure imgf000347_0001
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Figure imgf000349_0001

Figure imgf000350_0001

Figure imgf000351_0001
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Figure imgf000353_0001

Figure imgf000354_0001
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Figure imgf000356_0001
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Figure imgf000358_0003
Figure imgf000358_0002

Figure imgf000358_0001
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Figure imgf000360_0002

Figure imgf000360_0001
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Figure imgf000410_0001
Sequence Protein Sequence SEQ Polynucleotide Sequence SEQ Description ID ID

NO: NO:

ATGCAGTACATAAAAGCA AACTCGAAATTTATTGGC ATCCCTATGGGGTTACCTC AGTCTATCGCTCTTTCAAG CCTGATGGTCGCCCAGAT CTTAATGCAGTATATCAA AGCTAATTCCAAGTTTATT GGGATTCCTATGGGCCTCC CCCAAAGCATCGCCCTAT CTTCCCTGATGGTGGCTCA G Table 19: Illustrative Antigen, Epitope, Immune Enhancing Agents (IMs), and TAV Construct Sequences

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Figure imgf000426_0001
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Figure imgf000431_0001
Figure imgf000432_0001
Figure imgf000433_0001

Figure imgf000434_0001

Table 20: Illustrative Antigen, Epitope, Immune Enhancing Agents (IMs), and TAV Construct Sequences

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Figure imgf000490_0001

[000813] T e resu ts ste n Ta e 15 emonstrate t at a m nstrat on o mRNA encoding TAV IM fusion polypeptides can illicit detectable immune responses in mice with relative strengths greater than immune responses obtained with mRNA encoding TAVs only. Certain IMs preferentially enhanced the immune response to certain target proteins, and fusion to either the N-terminus or C-terminus provide superior enhancement of the immune reponse for certain targets. For example, mRNA encoding C-terminal Tet fusion proteins increased the strength of the immune response to TNF alpha as compared to mRNA encoding wild-type TNF alpha alone at day 28, but Tet failed to result in a detectable immune response at day 28 when fused to the C-terminus of IL-17A. In contrast, mRNA encoding STF2d fused to the C- terminus of IL-17A resulted in a strong antibody response at day 28, while C-terminal fusion of STF2d to TNF alpha failed to result in a detectable antibody response, and resulted in a low but detectable antibody response when paired with PCSK9 or GDF8. In another example, mRNA encoding C-terminal Tet fused to TNF alpha resulted in a strong immune response at day 28, while mRNA encoding N-terminal Tet fused to TNF alpha failed to result in a detectable immune response.

[000814] As shown in Table 15, vaccination in mice with mRNAs encoding TNF alpha IM fusion polypeptides resulted in stronger antibody responses in mice at day 28 than mRNAs encoding wild-type TNF alpha alone (i.e., with no IM) or vaccination with wild-type mouse TNF alpha polypeptides, however, this was not seen with all mRNAs tested. Vaccination with mRNA encoding wild-type mouse TNF alpha or vaccination with mouse wild-type TNF alpha polypeptide did not result in a detectable antibody response at day 28. Vaccination with mRNA encoding TNF alpha fused to PR8M2x3, HA307-318x3, MBP, and Tet at the C-terminus resulted in detectable antibody responses as measured by dilution factors at day 28. No antibody responses were detected in serum from mice injected with any of the mRNAs encoding TNF alpha fusion polypeptides with N-terminal IMs that were tested.

[000815] Antibody responses were also quantified by measuring the concentration of antigen specific antibody present in serum (FIGS.15-17). Similar to what was observed by measuring dilution factor, vaccination with mRNA encoding wild-type mouse TNF alpha or vaccinations with mouse wild-type TNF alpha polypeptide did not result in a detectable antibody response at day 7, day 14, or day 28 as measured by the concentrations of anti-TNF alpha antibody present in serum (FIGS.16B and 16C). Further, increased anti-TNF alpha antibody production was observed by day 28 in serum collected from mice vaccinated with mRNA encoding TNF alpha fused to PR8M2x3, HA307-318x3, MBP, and Tet at the C-terminus (FIGS.16D-16G). In contrast to the dilution factor data, an antibody response was detected by day 28 in serum obtained from mice vaccinated with mRNA encoding TNF alpha fused to PR8M2x3 and MBP at the N-terminus. In these instances, mRNAs encoding the C- terminal IM TNF alpha fusion polypeptide induced a greater antibody response than corresponding mRNAs encoding TNF alpha fusion polypeptides with the same IM at the N-terminus (FIGS.16E and 16F).

[000816] Vaccination in mice with mRNAs encoding IL-17A-IM fusion

polypeptides resulted in detectable antibody responses in mice at day 28, but as with TNF alpha, this effect was not observed with every mRNA tested. Vaccination with mRNA encoding wild-type mouse IL-17A resulted in a detectable antibody response, with the dilution factors of the five measured serum samples ranging from 8,000 to 64,000 (Table 15). Serum of four mice vaccinated with mouse wild-type IL-17A polypeptide had dilution factors of 2,000, while the other was measured at 64,000. Immune responses were observed following vaccination with mRNAs encoding IL- 17A fusion proteins with C-terminal PR8M2x3, STF2D, HA307-318x3,and MBP IMs, and N-terminal nHA307-318x3 and PR8M2x3 IMs. Notably, serum samples taken from mice vaccinated with mRNA encoding IL-17A fusion proteins with C- terminal PR8M2x3, STF2D, HA307-318x3,and MBP IMs displayed stronger anti-IL- 17A antibody responses than mRNA encoding wild-type IL-17A alone (i.e., no IMs), with all serum samples from these groups having detectable amounts of anti-IL-17A antibody at a dilution factor of 64,000.

[000817] Concentrations of anti-IL-17A in serum were also measured in serum samples (FIG.17). Vaccination with mRNA encoding wild-type mouse IL-17A resulted in a detectable increase of anti-IL-17A antibodies in serum taken at day 28, but not at earlier time points (FIG.17B). Similarly, vaccination with mouse wild-type IL-17A polypeptide resulted in a detectable increase of anti-IL-17A antibodies in serum taken at day 28, but not at day 7 or day 14 (FIG.17C). Increased anti-IL-17A antibody concentrations were observed at day 14 and day 28 in serum collected from mice vaccinated with mRNA encoding IL-17A fused to N-terminal or C-terminal HA307-318x3 or PR8M2x3, with the N-terminal IMs producing greater anti-IL-17A antibody responses at day 14 than the C-terminal IMs (FIGS.17D and 17E).

Vaccination with mRNAs encoding IL-17A fusion polypeptides with C-terminal MBP resulted in increased anti-IL-17A antibody levels at day 7, day 14, and day 28 (FIG.17F). Vaccination with mRNA encoding IL-17A fusion polypeptides with C- terminal STF2d resulted in increased anti-IL-17A antibodies at day 28 (FIG.17G). Vaccination with mRNA encoding IL-17A fusion polypeptide with C-terminal tet produced a detectable increase in anti-IL-17A antibody levels at day 28 (FIG.17H), while vaccination with mRNA encoding IL-17A fusion polypeptide with C-terminal TpDx3 or UnivThEpitopeX3 did not increase anti-IL-17A antibody levels at any of the time points measured (FIGS.17I and 17J).

[000818] Vaccination with mRNAs encoding PCSK9 IM fusion polypeptides resulted in detectable antibody responses in mice at day 28, but as above, this effect was not observed in every mRNA tested. Dilution factors ranging from 16,000 to 64,000 were measured in serum samples collected at day 28 from mice vaccinated with mouse recombinant wild-type PCSK9 polypeptide. Vaccination with mRNA encoding wild-type mouse PCSK9 without an IM was not tested in this screen.

Immune responses were observed following vaccinations with mRNAs encoding PCSK9 fusion proteins with C-terminal PR8M2x3, STF2d, and MBP, and with N- terminal Tet, PR8M2x3, and TpDx3 IMs. No detectable response was measured at day 28 in serum collected from mice vaccinated with mRNAs encoding PCSK9 fusion proteins with N-terminal UnivThEpitopeX3.

[000819] Concentrations of anti- PCSK9 were also measured in serum samples (FIG.15). Vaccination with mouse wild-type PCSK9 polypeptide detectable increase of anti-PCSK9 antibodies in serum taken at day 14 and day 28 (FIG.15B). Increased anti- PCSK9 antibody levels were observed at day 28 in serum collected from mice vaccinated with mRNA encoding PCSK9 fused to N-terminal or C-terminal Tet, HA307-318x3, and PR8M2x3, with the N-terminal IMs producing greater anti-IL- 17A antibody responses at day 14 than the C-terminal IMs (FIGS.17C-17E).

Vaccination with mRNAs encoding IL-17A fusion polypeptides with C-terminal MBP resulted in increased anti-IL-17A antibody levels at day 7, day 14, and day 28 (FIG.17F). Vaccination with mRNA encoding IL-17A fusion polypeptides with C- terminal STF2d resulted in increased anti-IL-17A antibodies at day 28 (FIG.17G). Vaccination with mRNA encoding IL-17A fusion polypeptide with C-terminal tet produced a modest increase in anti-IL-17A antibody levels at day 28 (FIG.17H), while vaccination with mRNA encoding IL-17A fusion polypeptide with C-terminal TpDx3 or UnivThEpitopeX3 did not increase anti-IL-17A antibody levels at any of the time points measured (FIGS.17I and 17J).

[000820] Vaccination with single or multipole doses of mRNAs encoding GDF8 IM fusion constructs did not result in immune responses with comparable strength to vaccination with wild-type GDF8 polypeptide (FIG.18). While no detectable immune response was observed at day 28 following single or multiple dose vaccinations with mRNA encoding wild-type GDF8, an immune response with dilution factors ranging from 1600 to 3200 was observed for both single and multiple dose conditions for mRNA encoding GDF8 N-terminal Tet fusion polypeptides (FIG. 18F). This demonstrates that mRNA encoding TAV IM fusion constructions can increase improve strength of anti-GDF8 antibody response as compared to mRNAs encoding GDF8 TAVs alone. [000821] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

[000822] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

Claims

Claims 1. An mRNA comprising:
(a) a first region comprising a sequence encoding one or more of an antigen polypeptide; and
(b) a second region comprising a sequence encoding one or more
immunomodulatory polypeptide,
wherein the mRNA comprises a modified nucleobase.
2. The mRNA of claim 1, wherein the mRNA further comprises a third region between the first region and the second region, wherein the third region comprises a sequence encoding a spacer or a cleavage site.
3. The mRNA of claim 1, wherein the spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site.
4. The mRNA of any one of claims 1-3, wherein said first region comprises two or more sequences encoding antigen polypeptides.
5. The mRNA of claim 4, wherein said two or more antigen polypeptides
comprise the same amino acid sequences.
6. The mRNA of claim 4, wherein said two or more antigen polypeptides
comprise different amino acid sequences.
7. The mRNA of any one of claims 4-6, wherein the first region further
comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more antigen polypeptides.
8. The mRNA of claims 7, wherein the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
9. The mRNA of any one of claims 1-8, wherein the second region comprises two or more sequences encoding immunomodulatory polypeptides.
10. The mRNA of claim 9, wherein the two or more immunomodulatory
polypeptides are the same.
11. The mRNA of claim 9, wherein the two or more immunomodulatory
polypeptides are different.
12. The mRNA of any one of claims 9-11, wherein the second region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides.
13. The mRNA of claim 12, wherein the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
14. The mRNA of any one of claims 1-13, wherein the immunomodulatory
polypeptide is selected from the group consisting of: GM-CSF, IL2, IL12, IL15, IL21, IL23, soluble LAG3, agonist CD28, anti-PD1, anti-PDL1/2, anti- OX40/OX40L, anti-GITR/GITRL, and anti-TIM3.
15. The mRNA of any one of claims 1-13, wherein the one or more
immunomodulatory polypeptide is an immune enhancing polypeptide (IM).
16. The mRNA of claim 15, wherein the immune enhancing polypeptide is
selected from the group consisting of: mannose binding protein, flagellin derived immunogens, T cell epitopes from Tetanus toxin, T cell epitope of M2 protein of H1N1 Puerto Rico/8, epitope from influenza HA antigen, universal T helper epitope, and a chimeric MHC class II peptide with epitopes from tetanus toxoid and diphtheria toxoid.
17. The mRNA of claim 16, wherein the immune enhancing polypeptide is T cell epitope of M2 protein of H1N1 Puerto Rico/8 or mannose binding protein.
18. The mRNA of any one of claims 1-16, wherein the mRNA encodes a fusion polypeptide comprising a scaffold polypeptide, the first region, and the second region, wherein the first region and the second region are inserted into the scaffold polypeptide.
19. The mRNA of any one of claims 1-18, wherein the mRNA further comprises a fourth region comprising a sequence encoding a dendritic cell targeting polypeptide.
20. The mRNA of claim 19, wherein the dendritic cell targeting polypeptide is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers.
21. The mRNA of claim 20, wherein the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.
22. The mRNA of any one of claims 1-21, wherein the antigen polypeptide is a proprotein convertase subtilisin/kexin type 9 (PCSK9) polypeptide.
23. The mRNA of claim 22, wherein the one or more PCSK9 polypeptide
comprises a human or murine PCSK9 polypeptide or fragment or variant thereof.
24. The mRNA of claim 22 or claim 23, wherein the one or more PCSK9
polypeptide comprises a PCSK9 polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Figure 14 or Tables 1, 2, 14, 15, or 17-20.
25. The mRNA of claim 24, wherein the one or more PCSK9 polypeptide
comprises a PCSK9 epitope comprising an amino acid sequence selected from the group consisting of: FSAKDVINEAWFPED; KDVINEAWFPEDQRV; DVINMAWFPEDQQVL; and NMAWFPEDQQVLTPN, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity.
26. The mRNA of claim 22-25, wherein the mRNA encodes a fusion protein comprising the one or more PCSK9 polypepide or fragment or variant thereof and an N-terminal immune enhancing polypeptide selected from the group consisting of Tet, PR8M2x3, and TpDx3.
27. The mRNA of any one of claims 1-21, wherein the antigen polypeptide is a tumor necrosis factor alpha (TNF alpha) polypeptide.
28. The mRNA of claim 27, wherein the one or more TNF alpha polypeptide comprises a human or murine TNF alpha polypeptide or fragment or variant thereof.
29. The mRNA of claim 27 or claim 28, wherein the one or more TNF alpha polypeptide comprises a TNF alpha polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 3 or 4, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity.
30. The mRNA of any of claims 27-29, wherein mRNA encodes a fusion protein comprising the one or more TNF alpha polypepide or fragment or variant thereof and a C-terminal immune enhancing polypeptide selected from the group consisting of PR8M2x3, HA307-318x3, and MBP.
31. The mRNA of any one of claims 1-21, wherein the antigen polypeptide is an interleukin-17A (IL-17A) polypeptide.
32. The mRNA of claim 31, wherein the one or more IL-17A polypeptide
comprises a human or murine IL-17A polypeptide or fragment or variant thereof.
33. The mRNA of claim 31 or claim 32, wherein the one or more IL-17A polypeptide comprises a IL-17A polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 5 or 6, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity.
34. The mRNA of any of claims 31-33, wherein the mRNA encodes a fusion protein comprising the at least one IL-17A polypepide or fragment or variant thereof and a C-terminal immune enhancing polypeptide selected from the group consisting of PR8M2x3, STF2D, HA307-318x3, and MBP.
35. The mRNA of any one of claims 1-21, wherein the antigen polypeptide is a growth differentiation factor 8 (GDF8) polypeptide.
36. The mRNA of claim 35, wherein the one or more GDF8 polypeptide
comprises a human or murine GDF8 polypeptide or fragment or variant thereof.
37. The mRNA of claim 35 or claim 36, wherein the one or more GDF8
polypeptide comprises a GDF8 polypeptide or fragment or variant thereof comprising an amino acid sequence set forth in any one of Tables 7 or 8, and fragments thereof comprising at least 8, at least 10, or least 12 amino acid residues, and variants thereof having at least 90% or at least 95% amino acid sequence identity.
38. The mRNA of any of claims 35-37, wherein the mRNA encodes a fusion protein comprising the one or more GDF8 polypepide or fragment or variant thereof and a C-terminal immune enhancing polypeptide comprising tet.
39. A lipid nanoparticle comprising the mRNA of any one of claims 1-38.
40. The lipid nanoparticle of claim 39, further comprising an immunomodulatory agent or moiety.
41. The lipid nanoparticle of claim 40, wherein the immunomodulatory agent or moiety enhances an immune response.
42. The lipid nanoparticle of any one of claims 39-41, further comprising a
dendritic cell targeting agent or moiety.
43. The lipid nanoparticle of claim 42, wherein the dendritic cell targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers.
44. The lipid nanoparticle of claim 43, wherein the dendritic cell surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.
45. A pharmaceutical composition comprising an mRNA of any one of claims 1- 38 or a lipid nanoparticle of any one of claims 39-44, and a pharmaceutically acceptable carrier, diluent or excipient.
46. The pharmaceutical composition of claim 45, comprising an
immunomodulatory agent or moiety.
47. The pharmaceutical composition of claim 46, wherein the immunomodulatory agent or moiety enhances the immune response.
48. The pharmaceutical composition of any one of claims 45-47, comprising a dendritic cell targeting agent or moiety.
49. The pharmaceutical composition of claim 48, wherein the dendritic cell
targeting agent or moiety is selected from the group consisting of a polypeptide encoding an antibody, a polypeptide encoding an antibody fragment, and a peptide that targets one or more dendritic cell surface markers.
50. The pharmaceutical composition of claim 49, wherein the dendritic cell
surface marker is selected from the group consisting of DEC205, DC-SIGN, CD11c, DCIR2, Dectin-1/2, CD80/86, F4/80-like receptor, CIRE, mannose receptor, and CD36.
51. A method of inducing an immune response to an antigen polypeptide in a cell, tissue or organism, comprising contacting said cell, tissue or organism with the mRNA of any one of claims 1-38, the lipid nanoparticle of any one of claims 39-44, or the pharmaceutical composition of any one of claims 45-50.
52. The method of claim 59, wherein the antigen polypeptide is endogenous to the cell, tissue or organism.
53. The method of claim 51 or claim 52, wherein the antigen is a PCSK9
polypeptide.
54. The method of claim 51 or claim 52, wherein the antigen is a TNF alpha
polypeptide.
55. The method of claim 51 or claim 52, wherein the antigen is an IL-17A
polypeptide.
56. The method of claim 51 or claim 52, wherein the antigen is a GDF8
polypeptide.
57. A method of treating or preventing hypercholesterolaemia or atherosclerosis in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition of any one of claims 45-50, wherein the at least one polypeptide is a PCSK9 polypeptide.
58. The method of claim 57, wherein the hypercholesterolaemia is a familial hypercholesterolaemia.
59. A method of treating or preventing an inflammatory disease, an autoimmune disease, or a cancer in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition of any one of claims 47-50, wherein the at least one polypeptide is a TNF alpha polypeptide.
60. A method of treating or preventing an inflammatory disease, an autoimmune disease, or a cancer in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition of any one of claims 47-50, wherein the at least one polypeptide is an IL-17A polypeptide.
61. A method of treating or preventing a disease associated with muscle loss in a subject in need thereof, comprising providing to the subject an effective amount of a pharmaceutical composition of any one of claims 47-50, wherein the at least one polypeptide is a GDF8 polypeptide.
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