Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
  • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55544—Bacterial toxins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

• 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).
• TAVs targeted adaptive vaccines
• 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.
• 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.
• PAP prostatic acid phosphatase
• 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.
• Vaccine production used in the art has several stages, including the generation of antigens, antigen purification and inactivation, and vaccine formulation.
• 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.
• TAVs targeted adaptive vaccines
• 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.
• the mRNA comprises the second region.
• 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.
• the spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site.
• first region comprises two or more sequences encoding polypeptides, which may comprise the same or different amino acid sequences.
• the first region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more polypeptides.
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• the one or more polypeptide comprises a human or murine polypeptide or fragment or variant thereof.
• 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).
• PCSK9 proprotein convertase subtilisin/kexin type 9
• IL-17A interleukin-17A
• TNF alpha tumor necrosis factor alpha
• GDF8 growth differentiation factor 8
• the second region of the mRNA comprises two or more sequences encoding immunomodulatory polypeptides, which may be the same or different.
• the second region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides.
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• 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.
• the one or more immunomodulatory polypeptide is an immune enhancing polypeptide.
• 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.
• the immune enhancing polypeptide is T cell epitope of M2 protein of H1N1 Puerto Rico/8 or mannose binding protein.
• 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.
• the mRNA further comprises a fourth region comprising a sequence encoding a dendritic cell targeting polypeptide.
• 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.
• 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.
• DEC205 refers to CD-205;
• DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin
• DCIR2 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
• 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.
• the present invention includes a lipid
• nanoparticle comprising any of the aforementioned mRNAs.
• the lipid nanoparticle further comprises an immunomodulatory agent or moiety, including any of those described above.
• the immunomodulatory agent or moiety enhances an immune response.
• the lipid nanoparticle further comprising a dendritic cell targeting agent or moiety.
• 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.
• 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.
• the present invention includes a pharmaceutical composition comprising any of the aforementioned mRNAs or lipid nanoparticles, and a pharmaceutically acceptable carrier, diluent or excipient.
• the pharmaceutical composition further comprises an immunomodulatory agent or moiety.
• the pharmaceutical composition further comprises an immunomodulatory agent or moiety.
• the pharmaceutical composition comprises a dendritic cell targeting agent or moiety.
• 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.
• 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.
• 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.
• the PCSK9 polypeptide is endogenous to the cell, tissue or organism.
• 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.
• the subject is treated for familial hypercholesterolaemia.
• 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.
• 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.
• the spacer or cleavage site is a 2A peptide or a cathepsin S cleavage site.
• the first region comprises two or more sequences encoding antigen polypeptides.
• the two or more antigen polypeptides comprise the same amino acid sequences.
• the two or more antigen polypeptides comprise different amino acid sequences.
• the first region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more antigen polypeptides.
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• the second region comprises two or more sequences encoding immunomodulatory polypeptides.
• the two or more immunomodulatory polypeptides are the same.
• the two or more immunomodulatory polypeptides are different.
• the second region further comprises a sequence encoding a linker or a cleavage site between the sequences encoding the two or more immunomodulatory polypeptides.
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• 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.
• the one or more immunomodulatory polypeptide is an immune enhancing polypeptide.
• 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.
• the immune enhancing polypeptide is T cell epitope of M2 protein of H1N1 Puerto Rico/8 or mannose binding protein.
• 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.
• the mRNA further comprises a fourth region comprising a sequence encoding a dendritic cell targeting polypeptide.
• 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.
• 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.
• the antigen polypeptide is a PCSK9 polypeptide.
• the one or more PCSK9 polypeptide comprises a human or murine PCSK9 polypeptide or fragment or variant thereof.
• 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.
• the antigen polypeptide is a PCSK9 polypeptide.
• 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.
• 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.
• 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.
• 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.
• 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.
• the antigen polypeptide is a tumor necrosis factor alpha (TNF alpha) polypeptide.
• TNF alpha tumor necrosis factor alpha
• the one or more TNF alpha polypeptide comprises a human or murine TNF alpha polypeptide or fragment or variant thereof.
• 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.
• 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.
• the antigen polypeptide is an interleukin-17A (IL-17A) polypeptide.
• one or more IL-17A polypeptide comprises a human or murine IL-17A polypeptide or fragment or variant thereof.
• 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.
• 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.
• the antigen polypeptide is a growth
• the one or more GDF8 polypeptide comprises a human or murine GDF8 polypeptide or fragment or variant thereof.
• 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.
• 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.
• lipid nanoparticle comprising the mRNA described herein.
• the lipid nanoparticle further comprising an immunomodulatory agent or moiety.
• the immunomodulatory agent or moiety enhances an immune response.
• the lipid nanoparticle comprises a dendritic cell targeting agent or moiety.
• 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.
• 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.
• compositions comprising an mRNA or a lipid nanoparticle described herein, and a pharmaceutically acceptable carrier, diluent or excipient.
• the composition comprises an immunomodulatory agent or moiety.
• the immunomodulatory agent or moiety enhances the immune response.
• the composition comprises a dendritic cell targeting agent or moiety.
• 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.
• 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.
• 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.
• the antigen polypeptide is endogenous to the cell, tissue or organism.
• the antigen is a PCSK9 polypeptide.
• the antigen is a TNF alpha polypeptide.
• the antigen is an IL-17A polypeptide.
• the antigen is a GDF8 polypeptide.
• 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.
• the hypercholesterolaemia is a familial hypercholesterolaemia.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• FIG.4 is a schematic of a series of chimeric polynucleotides illustrating various patterns of positional modifications based on the formula: 5 ' [A n ] x _Ll- [B 0 ] y _L2-[C p ] 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
• 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.
• FIG.6 is a schematic of a circular polynucleotide construct of the present invention.
• FIG.7 is a schematic of a circular polynucleotide construct of the present invention.
• FIG.8 is a schematic of a circular polynucleotide construct of the present invention comprising at least one spacer region.
• 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.
• FIG.10 is a schematic showing the components of one illustrative embodiment of targeted adaptive vaccines of the present invention.
• 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.
• the top line at day 40 corresponds to human (PCSK9) TAV
• the bottom line corresponds to the PBS control.
• the top line at day 40 corresponds to human (PCSK9) TAV
• the middle line corresponds to the murine (PCSK9) control
• the bottom line corresponds to the PBS control.
• 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.
• the top line corresponds to the PBS control
• the middle line corresponds to the murine (PCSK9) control
• the bottom line corresponds to the human (PCSK9) TAV.
• 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).
• the top line at day 40 corresponds to the murine (PCSK9) control
• the middle line corresponds to the PBS control
• the bottom line corresponds to human (PCSK9) TAV.
• FIG.14 provides the identification number and amino acid sequences of the overlapping human PCSK9 and mouse PCSK9 peptides used for mapping immunogenic epitopes.
• FIGS.15A-15I are graphs showing the concentration of anti-PCSK9 antibodies ( ⁇ g/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.
• IM immunogenicity enhancing polypeptide
• 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
• FIGS.16A-16J are graphs showing the concentration of anti-TNF alpha antibodies ( ⁇ g/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.
• 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).
• FIGS.17A-17J are graphs showing the concentration of anti-IL-17A antibodies ( ⁇ g/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).
• 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).
• 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 C-terminal flagellin derived immunogen
• FIG.18E
• the present invention is directed to polynucleotides encoding targeted adaptive vaccines (TAVs).
• TAVs targeted adaptive vaccines
• 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).
• PCSK9 proprotein convertase subtilisn kexin type 9
• TAV tumor necrosis factor alpha
• IL-17A interleukin-17A
• GDF8 Growth/differentiation factor 8
• Particular embodiments are directed to polynucleotides encoding PCSK9 targeted adaptive vaccines (TAVs), e.g., mRNAs encoding PCSK9 TAVs.
• TAVs PCSK9 targeted adaptive vaccines
• 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.
• 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.
• 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
• diseases or conditions that may be treated by reducing endogenous GDF8, such as muscular dystrophy such as muscular dyst
• RNA ribonucleic acid
• One beneficial outcome is to cause intracellular translation of the nucleic acid and production of at least one encoded peptide or polypeptide of interest.
• 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.
• the present invention provides compositions comprising polynucleotides encoding one or more targeted adaptive vaccines (TAVs).
• TAVs targeted adaptive vaccines
• the encoded TAVs comprise: (a) an antigen; and, optionally, (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety.
• the encoded TAVs comprise: (a) an antigen; and (b) a dendritic cell targeting agent or moiety and/or an immunomodulatory agent or moiety.
• the antigen and the dendritic cell targeting agent or moiety and/or immunomodulatory agent or moiety are encoded by the same polynucleotide.
• the antigen is a PCSK9 polypeptide or a fragment or variant thereof.
• 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.
• 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.
• 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.
• the TAV polynucleotide e.g., mRNA, encodes the antigen and the immunomodulatory agent or moiety.
• 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.
• the antigen is a PCSK9 polypeptide or a fragment or variant thereof.
• the TAV compositions may be formulated in any suitable delivery formulation or in simple saline.
• the TAV compositions e.g., TAV mRNAs
• the TAV composition or the LNP comprising a TAV composition is present in a
• composition further comprising one or more pharmaceutically acceptable carriers, diluents, or excipients.
• pharmaceutical composition comprises an adjuvant.
• 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.
• a TAV composition of the invention encodes one or more PCSK9 epitopes, e.g., antigenic epitopes.
• 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.
• a TAV composition of the invention encodes one or more TNF alpha epitopes, e.g., antigenic epitopes.
• 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.
• a TAV composition of the invention encodes one or more IL-17A epitopes, e.g., antigenic epitopes.
• 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.
• a TAV composition of the invention encodes one or more GDF8 epitopes, e.g., antigenic epitopes.
• the TAV compositions of the invention are polynucleotides.
• the polynucleotide is an mRNA encoding the TAV.
• the TAV-encoding mRNAs are chemically modified. mRNAs comprising one or more chemical modifications may be referred to herein as modified mRNAs (mmRNAs).
• 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.
• 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.
• the immunomodulatory agent or moiety is encoded on the same polynucleotide as the antigen.
• the immunomodulatory agent or moiety is located N- or C-terminal to the antigen.
• two or more immunomodulatory agents or moieties are included, which may be the same or different.
• 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.
• 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
• 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.
• 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).
• VVR cathepsin cleavage site
• the immunomodulatory agent or moiety may comprise a second antigen, such as a bacterial or viral protein or fragment thereof to enhance antigenicity.
• a second antigen such as a bacterial or viral protein or fragment thereof to enhance antigenicity.
• the bacterial or viral protein fragment is encoded on the same polynucleotide as the TAV antigen.
• reversing agents and methods are provided which function to turn off the TAV once the desired effect is achieved.
• these reversing agents may be selected from Bortezomib
• 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.
• 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.
• 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.
• the polynucleotides are preferably modified in a manner as to avoid the deficiencies of or provide improvements over other molecules of the art.
• TAVs Targeted Adaptive Vaccines
• 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.
• the polypeptide is an antigen, e.g., a vaccine antigen.
• 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.
• 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.
• Polynucleotides of the present invention may encode at least one polypeptide of interest, e.g., an antigen.
• polypeptide of interest e.g., an antigen.
• a selection of polypeptides of interest or "Targets" ofthe present invention are listed in Table 16 below.
• 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.
• a particular species of mammal e.g., a human
• 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.
• 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.
• TAVs may further comprise a reversing agent to turn off the TAV.
• 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.
• 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.
• 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.
• a dendritic cell targeting agent or moiety or an immunomodulatory agent or moiety.
• 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.
• 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.
• 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.
• an antigen such as a PCSK9 antigen, a TNF alpha antigen, an IL-17A antigen, or a GDF8 antigen
• the various components of a TAV are present in the same formulation or pharmaceutical composition.
• one or more component is present in a lipid nanoparticle (LNP) or other microparticle or nanoparticle.
• LNP lipid nanoparticle
• 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.
• aan antigen e.g., a PCSK9, TNF alpha, IL-17A, or GDF8 polypeptide
• an immunomodulatory polypeptide enhances an immune response, e.g., to the encoded antigen.
• 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.
• 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.
• 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.
• TAV polynucleotides of the invention encode two or more polypeptides or antigens, which may be the same or different.
• the two or more polypetides or antigens are derived from the same polypeptide.
• they may be two or more different antigenic fragments of a polypeptide.
• 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.
• 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.
• the spacer or cleavage site is a 2A peptide or a cathepsin cleavage site.
• the TAV polynucleotides encode two or more PCSK9 polypeptides or PCSK9 antigens, which may be the same or different.
• the TAV polynucleotides encode two or more TNF alpha polypeptides or TNF alpha antigens, which may be the same or different.
• 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.
• 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.
• the first region of the mRNA comprises two or more sequences encoding GDF8 antigens.
• the two or more sequences are both antigens that induce an immune response.
• 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.
• 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.
• a polypeptide may include portions of two or more overlapping peptides depicted in any of these tables or described elsewhere herein.
• a polypeptide comprises 15-25 or 18-20 amino acid residues, comprising overlapping regions of two, three or four peptides described herein.
• 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.
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• 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.
• the one or more immunomodulatory polypeptide is an immune enhancing polypeptide.
• 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.
• the mRNA comprises a further or alternative, e.g., fourth, region comprising a sequence encoding a dendritic cell targeting polypeptide.
• the polynucleotide e.g., mRNA
• the linker or cleavage site is a 2A peptide or a cathepsin cleavage site.
• 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.
• 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.
• 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.
• 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.
• 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.
• the first region and, optionally, the second region are inserted into the scaffold polypeptide.
• polynucleotide or mRNA encodes a fusion polypeptide comprising a scaffold polypeptide into which are inserted one or more antigens and/or immune enhancing polypeptides.
• 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.
• the one or more antigens are one or more PCSK9 antigens.
• the one or more antigens are one or more TNF alpha antigens.
• the one or more antigens are one or more IL- 17A antigens.
• the one or more antigens are one or more GDF8 antigens.
• 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.
• 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
• the various components of the TAV may be located in various positions upstream or downstream of each other.
• the sequence encoding the antigen is located upstream of or 5’ to the sequence encoding the immune modulating polypeptide.
• 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.
• sequences encoding antigens may be present both upstream and downstream of one or more sequence encoding an immune modulating polypeptide.
• sequences encoding immune modulating polypeptides may be present both upstream and downstream of one or more sequence encoding an antigen.
• Familial hypercholesterolaemia 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.
• PCSK9 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.
• 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.
• 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.
• the encoded PCSK9 polypeptides are capable of eliciting an immune response to an endogenout PCSK9 protein in a mammal, e.g. a human.
• 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.
• 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.
• an immune enhancing polypeptide polypeptide e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8
• T cell epitope from tetanus toxin e.g., T cell epitope of M2 protein of H1N1 Puerto Rico/8
• T cell epitope from tetanus toxin e.g.
• mRNA encoding a fusion peptide comprising an antigen and a mannose binding protein 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.
• 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.
• the mRNA of the present invention encodes two or more different epitopes.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• the PCSK9 polynucleotide encodes a PCSK9 antigen that is a C terminal catalytic domain fragment of a PCSK9 polypeptide.
• 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.
• 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:
• 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.
• embodiments of the present invention include polynucleotides, e.g., mRNAs, encoding any of these PCSK9 peptides or variants or fragments thereof;
• 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.
• the method is practiced in vitro or in vivo, e.g., in a mammal.
• 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.
• multiple PCSK9 antigens can be encoded by a single TAV polynucleotide.
• 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.
• 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.
• 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.
• Tumor Necrosis Factor 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
• TNF alpha soluble homotrimeric TNF alpha
• TACE metalloprotease TNF- ⁇ converting enzyme
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• TAVs may further comprise a reversing agent to turn off the TAV.
• 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.
• the TAV polynucleotides deliver a TNF alpha antigen and an immunomodulatory agent or moiety to the cell, tissue or subject.
• 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.
• TAV polynucleotides comprise a polynucleotide encoding a TNF alpha antigen and an immunomodulatory agent or moiety.
• the polynucleotide is an mRNA, such as a modified mRNA.
• 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.
• IM immune enhancing polypeptide
• 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
• immunomodulatory polypeptide enhances an immune response, e.g., to the encoded TNF alpha antigen.
• 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.
• 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.
• 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.
• 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.
• 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.
• the TNF alpha polynucleotide encodes a TNF alpha antigen that is a C-terminal catalytic domain fragment of a TNF alpha polypeptide.
• 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.
• 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;
• 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.
• the method is practiced in vitro or in vivo, e.g., in a mammal.
• 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.
• the antigen comprises one or more sequences shown in Table 4.
• 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
• multiple TNF alpha antigens can be encoded by a single TAV polynucleotide.
• 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.
• 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.
• 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.
• 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 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.
• Interleukin-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.
• IL-17 interleukin-17 family consists of a subset of cytokines that participate in both acute and chronic inflammatory responses.
• 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 ⁇ 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.
• 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.
• 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.
• 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
• 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,.
• 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.
• a TAV polynucleotide comprises a
• polynucleotide e.g., an mRNA encoding both an IL-17A polypeptide and, optionally, an immunomodulatory polypeptide.
• the polynucleotide e.g., an mRNA encoding both an IL-17A polypeptide and, optionally, an immunomodulatory polypeptide.
• immunomodulatory polypeptide enhances an immune response, e.g., to the encoded IL-17A antigen.
• 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
• 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.
• TAVs may further comprise a reversing agent to turn off the TAV.
• 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.
• the TAV polynucleotides deliver a IL-17A antigen and an
• 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.
• 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.
• the polynucleotide is an mRNA, such as a modified mRNA.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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
• the method is practiced in vitro or in vivo, e.g., in a mammal.
• 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.
• 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.
• multiple IL-17A antigens can be encoded by a single TAV polynucleotide.
• 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.
• 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.
• 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.
• 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.
• GDF8 Growth and Differentiation Factor
• TGF- ⁇ Transforming Growth Factor-beta
• 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.
• 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
• GDF8 knockout mice have approximate twice the muscle mass as normal mice.
• 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.
• 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.
• 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.
• 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.
• TAVs may further comprise a reversing agent to turn off the TAV.
• the encoded GDF8 polypeptides are capable of eliciting an immune response to an endogenout GDF8 protein in a mammal, e.g. a human.
• 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.
• the TAV polynucleotides deliver a GDF8 antigen and an immunomodulatory agent or moiety to a cell, tissue or subject.
• 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.
• 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.
• the polynucleotide is an mRNA, such as a modified mRNA.
• a TAV polynucleotides comprises a
• polynucleotide e.g., an mRNA encoding both a GDF8 polypeptide and, optionally, an immunomodulatory polypeptide.
• the immunomodulatory polypeptide enhances an immune response, e.g., to the encoded GDF8 antigen.
• GDF8 polynucleotides of the present invention may encode any GDF8 polypeptide, or fragment or variant thereof, including antigenic epitopes of a GDF8 polypeptide.
• 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.
• 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.
• GDF8 polynucleotides of the present invention may encode any GDF8 polypeptide, or fragment or variant thereof, including antigenic epitopes of a GDF8 polypeptide.
• 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.
• 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.
• 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.
• 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.
• 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
• the GDF8 fragment is 1-60, 60-80 amino acids, 30-70 amino acids, or greater, in length.
• 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.
• 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.
• the method is practiced in vitro or in vivo, e.g., in a mammal.
• 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.
• multiple GDF8 antigens can be encoded by a single TAV polynucleotide.
• 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
• 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.
• 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.
• the antigen comprises one or more sequences shown in Table 8.
• the antigen comprises an amino acid sequence of one or more of DFGLDCDEHSTESRCCRYPL;
• WIIAPKRYKANYCSGECEFV ECEFVFLQKYPHTHLVHQAN
• VHQANPRGSAGPCCTPTKMS PTKMSPINMLYFNGKEQIIY;
• polypeptide means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds.
• the polypeptides of interest are antigens encoded by the polynucleotides as described herein.
• 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.
• 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.
• polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a
• 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.
• 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.
• “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.
• polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.
• 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.
• compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives.
• derivatives are 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.
• sequence tags or amino acids such as one or more lysines
• Sequence tags can be used for peptide purification or localization.
• Lysines can be used to increase peptide solubility or to allow for biotinylation.
• 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
• 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.
• the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation.
• a“consensus” sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.
• protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest of this invention.
• any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
• a reference protein 10 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length.
• 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.
• 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.
• At least one polypeptide of interest may be an antigen or fragment thereof, or any component of a targeted adaptive vaccine.
• variant polypeptides have a certain identity with a reference polypeptide sequence.
• 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.
• Reference molecules may share a certain identity with the designed molecules (polypeptides or polynucleotides).
• identity 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:
• the encoded polypeptide variant may have the same or a similar activity as the reference polypeptide.
• the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide.
• 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. Schulffer, 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.”
• BLAST algorithm 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.
• TAV polynucleotides of the present invention may encode or include a dendritic cell targeting agent or moiety.
• antigens of the present invention e.g., PCSK9 antigens, TNF alpha antigens, IL-17A antigens, and GDF8 antigens
• a TAV composition of the invention in combination with a dendritic cell targeting agent or moiety.
• the antigen is fused to a dendritic cell targeting agent or moiety.
• 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).
• 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.
• 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.
• an antigen e.g., a PCSK9 antigen, a TNF alpha antigen, a IL-17A antigen, or a GDF8 antigen.
• 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).
• 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.
• the polynucleotide encodes both the antigen and the immunomodulatory agent or moiety.
• the immunomodulatory agent or moiety is an immunogenicity enhancing polypeptide also referred to herein as an immunogenicity enhancing motif (IM).
• 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.
• 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.
• 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.
• 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.
• the immunomodulatory agent or moiety may comprise a bacterial or viral protein or fragment thereof to enhance antigenicity.
• the immunomodulatory agent may comprise a viral vaccine or a viral cell surface protein or epitope.
• 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.
• the bacterial or viral protein fragment is encoded on the same polynucleotide as the TAV antigen as a poly-cistronic transcript.
• the TAV construct may comprise a non-mammalian lead-in sequence.
• 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.
• 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.
• 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.
• 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.
• FDA US Food and Drug Administration
• bortezomib 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.
• AMR antibody mediated rejection
• 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.
• 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.
• SLE systemic lupus erythematosus
• 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).
• anti-CD20 antibodies may be used as a reversing agent to turn off TAV.
• the reversal may occur through elimination of antibodies generated by TAV via B cell depletion.
• 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).
• Rituximab inhibits activated B cells by binding to CD20 on the B cell surface, leading to their rapid elimination from the circulation.
• Rituximab may be used as a reversing agent to turn off TAV.
• Adjuvants or immune potentiators may also be administered with or in combination with one or more TAV compositions of the invention.
• 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.
• Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.
• 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
• Adjuvants for nucleic acid vaccines 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.
• 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.
• 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.
• peptides or polypeptides may serve as an antigen or antigenic molecule.
• 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 ⁇ - D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′-amino
• EDA ethylene nucleic acids
• CeNA cyclohexenyl nucleic acids
• 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
• 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.
• 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.
• the polynucleotides of the present invention that are circular are known as“circular polynucleotides” or“circP.”
• “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.
• 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, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000
• 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).
• a region may be referred to as a“coding region” or “region encoding.”
• the polynucleotides of the present invention is a messenger RNA or functions as a messenger RNA.
• messenger RNA 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.
• mRNA messenger RNA
• the polynucleotides of the present invention may be structurally modified or chemically modified.
• 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.
• the polynucleotide“ATCG” may be chemically modified to“AT-5meC-G”.
• the same polynucleotide may be structurally modified from“ATCG” to“ATCCCG”.
• 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.”
• the polynucleotides of the present invention 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.
• 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).
• 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.
• the 2A peptide may have the protein sequence: GSGATNFSLLKQAGDVEENPGP, fragments or variants thereof.
• the 2A peptide cleaves between the last glycine and last proline.
• the polynucleotides of the present invention may include a polynucleotide sequence encoding the 2A peptide having the protein sequence GSGATNFSLLKQAGDVEENPGP fragments or variants thereof.
• 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.
• this sequence may be used to separate the coding region of two or more polypeptides of interest.
• 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.
• 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.
• 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.
• the polynucleotides of the present invention may include a sequence encoding a cleavage site, such as, e.g., a cathepsin S cleavage site.
• 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.
• the polynucleotides are codon optimized, e.g. for expression in human cells or host cells for recombinant production.
• 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.
• the mRNA is an IVT polynucleotide.
• 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.
• 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.
• 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
• 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.
• the one or more polypeptides or antigens are one or more PCSK9 polypeptides or antigens.
• the one or more polypeptides or antigens are one or more TNF alpha polypeptides or antigens.
• the one or more polypeptides or antigens are one or more IL- 17A polypeptides or antigens.
• the one or more polypeptides or antigens are one or more GDF8 polypeptides or antigens.
• the polynucleotide comprises a sequence encoding an
• 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.
• 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.
• 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.
• the cDNAs comprise a sequence set forth herein, e.g., in Table 14, or comprise a complement thereof.
• 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.
• Figure 1 shows a primary construct 100 of an IVT polynucleotide of the present invention.
• “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.
• 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.
• UTR untranslated region
• 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, TERZAK TM 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,
• 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).
• first operational region 105 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.
• second operational region 107 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.”
• 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.
• 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.
• 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).
• 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, from 500 to 2,000, from 500 to 3,000, from 500 to
• 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).
• 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).
• the length may be determined in units of or as a function of polyA Binding Protein binding.
• 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.
• the capping region of the IVT polynucleotide may comprise a single cap or a series of nucleotides forming the cap.
• 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.
• the cap is absent.
• 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.
• 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.”
• the IVT polynucleotides of the present invention 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.
• 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).
• 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.
• 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.
• IVT polynucleotides such as, but not limited to, primary constructs
• formulations and compositions comprising IVT polynucleotides are described in
• polynucleotides of the foregoing are considered useful as a polypeptide of interest or antigen of the TAVs of the present invention.
• 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.
• the chimeric polynucleotides which are modified mRNA molecules of the present invention are termed“chimeric modified mRNA” or“chimeric mRNA.”
• 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.
• 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.
• 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.
• UTRs untranslated regions
• 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.
• Pattern chimeras may vary in their chemical modification by degree (such as those described above) or by kind (e.g., different modifications).
• 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”.
• 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.
• 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.
• 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.
• Percent chimeras 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.
• 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.
• 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.
• 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.
• 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.
• polynucleotide which is not chimeric is the canonical pseudouridine/5-methyl cytosine modified polynucleotide of the prior art.
• IVT in vitro transcription
• 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.
• the chimeric polynucleotides of the present invention may be structurally modified or chemically modified.
• the polynucleotides may be referred to as“modified chimeric polynucleotides.”
• 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.
• the regions or parts of the chimeric polynucleotides of the present invention may be separated by a linker or spacer moiety.
• linkers or spaces may be nucleic acid based or non-nucleosidic.
• 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.
• chimeric polynucleotides of the present invention may comprise a region or part which is not positionally modified or not chimeric as defined herein.
• 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.
• 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.
• chimeric polynucleotides encode one or more polypeptides of interest.
• the chimeric polynucleotides are substantially non-coding.
• the chimeric polynucleotides have both coding and non-coding regions and parts.
• Figure 2 illustrates the design of certain chimeric polynucleotides of the present invention when based on the scaffold of the polynucleotide of Figure 1.
• 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.
• 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.
• 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.
• the cap is absent.
• the present invention contemplates chimeric polynucleotides which are circular or cyclic.
• 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.
• the present invention contemplates polynucleotides which are circular or cyclic.
• 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.
• 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.
• 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.
• Figure 6 shows a representative circular construct 200 of the circular polynucleotides of the present invention.
• 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.
• 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.
• the“first region” may be referred to as a “coding region,” a“non-coding region” or“region encoding” or simply the“first region.”
• 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.
• 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.
• first operational region 205 Bridging the 5′ terminus of the first region 202 and the first flanking region 104 is a first operational region 205.
• this operational region may comprise a start codon.
• the operational region may alternatively comprise any translation initiation sequence or signal including a start codon.
• a second operational region 207 Bridging the 3′ terminus of the first region 202 and the second flanking region 106 is a second operational region 207.
• 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.
• 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.”
• 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.
• 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.
• 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.
• 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.
• 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.
• alkylating agents phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.
• biotin e.g., aspirin, vitamin E, folic acid
• 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.
• 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.
• polynucleotides of the present invention which encode an antigen are conjugated to one or more dendritic cell markers.
• 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.
• 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.
• 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.
• 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.
• polynucleotides of the present invention, including mRNAs comprise one or more of the following regions, e.g., untranslated regions.
• UTRs Untranslated Regions
• polynucleotides of the present invention may comprise one or more regions or parts which act or function as an untranslated region.
• polynucleotides are designed to encode at least one polypeptide of interest, the polynucleotides may comprise one or more of these untranslated regions.
• UTRs wild type untranslated regions
• 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.
• 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
• 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.
• liver- expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII
• non-UTR sequences may also be used as regions or subregions within the polynucleotides.
• 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.
• 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.
• any UTR from any gene may be incorporated into the regions of the polynucleotide.
• multiple wild-type UTRs of any known gene may be utilized.
• 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.
• a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.
• 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.
• 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.
• a double, triple or quadruple UTR such as a 5′ or 3′ UTR may be used.
• a“double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
• 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.
• 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.
• flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property.
• 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.
• 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.
• the untranslated region may also include translation enhancer elements (TEE).
• TEE translation enhancer elements
• the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art.
• AU rich elements 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.
• AREs 3′ UTR AU rich elements
• 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.
• 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.
• microRNAs 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
• 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.
• 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.
• 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.
• A adenine
• the bases of the microRNA seed have complete complementarity with the target sequence.
• microRNA target sequences 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:
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• Transfection experiments can be conducted in relevant cell lines, using engineered polynucleotides and protein production can be assayed at various time points post-transfection.
• 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.
• 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.
• CBP mRNA Cap Binding Protein
• the cap further assists the removal of 5′ proximal introns removal during mRNA splicing.
• 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.
• 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 ⁇ -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 ⁇ -methyl- phosphonate and seleno-phosphate nucleotides.
• Additional modifications include, but are not limited to, 2′-O-methylation of the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2′-hydroxyl group of the sugar ring.
• Multiple distinct 5′-cap structures can be used to generate the 5′-cap of a nucleic acid molecule, such as a polynucleotide which functions as an mRNA molecule.
• Cap analogs which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5′-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non- enzymatically) or enzymatically synthesized and/or linked to the polynucleotides of the invention.
• 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 (m 7 G-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.
• 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, m 7 Gm-ppp-G).
• the cap is a dinucleotide cap analog.
• 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.
• the cap is a cap analog is a N7-(4- chlorophenoxyethyl) substituted dicucleotide form of a cap analog known in the art and/or described herein.
• Non-limiting examples of a N7-(4-chlorophenoxyethyl) substituted dicucleotide form of a cap analog include a N7-(4-chlorophenoxyethyl)- G(5’)ppp(5’)G and a N7-(4-chlorophenoxyethyl)-m 3’-O G(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.
• a cap analog of the present invention is a 4-chloro/bromophenoxyethyl analog.
• 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.
• 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.
• 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).
• 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.
• Cap1 structure Such a structure is termed the Cap1 structure.
• 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).
• 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.
• 5′ terminal caps may include endogenous caps or cap analogs.
• 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.
• 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.
• BYDV-PAV barley yellow dwarf virus
• JSRV Jaagsiekte sheep retrovirus
• Enzootic nasal tumor virus See e.g., International Pub. No. WO2012129648; herein incorporated by reference in its entirety
• Transfection experiments can be conducted in relevant cell lines at and protein production can be assa
• IRES 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”).
• multicistronic nucleic acid molecules When polynucleotides are provided with an IRES, further optionally provided is a second translatable region.
• 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).
• picornaviruses e.g. FMDV
• CFFV pest viruses
• PV polio viruses
• ECMV encephalomyocarditis viruses
• FMDV foot-and-mouth disease viruses
• HCV hepatitis C viruses
• CSFV classical swine fever viruses
• MLV murine leukemia virus
• SIV simian immune deficiency viruses
• CrPV cricket paralysis viruses
• a long chain of adenine nucleotides may be added to a polynucleotide such as an mRNA molecule in order to increase stability.
• a polynucleotide such as an mRNA molecule
• 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
• PolyA tails may also be added after the construct is exported from the nucleus.
• 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).
• 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.
• mRNAs are distinguished by their lack of a 3 ⁇ 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;
• SLBP stem–loop binding protein
• the length of a poly-A tail when present, is greater than 30 nucleotides in length.
• 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).
• 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 from about 30 to
• 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.
• 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.
• 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.
• engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression.
• 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.
• 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.
• the G-quartet is incorporated at the end of the poly-A tail.
• the resultant polynucleotide is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production from an mRNA equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone. Start codon region
• the polynucleotides of the present invention may have regions that are analogous to or function like a start codon region.
• 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.
• the translation of a polynucleotide begins on the alternative start codon ACG.
• polynucleotide translation begins on the alternative start codon CTG or CUG.
• the translation of a polynucleotide begins on the alternative start codon GTG or GUG.
• Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and/or the structure of the polynucleotide. (See e.g., Matsuda and Mauro PLoS ONE, 20105:11; the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and/or structure of a polynucleotide.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the polynucleotides of the present invention include the stop codon TGA and one additional stop codon.
• the addition stop codon may be TAA.
• the polynucleotides of the present invention include three stop codons.
• 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.
• 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.
• the polynucleotides encode at least one polypeptide of interest, e.g., an antigen.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• GDF Growth/differentiation factors
• Members of the GDF family include GDF1, GDF2, GDF3, GDF4, GDF5, GDF6, GDF8 (myostatin), GDF9, GDF10, GDF11, and GDF15.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the 5’UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases.
• the 5’UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.
• 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.
• 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).
• NTP nucleotide triphosphate
• 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.
• 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.
• the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides.
• 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.
• the guanine bases 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.
• the polynucleotide may include at least one substitution and/or insertion upstream of the start codon.
• 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.
• 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.
• 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).
• 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.
• the polynucleotides of the present invention may include at least one post transcriptional control modulator.
• post transcriptional control modulators may be, but are not limited to, small molecules, compounds and regulatory sequences.
• post transcriptional control may be achieved using small molecules identified by PTC Therapeutics Inc. (South Plainfield, NJ) using their GEMS TM (Gene Expression Modulation by Small-Moleclues) screening technology.
• 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.
• 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.
• 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.
• the compound may bind to a region of the 28S ribosomal RNA in order to modulate premature translation termination (See e.g.,
• polynucleotides of the present invention may include at least one post transcription control modulator to alter protein expression.
• the expression of VEGF may be regulated using the compounds described in or a compound found by the methods described in International
• the polynucleotides of the present invention may include at least one post transcription control modulator to control translation.
• the post transcription control modulator may be a RNA regulatory sequence.
• 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
• 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.
• 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.
• the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.
• regions of the polynucleotide 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.
• UTRs untranslated regions
• Kozak sequences oligo(dT) sequence
• detectable tags may include multiple cloning sites which may have XbaI recognition.
• 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.
• the polynucleotides components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
• a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
• 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.
• 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.
• 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.
• 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
• 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.
• a Syn5 promoter may be used in the synthesis of the polynucleotides.
• the Syn5 promoter may be 5’-ATTGGGCACCCGTAAGGG-3’ as described by Zhu et al.
• 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.
• 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.
• 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.
• 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
• 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.
• HNAs hexitol nucleic acids
• mRNAs Short messenger RNAs with hexitol residues in two codons have been constructed (Lavrik et al.,
• 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.
• the polynucleotides of the present invention may be quantified in exosomes or when derived from one or more bodily fluid.
• 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 umbil
• 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.
• 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
• 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.
• ELISA enzyme linked immunosorbent assay
• the polynucleotide may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
• UV/Vis ultraviolet visible spectroscopy
• 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).
• 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 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 LNA TM oligo-T capture probes
• 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).
• 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).
• 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.
• a purified polynucleotide e.g., DNA and RNA
• a purified polynucleotide 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.
• 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.
• polynucleotides may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. III. Modifications
• Polynucleotides of the present invention may comprise one or more modification, including any of those described herein.
• a polynucleotide such as a modified mRNA, chimeric polynucleotide, IVT
• 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.
• the modifications may be various distinct modifications.
• the regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
• a modified polynucleotide, introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide.
• 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.
• the modified nucleobase is a modified cytosine.
• exemplary nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo- cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, 2-thio- cytidine (s 2 C), 2-thio-5-methyl-cytidine.
• the modified nucleobase is a modified adenine.
• exemplary nucleobases and nucleosides having a modified adenine include 7-deaza- adenine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A).
• the modified nucleobase is a modified guanine.
• exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 7-deaza- guanosine, 7-cyano-7-deaza-guanosine (preQ 0 ), 7-aminomethyl-7-deaza-guanosine (preQ 1 ), 7-methyl-guanosine (m 7 G), 1-methyl-guanosine (m 1 G), 8-oxo-guanosine, 7- methyl-8-oxo-guanosine.
• the polynucleotides of the TAVs can include any useful linker between the nucleosides.
• Such linkers, including backbone modifications are given in Table 4.
• 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).
• modifications e.g., one or more modifications
• RNAs ribonucleic acids
• DNAs deoxyribonucleic acids
• TAAs threose nucleic acids
• GNAs glycol nucleic acids
• PNAs peptide nucleic acids
• LNAs locked nucleic acids
• the polynucleotides of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
• 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.
• the invention provides a polynucleotide containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
• 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
• the present invention also includes building blocks, e.g., modified ribonucleosides, and modified ribonucleotides, of polynucleotide molecules.
• building blocks e.g., modified ribonucleosides, and modified ribonucleotides
• 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.
• modified nucleosides and nucleotides 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.
• a polynucleotide e.g., RNA or mRNA, as described herein
• 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 C 1-6 alkyl; optionally substituted C 1-6 alkoxy;
• 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” nucle
• RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen.
• 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 ⁇ -L-threofuranosyl-(3′ ⁇ 2′)) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone).
• GAA glycol nucleic acid
• R-GNA or S-GNA threose nucleic acid
• TAA threose nucleic acid
• PNA peptide nucleic acid
• the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
• 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.
• 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”).
• organic base e.g., a purine or pyrimidine
• 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.
• 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.
• non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
• the modified nucleosides and nucleotides can include a modified nucleobase.
• nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil.
• nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine.
• modified nucleobases are taught in International Application Number
• an mRNA of the invention comprises one or more modified nucleobases, nucleosides, or nucleotides (termed“modified mRNAs” or “mmRNAs”).
• 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.
• 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.
• the modified nucleobase is a modified uracil.
• nucleobases and nucleosides having a modified uracil include
• pseudouridine pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio- 5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5- carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-car
• 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 (m 3 C), N4-acetyl- cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl- cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2- thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-
• 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 (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenine
• N6-hydroxynorvalylcarbamoyl-adenosine hn 6 A
• 2-methylthio- N6-hydroxynorvalylcarbamoyl-adenosine ms 2 hn 6 A
• N6-acetyl-adenosine ac 6 A
• 7- methyl-adenine 2-methylthio-adenine, 2-methoxy-adenine, ⁇ -thio-adenosine, 2′-O- methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m 6 Am), N6,N6,2′-O-trimethyl- adenosine (m 6
• the modified nucleobase is a modified guanine.
• exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl- wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o 2 yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza- guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ 0 ), 7-aminomethyl-7
• N2,7-dimethyl-guanosine (m 2,7 G), N2, N2,7-dimethyl- guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, ⁇ -thio- guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m 2
• 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.)
• the modified nucleobase is pseudouridine ( ⁇ ), N1- methylpseudouridine (m 1 ⁇ ), 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.
• an mRNA of the invention includes a combination of one or more of the aforementioned modified nucle
• the modified nucleobase is 1-methyl-pseudouridine (m 1 ⁇ ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine ( ⁇ ), ⁇ - thio-guanosine, or ⁇ -thio-adenosine.
• 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]
• the mRNA comprises pseudouridine ( ⁇ ).
• the mRNA comprises pseudouridine ( ⁇ ) and 5-methyl-cytidine (m 5 C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m 1 ⁇ ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m 1 ⁇ ) and 5-methyl- cytidine (m 5 C). In some embodiments, the mRNA comprises 2-thiouridine (s 2 U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m 5 C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo 5 U).
• the mRNA comprises 5-methoxy-uridine (mo 5 U) and 5-methyl- cytidine (m 5 C). In some embodiments, the mRNA comprises 2’-O-methyl uridine. In some embodiments, the mRNA comprises 2’-O-methyl uridine and 5-methyl-cytidine (m 5 C). In some embodiments, the mRNA comprises comprises N6-methyl-adenosine (m 6 A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C).
• an mRNA of the invention may be modified in a coding region (e.g., an open reading frame encoding a polypeptide).
• a coding region e.g., an open reading frame encoding a polypeptide.
• an mRNA may be modified in regions besides a coding region.
• a 5′-UTR and/or a 3′-UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications.
• nucleoside modifications may also be present in the coding region.
• 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 include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.
• 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.
• 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.
• 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
• polynucleotides of the invention may be synthesized to comprise the combinations or single modifications of Tables 5 or 6.
• 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.
• 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.
• 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.
• the present invention provides TAVs and TAV pharmaceutical compositions and complexes optionally in combination with one or more
• 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 21 st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
• compositions are administered to humans, human patients or subjects.
• active ingredient generally refers to the TAVs or the polynucleotides contained therein to be delivered as described herein.
• compositions 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.
• Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology.
• 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.
• compositions 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.
• 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.
• 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.
• 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.
• 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.
• the polynucleotides of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
• 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.
• 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.
• 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.
• 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.
• the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
• 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.
• the formulations described herein may contain at least one polynucleotide.
• the formulations may contain 1, 2, 3, 4 or 5 polynucleotides.
• the formulations described herein may comprise more than one type of polynucleotide.
• the formulation may comprise a chimeric polynucleotide in linear and circular form.
• the formulation may comprise a circular polynucleotide and an IVT polynucleotide.
• the formulation may comprise an IVT polynucleotide, a chimeric polynucleotide and a circular polynucleotide.
• compositions 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, 21 st Edition, A. R. Gennaro, Lippincott, Williams ⁇ Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
• any 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.
• 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.
• 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.
• 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).
• particle size Akinc et al., Mol Ther.2009 17:872-879; herein incorporated by reference in its entirety.
• 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
• C12- 200 including derivatives and variants
• MD1 can be tested for in vivo activity.
• 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.
• 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).
• 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.
• a polynucleotide formulated with a lipidoid for systemic intravenous administration can target the liver.
• 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.
• an intravenous formulation using a C12-200 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).
• an MD1 lipidoid-containing formulation may be used to effectively deliver polynucleotides to hepatocytes in vivo.
• 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.
• 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; 8 th 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.
• 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.
• the component molar ratio may include, but is not limited to, 50% C12-200, 10%
• 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.
• 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 Liposomes, Lipoplexes, and Lipid Nanoparticles
• the TAVs of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.
• 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.
• MLV multilamellar vesicle
• SUV small unicellular vesicle
• LUV large unilamellar vesicle
• 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.
• 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.
• 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,
• 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).
• DOXIL® 1,2-dioleyloxy-N,N- dimethylaminopropane
• DiLa2 liposomes from Marina Biotech (Bothell, WA)
• 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.
• 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.
• SPLP stabilized
• the liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide.
• 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.
• DSPC disteroylphosphatidyl choline
• DODMA 1,2-dioleyloxy-N,N-dimethylaminopropane
• 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.
• DSDMA 1,2-distearloxy- N,N-dimethylaminopropane
• DODMA 1,2-dilinolenyloxy- 3-dimethylaminopropane
• 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.
• 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%.
• formulations may comprise from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about 15.0% DSPC.
• 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.
• liposomes may be formulated for targeted delivery.
• 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.
• 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).
• the TAVs may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed.
• the emulsion may be made by the methods described in International Publication No. WO201087791, herein incorporated by reference in its entirety.
• 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.
• polynucleotides encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No.
• 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
• 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
• 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.
• the TAVs may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
• 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.
• the liposome may have a N:P ratio of greater than 20:1 or less than 1:1.
• 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.
• 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.
• 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).
• DOPE dioleoyl phosphatidylethanolamine
• 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.
• 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.
• the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA.
• changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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)
• the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos.
• the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733,
• 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.
• 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-N-N-dimemylhexacosa-17,20- dien-9-amine, (1Z,
• the lipid may be a cleavable lipid such as those described in International Publication No. WO2012170889, herein incorporated by reference in its entirety.
• 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.
• the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos.
• the cationic lipid may be a trialkyl cationic lipid.
• trialkyl cationic lipids and methods of making and using the trialkyl cationic lipids are described in International Patent Publication No.
• 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.
• 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.
• the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
• the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component.
• the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol.
• the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.
• 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:
• the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or
• TAVs described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or
• 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.
• 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.
• 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.
• the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle described in US Patent No.
• 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.
• the LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or
• modified RNA described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or
• LNP formulations described herein may comprise a polycationic composition.
• 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.
• 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.
• the LNP formulations described herein may additionally comprise a permeability enhancer molecule.
• permeability enhancer molecules are described in US Patent Publication No. US20050222064; the content of which is herein incorporated by reference in its entirety.
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the conjugate may be the membrane protein CD47 (e.g., see Rodriguez et al .
• 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.
• the nanoparticle may comprise PEG and a conjugate of CD47 or a derivative thereof.
• the nanoparticle may comprise both the“self” peptide described above and the membrane protein CD47.
• 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.
• TAV pharmaceutical compositions comprising the polynucleotides of the present invention and a conjugate which may have a degradable linkage.
• conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.
• 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.
• the nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a TAV.
• 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).
• Nanoparticle formulations of the present invention may be coated with a surfactant or polymer in order to improve the delivery of the particle.
• 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.
• 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.
• the lipid nanoparticles of the present invention may be hydrophilic polymer particles.
• hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in US Patent Publication No. US20130210991, the contents of which are herein
• the lipid nanoparticles of the present invention may be hydrophobic polymer particles.
• 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.
• 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.
• the internal ester linkage may be located on either side of the saturated carbon.
• an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
• a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
• 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.
• the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
• 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).
• oral e.g., the buccal and esophageal membranes and tonsil tissue
• ophthalmic e.g., gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum)
• nasal, respiratory e.g., nasal, pharyngeal, tracheal and bronchial
• 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.
• PEG polyethylene glycol
• 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.
• 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.
• 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.
• 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 (
• 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).
• 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
• poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer
• 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.
• 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).
• 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).
• 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, th
• 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.
• 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.
• 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.
• hypotonic formulations may be found in International Patent Publication No. WO2013110028, the contents of which are herein incorporated by reference in its entirety.
• 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).
• the TAV is formulated as a lipoplex, such as, without limitation, the ATUPLEX TM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT TM 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.
• a lipoplex such as, without limitation, the ATUPLEX TM system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT TM 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;
• 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.
• 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
• the TAV is formulated as a solid lipid nanoparticle.
• a solid lipid nanoparticle 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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,
• therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.
• the TAV therapeutic nanoparticles may be formulated to be target specific.
• the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518; herein incorporated by reference in its entirety).
• the therapeutic nanoparticles may be formulated to be cancer specific.
• the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725,
• 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.
• 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,
• the TAV compositions may be encapsulated in, linked to and/or associated with zwitterionic lipids.
• 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.
• the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.
• 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.
• 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.
• the nanoparticle may be formulated by the methods described in U.S. Pub. No.
• 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.
• 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
• 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
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high density nucleic acid layer.
• 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.
• 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[ ⁇ -(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross
• 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 .
• 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.
• 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.
• the TAV compositions of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No.
• 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).
• 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).
• 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.
• 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).
• 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).
• 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),
• 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
• 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.
• 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.
• 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),
• 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.
• the poly(amine-co-esters) may be the polymers described in and/or made by the methods described in International Publication No
• 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.
• 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.
• 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.
• the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines.
• LPEI linear polyethyleneimine
• 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.
• the TAV compositions of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains.
• Degradable 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.
• the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
• 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.
• 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.
• 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.
• 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.
• 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.
• the polymers described herein may be conjugated to a lipid-terminating PEG.
• PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG.
• PEG conjugates for use with the present invention are described in International Publication No.
• 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.
• the TAV compositions disclosed herein may be mixed with the PEGs or the sodium phosphate/sodium carbonate solution prior to administration.
• a polynucleotides encoding a protein of interest may be mixed with the PEGs and also mixed with the sodium phosphate/sodium carbonate solution.
• 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.
• the TAV compositions described herein may be conjugated with another compound.
• 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.
• 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.
• TAV compositions described herein may be conjugated and/or encapsulated in gold-nanoparticles.
• a gene delivery composition may include a nucleotide sequence and a poloxamer.
• the TAVs of the present invention may be used in a gene delivery composition with the poloxamer described in U.S. Pub. No. 20100004313.
• 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
• 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.
• 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.
• the polyplex may be formed using the noval alpha-aminoamidine polymers described in International Publication No.
• 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.
• the polyplex comprises two or more cationic polymers.
• the catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
• 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.
• 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;
• 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).
• hydrophilic- hydrophobic polymers e.g., PEG-PLGA
• hydrophobic polymers e.g., PEG
• hydrophilic polymers International Pub. No. WO20120225129
• the TAV compositions of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide.
• 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
• compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space.
• a cell penetrating agent e.g., liposomes
• 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;
• 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.
• 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.
• 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
• 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).
• 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
• 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.
• 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 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, polyeth
• 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).
• the conjugate of the present invention may function as a carrier for the TAVs of the present invention.
• 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).
• polyamino acids examples 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.
• 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.
• 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 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.
• 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.
• 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.
• the targeting group is an aptamer.
• the aptamer can be unmodified or have any combination of modifications disclosed herein.
• the TAV compositions disclosed herein may be formulated as self-assembled nanoparticles.
• 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 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.
• 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.
• 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.
• suspensions may comprise co- solvents including, but not limited to ethanol, glycerol and/or propylene glycol.
• 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( ⁇ -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.
• 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.
• the formulation may be a microemulsion comprising microparticles and TAV compositions.
• 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.
• 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.
• compositions 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, 21 st Edition, A. R.
• 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.
• 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.
• 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.
• 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.
• 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).
• chelants such as, for example, DTPA or DTPA-bisamide
• calcium chelate complexes as for example calcium DTPA, CaNaDTPA-bisamide
• calcium or sodium salts for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate
• antioxidants and suspending agents can be used.
• pharmaceutical compositions may comprise cyroprotectants.
• 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.
• 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.
• 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.
• 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.
• the TAV compositions of the present invention may be delivered to a cell naked.
• “naked” refers to delivering TAV compositions free from agents which promote transfection.
• 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.
• 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.
• 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.
• 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
• 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 cistern
• 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), n
• Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
• 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.
• oral compositions can be any suitable for example, oral compositions, solubilizing agents and e
• compositions are mixed with solubilizing agents such as CREMOPHOR ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or
• 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).
• FDA US Food and Drug Administration
• 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.
• 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.
• 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.
• 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.
• 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 can be used, 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.
• 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.
• TAV compositions can be delivered to the skin by several different approaches known in the art.
• 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.
• 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.
• an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
• Topically-administrable formulations may, for example, comprise from about 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.
• compositions of the present invention may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
• 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.
• 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.
• TAV compositions of the present invention may also be administered with an immunologic-enhancing drug, such as levamisole, isoprinosine and Zadaxin.
• an immunologic-enhancing drug such as levamisole, isoprinosine and Zadaxin.
• compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.
• 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.
• therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions.
• agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually.
• the levels utilized in combination will be lower than those utilized individually.
• the combinations, each or together may be administered according to the split dosing regimens described herein.
• 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.
• 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
• 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.
• 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).
• multiple administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
• split dosing regimens such as those described herein may be used.
• TAV compositions may be administered in split-dose regimens.
• 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.
• 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.
• a“total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.
• 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.
• 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 e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous.
• 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.
• 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.
• 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.
• the rate of polynucleotides release can be controlled.
• 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.
• 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.
• multi-dose administration may comprise repeat- dose administration.
• 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.
• subjects may display a repeat-dose response.
• 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.
• a response may be the expression of a protein in response to a repeat-dose comprising TAV.
• 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.”
• the TAV compositions of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine.
• 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.
• the active therapeutic agents of the invention include TAV compositions, cells containing TAV compositions or polypeptides translated from the
• 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).
• 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.
• 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.
• 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.
• a polypeptide antigen e.g., a polypeptide PCSK9, TNF alpha, IL-17A, or GDF8 antigen
• 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.
• 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.
• 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.
• 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.
• 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.
• the TAV compositions disclosed herein encode an antigenic polypeptide and act as a vaccine when provided to a subject.
• 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.
• 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
• 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.
• RNA is susceptible to RNase degradation and other natural decomposition in the cytoplasm of cells.
• 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.
• 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.
• 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.
• the TAV compositions of the invention may encode an immunogen.
• the delivery of the polynucleotides encoding an immunogen may activate the immune response.
• 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).
• 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).
• 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.
• the polynucleotides of the invention may be used for a vaccine in combination with an inhibitor which can impair antigen presentation (see
• a formulation of the TAV polynucleotides of the invention may further comprise an amphipathic and/or immunogenic amphipathic peptide.
• 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
• the TAV polynucleotides of the invention may be immunostimultory.
• 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- ⁇ , INF- ⁇ , GM-CFS, LT- ⁇ , or growth factors such as hGH.
• 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- ⁇ , INF- ⁇ , GM-CFS, LT- ⁇ , or growth factors such as hGH.
• a cytokine that promotes the immune
• 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.
• 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).
• 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.
• the TAV polynucleotides of the invention may be administrated with other prophylactic or therapeutic compounds.
• the prophylactic or therapeutic compound may be an adjuvant or a booster.
• 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
• the TAV polynucleotides of the invention may be administered intranasally similar to the administration of live vaccines.
• the polynucleotide may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.
• the polynucleotide encode at least one antigen, at least one dendritic cell targeting agent or moiety and at least one
• 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.
• 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.
• 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.
• 5’cap analogs are described in US Patent Publication No. US20120195917, the contents of which are herein incorporated by reference in its entirety.
• 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.
• 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.
• an mRNA encoding a PCSK9 antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided.
• 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.
• the pharmaceutical composition comprises an mRNA encoding a PCSK9 antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide.
• the dendritic cell targeting agent or immune enhancing polypeptide is also encoded by the mRNA.
• the disease or disorder is hypercholesterolemia or atherosclerosis.
• 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.
• a TNF alpha antigen e.g., an endogenous TNF alpha polypeptide
• an mRNA encoding a TNF alpha antigen and, optionally, a dendritic cell targeting agent or immune enhancing polypeptide is provided.
• 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.
• TNF alpha associated disease 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,

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