US20190351039A1 - Immunomodulatory therapeutic mrna compositions encoding activating oncogene mutation peptides - Google Patents

Immunomodulatory therapeutic mrna compositions encoding activating oncogene mutation peptides Download PDF

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US20190351039A1
US20190351039A1 US16/482,473 US201816482473A US2019351039A1 US 20190351039 A1 US20190351039 A1 US 20190351039A1 US 201816482473 A US201816482473 A US 201816482473A US 2019351039 A1 US2019351039 A1 US 2019351039A1
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Eric Yi-Chun Huang
Sze-Wah TSE
Jared IACOVELLI
Kristine MCKINNEY
Nicholas Valiante
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ModernaTx Inc
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ModernaTx Inc
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Assigned to MODERNATX, INC. reassignment MODERNATX, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: VALIANTE, NICHOLAS, TSE, Sze-Wah, IACOVELLI, Jared, HUANG, ERIC YI-CHUN, MCKINNEY, Kristine
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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5015—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the ability to modulate an immune response is beneficial in a variety of clinical situations, including the treatment of cancer and pathogenic infections, as well as in potentiating vaccine responses to provide protective immunity.
  • a number of therapeutic tools exist for modulating the function of biological pathways and/or molecules that are involved in diseases such as cancer and pathogenic infections. These tools include, for example, small molecule inhibitors, cytokines and therapeutic antibodies. Some of these tools function through modulating immune responses in a subject, such as cytokines that modulate the activity of cells within the immune system or immune checkpoint inhibitor antibodies, such as anti-CTLA-4 or anti-PD-L1 that modulate the regulation of immune responses.
  • vaccines have long been used to stimulate an immune response against antigens of pathogens to thereby provide protective immunity against later exposure to the pathogens. More recently, vaccines have been developed using antigens found on tumor cells to thereby enhance anti-tumor imunoresponsiveness.
  • antigen(s) used in the vaccine other agents may be included in a vaccine preparation, or used in combination with the vaccine preparation, to further boost the immune response to the vaccine. Such agents that enhance vaccine responsiveness are referred to in the art as adjuvants.
  • vaccine adjuvants examples include aluminum gels and salts, monophosphoryl lipid A, MF59 oil-in-water emulsion, Freund's complete adjuvant, Freund's incomplete adjuvant, detergents and plant saponins. These adjuvants typically are used with protein or peptide based vaccines. Alternative types of vaccines, such as RNA based vaccines, are now being developed.
  • immunomodulatory therapeutic compositions including lipid-based compositions such as lipid nanoparticles, which include an RNA (e.g., messenger RNA (mRNA)) that can safely direct the body's cellular machinery to produce a cancer protein or fragment thereof of interest, e.g., an activating oncogene mutation peptide.
  • RNA e.g., messenger RNA (mRNA)
  • mRNA messenger RNA
  • the RNA is a modified RNA.
  • the immunomodulatory therapeutic compositions, including mRNA compositions and/or lipid nanoparticles comprising the same are useful to induce a balanced immune response against cancers, comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.
  • the immunomodulatory therapeutic compositions including mRNA compositions and/or lipid nanoparticles of the disclosure may be utilized in various settings depending on the prevalence of the cancer or the degree or level of unmet medical need.
  • the immunomodulatory therapeutic compositions, including mRNA compositions and lipid nanoparticles of the disclosure may be utilized to treat and/or prevent a cancer of various stages or degrees of metastasis.
  • the immunomodulatory therapeutic compositions and lipid nanoparticles of the disclosure have superior properties in that they produce much larger antibody titers and produce responses earlier than alternative anti-cancer therapies including cancer vaccines.
  • RNA of the provided compositions are presented to the cellular system in a more native fashion.
  • the disclosure provides an immunomodulatory therapeutic composition, comprising: one or more mRNA each comprising an open reading frame encoding an activating oncogene mutation peptide, and optionally one or more mRNA each comprising an open reading frame encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject, wherein the immune response comprises a cellular or humoral immune response characterized by: (i) stimulating Type I interferon pathway signaling, (ii) stimulating NFkB pathway signaling, (iii) stimulating an inflammatory response, (iv) stimulating cytokine production, (v) stimulating dendritic cell development, activity or mobilization, and (vi) a combination of any of (i)-(v); and a pharmaceutically acceptable carrier.
  • the disclosure provides an immunomodulatory therapeutic composition, including mRNA compositions and/or lipid nanoparticles comprising the same, that enhances an immune response by, for example, stimulating Type I interferon pathway signaling, stimulating NFkB pathway signaling, stimulating an inflammatory response, stimulating cytokine production or stimulating dendritic cell development, activity or mobilization.
  • Enhancement of an immune response to an antigen of interest by an immune potentiator mRNA results in, for example, stimulation of cytokine production, stimulation of cellular immunity (T cell responses), such as antigen-specific CD8 + or CD4 + T cell responses and/or stimulation of humoral immunity (B cell responses), such as antigen-specific antibody responses.
  • T cell responses such as antigen-specific CD8 + or CD4 + T cell responses
  • B cell responses humoral immunity
  • the disclosure provides an immunomodulatory therapeutic composition wherein the activating oncogene mutation is a KRAS mutation.
  • the KRAS mutation is a G12 mutation.
  • the G12 KRAS mutation is selected from G12D, G12V, G12S, G12C, G12A, and G12R KRAS mutations.
  • the G12 KRAS mutation is selected from G12D, G12V, and G12C KRAS mutations.
  • the KRAS mutation is a G13 mutation.
  • the G13 KRAS mutation is a G13D KRAS mutation.
  • the disclosure provides an immunomodulatory therapeutic composition wherein the activating oncogene mutation is a H-RAS or N-RAS mutation.
  • the skilled artisan will select a KRAS mutation, a HLA subtype and a tumor type based on the guidance provided herein and prepare a KRAS vaccine for therapy.
  • the KRAS mutation is selected from: G12C, G12V, G12D, G13D.
  • the HLA subtype is selected from: A*02:01, C*07:01, C*04:01, C*07:02, HLA-A11 and/or HLA-C08.
  • the tumor type is selected from colorectal, pancreatic, lung (e.g., non-small cell lung cancer (NSCLC), and endometrioid.
  • NSCLC non-small cell lung cancer
  • the HRAS mutation is a mutation at codon 12, codon 13, or codon 61. In some embodiments, the HRAS mutation is a 12V, 61L, or 61R mutation.
  • the NRAS mutation is a mutation at codon 12, codon 13, or codon 61. In some embodiments, the NRAS mutation is a 12D, 13D, 61K, or 61R mutation.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the mRNA has an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides.
  • the concatemer comprises 3, 4, 5, 6, 7, 8, 9, or 10 activating oncogene mutation peptides. In some aspects, the concatemer comprises 4 activating oncogene mutation peptides.
  • the disclosure provides an immunomodulatory therapeutic composition, comprising: an mRNA comprising an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides, wherein the concatemer comprises KRAS activating oncogene mutation peptides G12D, G12V, G12C, and G13D; and one or more mRNA each comprising an open reading frame encoding a polypeptide that enhances an immune response to the KRAS activating oncogene mutation peptides in a subject.
  • the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C.
  • the concatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D.
  • Some embodiments of the present disclosure provide immunomodulatory therapeutic compositions that include an mRNA comprising an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides.
  • at least two of the peptide epitopes are separated from one another by a single Glycine.
  • the concatemer comprises 3-10 activating oncogene mutation peptides.
  • all of the peptide epitopes are separated from one another by a single Glycine.
  • at least two of the peptide epitopes are linked directly to one another without a linker.
  • the disclosure provides an immunomodulatory therapeutic composition, comprising: 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 activating oncogene mutation peptides; and one or more mRNA each comprising an open reading frame encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject.
  • the composition comprises 4 mRNAs encoding 4 activating oncogene mutation peptides.
  • the 4 mRNAs encode KRAS activating oncogene mutation peptides G12D, G12V, G12C, and G13D.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the activating oncogene mutation peptide comprises 10-30, 15-25, or 20-25 amino acids in length. In some aspects, the activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, the activating oncogene mutation peptide comprises 25 amino acids in length.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the mRNA encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject encodes a constitutively active human STING polypeptide.
  • the constitutively active human STING polypeptide comprises one or more mutations selected from the group consisting of V147L, N154S, V155M, R284M, R284K, R284T, E315Q, R375A, and combinations thereof.
  • the constitutively active human STING polypeptide comprises mutation V155M (e.g., having the amino acid sequence shown in SEQ ID NO: 1 or encoded by a nucleotide sequence shown in SEQ ID NO: 139 or 170). In some aspects the constitutively active human STING polypeptide comprises mutations V147L/N154S/V155M. In some aspects, the constitutively active human STING polypeptide comprises mutations R284M/V147L/N154S/V155M.
  • the constitutively active human STING polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-10 and 164.
  • the constitutively active human STING polypeptide is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 139-148, 165, 168, 170, 201-209 and 225.
  • the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site, such as for example set forth in SEQ ID NO: 149.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the mRNA encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject encodes a constitutitively active human IRF3 polypeptide.
  • the constitutively active human IRF3 polypeptide comprises an S396D mutation.
  • the constitutively active human IRF3 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 12 or is encoded by a nucleotide sequence set forth in SEQ ID NO: 151 or 212.
  • the constitutively active IRF3 polypeptide is a mouse IRF3 polypeptide, for example comprising an amino acid sequence set forth in SEQ ID NO: 11 or encoded by the nucleotide sequence shown in SEQ ID NO: 150 or 211.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the mRNA encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject encodes a constitutitively active human IRF7 polypeptide.
  • the constitutively active human IRF7 polypeptide comprises one or more mutations selected from the group consisting of S475D, S476D, S477D, S479D, L480D, S483D, S487D, and combinations thereof; deletion of amino acids 247-467; and combinations of the foregoing mutations and/or deletions.
  • the constitutively active human IRF7 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14-18. In one embodiment, the constitutively active human IRF7 polypeptide is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 153-157 and 214-218.
  • the disclosure provides an immune potentiator mRNA encoding a polypeptide selected from the group consisting of MyD88, TRAM, IRF1, IRF8, IRF9, TBK1, IKKi, STAT1, STAT2, STAT4, STAT6, c-FLIP, IKK ⁇ , RIPK1, TAK-TAB1 fusion, DIABLO, Btk, self-activating caspase-1 and Flt3.
  • a polypeptide selected from the group consisting of MyD88, TRAM, IRF1, IRF8, IRF9, TBK1, IKKi, STAT1, STAT2, STAT4, STAT6, c-FLIP, IKK ⁇ , RIPK1, TAK-TAB1 fusion, DIABLO, Btk, self-activating caspase-1 and Flt3.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the composition further comprises a cancer therapeutic agent.
  • the composition further comprises an inhibitory checkpoint polypeptide.
  • the inhibitory checkpoint polypeptide is an antibody or fragment thereof that specifically binds to a molecule selected from the group consisting of PD-1, PD-L1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3.
  • the composition further comprises a recall antigen.
  • the recall antigen is an infectious disease antigen.
  • the composition does not comprise a stabilization agent.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the mRNA is formulated in a lipid nanoparticle.
  • the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the inonizable amino lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethylaminobutyrate
  • the ionizable amino lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In some aspects, the ionizable amino lipid comprises a compound of Formula (I). In some aspects, the compound of Formula (I) is Compound 25.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein each mRNA includes at least one chemical modification.
  • the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine,
  • the disclosure provides an immunomodulatory therapeutic composition, including mRNA compositions and lipid-based compositions such as lipid nanoparticles, comprising: one or more mRNA each comprising an open reading frame encoding a KRAS activating oncogene mutation peptide, and optionally one or more mRNA each comprising an open reading frame encoding a constitutively active human STING polypeptide; and a pharmaceutically acceptable carrier.
  • the constitutively active human STING polypeptide comprises mutation V155M.
  • the constitutively active human STING polypeptide comprises an amino acid sequence shown in SEQ ID NO: 1.
  • the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the KRAS activating oncogene mutation peptide is selected from G12D, G12V, G12S, G12C, G12A, G12R, and G13D. In some aspects, the KRAS activating oncogene mutation peptide is selected from G12D, G12V, G12C, and G13D.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the mRNA comprises an open reading frame encoding a concatemer of two or more KRAS activating oncogene mutation peptides.
  • the concatemer comprises 3, 4, 5, 6, 7, 8, 9 or 10 KRAS activating oncogene mutation peptides.
  • the concatemer comprises 4 KRAS activating oncogene mutation peptides.
  • the concatemer comprises G12D, G12V, G12C, and G13D.
  • the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C.
  • the concatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the composition comprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 KRAS activating oncogene mutation peptides.
  • the composition comprises 4 mRNAs encoding 4 KRAS activating oncogene mutation peptides.
  • the 4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C, and G13D.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the KRAS activating oncogene mutation peptide comprises 10-30, 15-25, or 20-25 amino acids in length. In some aspects, the KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, the activating oncogene mutation peptide comprises 25 amino acids in length.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the mRNA has an open reading frame encoding a concatemer of two or more KRAS activating oncogene mutation peptides and the concatemer comprises an amino acid sequence selected from the group set forth in SEQ ID NOS: 42-47, 73 and 137.
  • the mRNA encoding the concatemer comprises a nucleotide sequence selected from the group set forth in SEQ ID NOS: 129-131, 133, 138, 167, 169, 193-195 and 197-198.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein the composition comprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 KRAS activating oncogene mutation peptides, and wherein the KRAS activating oncogene mutation peptides comprise an amino acid sequence selected from the group set forth in SEQ ID NOs: 36-41, 72 and 125.
  • the KRAS activating oncogene mutation peptides comprise the amino acid sequence set forth in SEQ ID NOs: 39-41 and 72.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises a nucleotide sequence selected from the group set forth in SEQ ID NOs: 126-128, 132, 190-192 and 196.
  • the disclosure provides an immunomodulatory therapeutic composition, including mRNA compositions and/or lipid nanoparticles comprising the same, comprising an mRNA construct encoding at least one mutant human KRAS antigen and a constitutively active human STING polypeptide, for example wherein the mRNA (e.g., a modified mRNA) encodes an amino acid sequence as set forth in any one of SEQ ID NOs: 48-71.
  • mRNA e.g., a modified mRNA
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein each mRNA is formulated in the same or different lipid nanoparticle.
  • each mRNA encoding a KRAS activating oncogene mutation peptide is formulated in the same or different lipid nanoparticle.
  • each mRNA encoding constitutively active human STING is formulated in the same or different lipid nanoparticle.
  • each mRNA encoding a KRAS activating oncogene mutation peptide is formulated in the same lipid nanoparticle and each mRNA encoding constitutively active human STING is formulated in a different lipid nanoparticle.
  • each mRNA encoding a KRAS activating oncogene mutation peptide is formulated in the same lipid nanoparticle and each mRNA encoding constitutively active human STING is formulated in the same lipid nanoparticle as each mRNA encoding a KRAS activating oncogene mutation peptide.
  • each mRNA encoding a KRAS activating oncogene mutation peptide is formulated in a different lipid nanoparticle and each mRNA encoding constitutively active human STING is formulated in the same lipid nanoparticle as each mRNA encoding each KRAS activating oncogene mutation peptide.
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing embodiments, wherein the immunomodulatory therapeutic composition is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the ionizable amino lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethylaminobutyrate
  • the ionizable amino lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In some aspects, the ionizable amino lipid comprises a compound of Formula (I). In some aspects, the compound of Formula (I) is Compound 25.
  • the lipid nanoparticle comprises Compound 25 (as the ionizable amino lipid), DSPC (as the phospholipid), cholesterol (as the sterol) and PEG-DMG (as the PEG-modified lipid).
  • the lipid nanoparticle comprises a molar ratio of about 20-60% Compound 25:5-25% DSPC:25-55% cholesterol; and 0.5-15% PEG-DMG.
  • the lipid nanoparticle comprises a molar ratio of about 50% Compound 25:about 10% DSPC:about 38.5% cholesterol:about 1.5% PEG-DMG (i.e., Compound 25:DSPC:cholesterol:PEG-DMG at about a 50:10:38.5:1.5 ratio). In one embodiment, the lipid nanoparticle comprises a molar ratio of 50% Compound 25:10% DSPC:38.5% cholesterol: 1.5% PEG-DMG (i.e., Compound 25:DSPC:cholesterol:PEG-DMG at a 50:10:38.5:1.5 ratio).
  • the disclosure provides an immunomodulatory therapeutic composition of any one of the foregoing or related embodiments, wherein each mRNA includes at least one chemical modification.
  • the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine
  • the disclosure provides a lipid nanoparticle comprising: an mRNA comprising an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the 4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C, and G13D; an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C.
  • the concatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D.
  • each KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, each KRAS activating oncogene mutation peptide comprises 25 amino acids in length.
  • the disclosure provides a lipid nanoparticle comprising: an mRNA comprising an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the 4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C, and G13D, and wherein the concatemer comprises the amino acid sequence set forth in SEQ ID NO:137; an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the mRNA encoding the concatemer of 4 KRAS activating oncogene mutation peptides comprises the nucleotide sequence set forth in SEQ ID NO: 138, SEQ ID NO: 167 or SEQ ID NO: 169.
  • the constitutively active human STING polypeptide comprises mutation V155M.
  • the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the mRNA encoding the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site.
  • the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.
  • the disclosure provides a lipid nanoparticle comprising:
  • a first mRNAs comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12D;
  • a second mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12V;
  • a third mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12C;
  • a fourth mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G13D;
  • a fifth mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the mRNAs are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 and 10:1. In one embodiment, the mRNAs are present at a KRAS:STING mass ratio of 5:1.
  • each KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, each KRAS activating oncogene mutation peptide comprises 25 amino acids in length.
  • the KRAS activating oncogene mutation peptides comprise the amino acid sequences set forth in SEQ ID NOs: 39-41 and 72.
  • the mRNAs encoding the KRAS activating oncogene mutation peptides comprise the nucleotide sequences set forth in SEQ ID NOs: 126-128, 132, 190-192 and 196.
  • the constitutively active human STING polypeptide comprises mutation V155M. In some aspects, the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1. In some aspects, the mRNA encoding the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site. In some aspects, the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.
  • the disclosure provides a lipid nanoparticle of any one of the foregoing embodiments, wherein the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% phopholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the inonizable amino lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
  • DLin-KC2-DMA 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
  • DLin-MC3-DMA dilinoleyl-methyl-4-dimethylaminobutyrate
  • the ionizable amino lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In some aspects, the ionizable amino lipid comprises a compound of Formula (I). In some aspects, the compound of Formula (I) is Compound 25.
  • the disclosure provides a lipid nanoparticle of any one of the foregoing embodiments, wherein each mRNA includes at least one chemical modification.
  • the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine
  • the disclosure provides a drug product comprising any of the foregoing or related lipid nanoparticles for use in cancer therapy, optionally with instructions for use in cancer therapy.
  • the disclosure provides a first lipid nanoparticle comprising: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12D; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the disclosure provides a second lipid nanoparticle comprising: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12V; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the disclosure provides a third lipid nanoparticle comprising an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12C; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the disclosure provides a fourth lipid nanoparticle comprising: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G13D; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • each KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, each KRAS activating oncogene mutation peptide comprises 25 amino acids in length.
  • the KRAS activating oncogene mutation peptide comprises the amino acid sequences set forth in SEQ ID NO: 39.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises the nucleotide sequence set forth in SEQ ID NOs: 126 or 190.
  • the KRAS activating oncogene mutation peptide comprises the amino acid sequences set forth in SEQ ID NO: 40.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises the nucleotide sequence set forth in SEQ ID NOs: 127 or 191.
  • the KRAS activating oncogene mutation peptide comprises the amino acid sequences set forth in SEQ ID NO: 72.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises the nucleotide sequence set forth in SEQ ID NOs: 132 or 196.
  • the KRAS activating oncogene mutation peptide comprises the amino acid sequences set forth in SEQ ID NO: 41.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises the nucleotide sequence set forth in SEQ ID NOs: 128 or 192.
  • the constitutively active human STING polypeptide comprises mutation V155M.
  • the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site.
  • the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.
  • the disclosure provides a drug product comprising any of the foregoing or related lipid nanoparticles for use in cancer therapy, optionally with instructions for use in cancer therapy. In some aspects, the disclosure provides a drug product comprising any of the foregoing first, second, third and fourth lipid nanoparticles, for use in cancer therapy, optionally with instructions for use in cancer therapy.
  • the disclosure provides a drug product comprising a first, second, third and fourth lipid nanoparticles, for use in cancer therapy, optionally with instructions for use in cancer therapy, wherein:
  • the first lipid nanoparticle comprises: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12D; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide;
  • the second lipid nanoparticle comprises: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12V; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide;
  • the third lipid nanoparticle comprises: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12C; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide; and
  • the fourth lipid nanoparticle comprises: an mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G13D; and an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide.
  • the disclosure provides a method for treating a subject, comprising: administering to a subject having cancer any of the foregoing or related immunomodulatory therapeutic compositions or any of the foregoing or related lipid nanoparticle.
  • the immunomodulatory therapeutic composition or lipid nanoparticle is administered in combination with a cancer therapeutic agent.
  • the immunomodulatory therapeutic composition or lipid nanoparticle is administered in combination with an inhibitory checkpoint polypeptide.
  • the inhibitory checkpoint polypeptide is an antibody or fragment thereof that specifically binds to a molecule selected from the group consisting of PD-1, PD-L1, TIM-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3.
  • the cancer is selected from cancer of the pancreas, peritoneum, large intestine, small intestine, biliary tract, lung, endometrium, ovary, genital tract, gastrointestinal tract, cervix, stomach, urinary tract, colon, rectum, and hematopoietic and lymphoid tissues.
  • the cancer is colorectal cancer.
  • the cancer is pancreatic cancer.
  • the cancer is lung cancer, such as non-small cell lung cancer (NSCLC).
  • the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer and lung cancer (e.g., NSCLC).
  • An mRNA (e.g., mmRNA) construct of the disclosure can comprise, for example, a 5′ UTR, a codon optimized open reading frame encoding the polypeptide, a 3′ UTR and a 3′ tailing region of linked nucleosides.
  • the mRNA further comprises one or more microRNA (miRNA) binding sites.
  • a modified mRNA construct of the disclosure is fully modified.
  • the mmRNA comprises pseudouridine ( ⁇ ), pseudouridine ( ⁇ ) and 5-methyl-cytidine (m 5 C), 1-methyl-pseudouridine (m 1 ⁇ ), 1-methyl-pseudouridine (m 1 ⁇ ) and 5-methyl-cytidine (m 5 C), 2-thiouridine (s 2 U), 2-thiouridine and 5-methyl-cytidine (m 5 C), 5-methoxy-uridine (mo 5 U), 5-methoxy-uridine (mo 5 U) and 5-methyl-cytidine (m 5 C), 2′-O-methyl uridine, 2′-O-methyl uridine and 5-methyl-cytidine (m 5 C), N6-methyl-adenosine (m 6 A) or N6-methyl-adenosine (m 6 A) and 5-methyl-cytidine (m 5 C).
  • the mmRNA comprises 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, or combinations thereof.
  • the mmRNA comprises 1-methyl-pseudouridine (m 1 ⁇ ), 5-methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine ( ⁇ ), ⁇ -thio-guanosine, or ⁇ -thio-adenosine, or combinations thereof.
  • the mmRNA comprises pseudouridine or a pseudouridine analog.
  • the mmRNA comprises N1-methylpseudouridine.
  • each mmRNA comprises fully modified N1-methylpseudouridine.
  • the dosage of the RNA polynucleotide in the immunomodulatory therapeutic composition is 1-5 ⁇ g, 5-10 ⁇ g, 10-15 ⁇ g, 15-20 ⁇ g, 10-25 ⁇ g, 20-25 ⁇ g, 20-50 ⁇ g, 30-50 ⁇ g, 40-50 ⁇ g, 40-60 ⁇ g, 60-80 ⁇ g, 60-100 ⁇ g, 50-100 ⁇ g, 80-120 ⁇ g, 40-120 ⁇ g, 40-150 ⁇ g, 50-150 ⁇ g, 50-200 ⁇ g, 80-200 ⁇ g, 100-200 ⁇ g, 100-300 ⁇ g, 120-250 ⁇ g, 150-250 ⁇ g, 180-280 ⁇ g, 200-300 ⁇ g, 30-300 ⁇ g, 50-300 ⁇ g, 80-300 ⁇ g, 100-300 ⁇ g, 40-300 ⁇ g, 50-350 ⁇ g, 100-350 ⁇ g, 200-350 ⁇ g, 300-350 ⁇ g, 320-400
  • the immunomodulatory therapeutic composition is administered to the subject by intradermal or intramuscular injection. In some embodiments, the immunomodulatory therapeutic composition is administered to the subject on day zero. In some embodiments, a second dose of the immunomodulatory therapeutic composition is administered to the subject on day twenty one.
  • a dosage of 25 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 10 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 30 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject.
  • a dosage of 75 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 150 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 300 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the immunomodulatory therapeutic composition administered to the subject.
  • the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.
  • the immunomodulatory therapeutic composition is chemically modified and in other embodiments the immunomodulatory therapeutic composition is not chemically modified.
  • the effective amount is a total dose of 1-100 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a dose of 25 ⁇ g administered to the subject a total of one or two times. In some embodiments, the effective amount is a dose of 100 ⁇ g administered to the subject a total of two times.
  • the effective amount is a dose of 1 ⁇ g-10 ⁇ g, 1 ⁇ g-20 ⁇ g, 1 ⁇ g-30 ⁇ g, 5 ⁇ g-10 ⁇ g, 5 ⁇ g-20 ⁇ g, 5 ⁇ g-30 ⁇ g, 5 ⁇ g-40 ⁇ g, 5 ⁇ g-50 ⁇ g, 10 ⁇ g-15 ⁇ g, 10 ⁇ g-20 ⁇ g, 10 ⁇ g-25 ⁇ g, 10 ⁇ g-30 ⁇ g, 10 ⁇ g-40 ⁇ g, 10 ⁇ g-50 ⁇ g, 10 ⁇ g-60 ⁇ g, 15 ⁇ g-20 ⁇ g, 15 ⁇ g-25 ⁇ g, 15 ⁇ g-30 ⁇ g, 15 ⁇ g-40 ⁇ g, 15 ⁇ g-50 ⁇ g, 20 ⁇ g-25 ⁇ g, 20 ⁇ g-30 ⁇ g, 20 ⁇ g-40 ⁇ g 20 ⁇ g-50 ⁇ g, 20 ⁇ g-25 ⁇ g, 20 ⁇ g-30 ⁇ g, 20 ⁇ g-40 ⁇ g 20 ⁇ g-50
  • the disclosure provides a composition (e.g., a vaccine) comprising an mRNA encoding a KRAS activating oncogene mutation peptide and an mRNA encoding a constitutively active human STING polypeptide wherein the mRNA encoding the KRAS activating oncogene mutation peptide and the mRNA encoding the constitutively active human STING polypeptide are present at a KRAS:STING mass ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or 20:1, or alternatively at a STING:KRAS mass ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or 1:20.
  • a composition e.g., a vaccine
  • the mRNAs are present at a mass ratio of 5:1 of mRNA encoding the KRAS activating oncogene mutation peptide to the mRNA encoding the constitutively active human STING polypeptide (KRAS:STING mass ratio of 5:1 or alternatively a STING:KRAS mass ratio of 1:5). In some aspects, the mRNAs are present at a mass ratio of 10:1 of mRNA encoding the KRAS activating oncogene mutation peptide to the mRNA encoding the constitutively active human STING polypeptide (KRAS:STING mass ratio of 10:1 or alternatively a STING:KRAS ratio of 1:10).
  • lipid nanoparticle comprising:
  • an mRNA comprising an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the 4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C, and G13D;
  • an mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide
  • mRNAs are present at a KRAS:STING mass ratio selected from the group consisting of of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.
  • the disclosure relates to a lipid nanoparticle comprising:
  • a first mRNAs comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12D;
  • a second mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12V;
  • a third mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12C;
  • a fourth mRNA comprising an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G13D;
  • a fifth mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide
  • first, second, third, fourth and fifth mRNAs are present at an KRAS:STING mass ratio selected from the group consisting of of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.
  • the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C. In some aspects, the concatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D. In some aspects, each KRAS activating oncogene mutation peptide comprises 20, 21, 22, 23, 24, or 25 amino acids in length. In some aspects, each KRAS activating oncogene mutation peptide comprises 25 amino acids in length. In some aspects, the concatemer comprises an amino acid sequence set forth in SEQ ID NO: 137.
  • the mRNA encoding the concatemer of 4 KRAS activating oncogene mutation peptides comprises the nucleotide sequence set forth in SEQ ID NO: 138, SEQ ID NO: 167 or SEQ ID NO: 169.
  • the constitutively active human STING polypeptide comprises mutation V155M.
  • the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the mRNA encoding the constitutively active human STING polypeptide comprises a 3′ UTR comprising at least one miR-122 microRNA binding site.
  • the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 139, SEQ ID NO: 168, or SEQ ID NO: 170.
  • the lipid nanoparticle comprises mRNAs present at an KRAS:STING mass ratio of 1:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 2:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 3:1.
  • the the mRNAs are present at a KRAS:STING mass ratio of 4:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 5:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 6:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 7:1.
  • the mRNAs are present at a KRAS:STING mass ratio of 8:1. In some aspects, the mRNAs are present at a KRAS:STING mass ratio of 9:1. In some aspects, the mRNAS are present at a KRAS:STING mass ratio of 10:1.
  • the disclosure pertains to a lipid nanoparticle comprising a modified mRNA of the disclosure.
  • the lipid nanoparticle is a liposome.
  • the lipid nanoparticle comprises a cationic and/or ionizable amino lipid.
  • the cationic and/or ionizable amino lipid is 2,2-dilinoleyl-4-methylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA).
  • the ionizable amino lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe). In some aspects, the ionizable amino lipid comprises a compound of Formula (I). In one embodiment, the ionizable amino lipid is Compound 25. In one embodiment, the lipid nanoparticle further comprises a targeting moiety conjugated to the outer surface of the lipid nanoparticle.
  • the disclosure pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising a modified mRNA of the disclosure or a lipid nanoparticle of the disclosure, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the disclosure pertains to a method for enhancing an immune response to an antigen(s) of interest, the method comprising administering to a subject in need thereof a mRNA composition of disclosure encoding an antigen(s) of interest and a polypeptide that enhances an immune response to the antigen(s) of interest, or lipid nanoparticle thereof, or pharmaceutical composition thereof, such that an immune response to the antigen of interest is enhanced in the subject.
  • enhancing an immune response in a subject comprises stimulating cytokine production (e.g., IFN- ⁇ or TNF- ⁇ ).
  • enhancing an immune response in a subject comprises stimulating antigen-specific CD8 + T cell activity, e.g., priming, proliferation and/or survival (e.g., increasing the effector/memory T cell population).
  • enhancing an immune response in a subject comprises stimulating antigen-specific CD4 + T cell activity (e.g., increasing helper T cell activity).
  • enhancing an immune response in a subject comprises stimulating B cell responses (e.g., increasing antibody production).
  • the disclosure provides methods for enhancing an immune response to an activating oncogene mutation peptide, wherein the subject is administered two different immune potentiator mRNA (e.g., mmRNA) constructs (wherein one or both constructs also encode, or are administered with an mRNA (e.g., mmRNA) construct that encodes, the activating oncogene mutation peptide), either at the same time or sequentially.
  • the subject is administered an immune potentiator mmRNA composition that stimulates dendritic cell development or activity prior to administering to the subject an immune potentiator mRNA composition that stimulates Type I interferon pathway signaling.
  • the disclosure provides methods of stimulating an immune response to a tumor in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a composition comprising at least one mRNA construct encoding a tumor antigen(s) and an mRNA construct encoding a polypeptide that enhances an immune response to the tumor antigen(s), or a lipid nanoparticle thereof, or a pharmaceutical composition thereof, such that an immune response to the tumor is stimulated in the subject.
  • the tumor is a liver cancer, a colorectal cancer, a pancreatic cancer, a non-small cell lung cancer (NSCLC), a melanoma cancer, a cervical cancer or a head or neck cancer.
  • composition comprising:
  • a first mRNA comprising an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C, and
  • a second mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide, wherein the constitutively active human STING polypeptide comprises mutation V155M,
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1;
  • the concatemer of 4 KRAS activating oncogene mutation peptides comprises the amino acid sequence set forth in SEQ ID NO: 137.
  • the first mRNA encoding the concatemer of 4 KRAS activating oncogene mutation peptides comprises the nucleotide sequence set forth in SEQ ID NO: 169.
  • the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 170.
  • the first mRNA comprises a 5′ UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176.
  • the second mRNA comprises a 5′ UTR comprising the nucleotide sequence set forth in SEQ ID NO: 176.
  • the second mRNA encoding the constitutively active human STING polypeptide comprises a 3′ UTR having a miR-122 microRNA binding site.
  • the miR-122 microRNA binding site comprises the nucleotide sequence shown in SEQ ID NO: 175.
  • the first mRNA and second mRNA each comprise a poly A tail.
  • the poly A tail comprises about 100 nucleotides.
  • the first and second mRNAs each comprise a 5′ Cap 1 structure. In some aspects, the first and second mRNAs each comprise at least one chemical modification. In some aspects, the chemical modification is N1-methylpseudouridine. In some aspects, the first mRNA is fully modified with N1-methylpseudouridine. In some aspects, the second mRNA is fully modified with N1-methylpseudouridine. In some aspects, the pharmaceutically acceptable carrier comprises a buffer solution.
  • composition comprising:
  • first and second mRNA are each fully modified with N1-methylpseudouridine
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1; and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises a buffer solution.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 1:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 2:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 3:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 4:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 5:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present KRAS:STING mass ratio of 6:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 7:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 8:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 9:1.
  • the disclosure provides a composition wherein the first and second mRNAs are present at a KRAS:STING mass ratio of 10:1.
  • the disclosure provides a composition which is formulated in a lipid nanoparticle.
  • the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle comprises a molar ratio of about 50% Compound 25:about 10% DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG.
  • the disclosure provides a composition which is formulated for intramuscular delivery.
  • the disclosure provides a lipid nanoparticle comprising:
  • a first mRNA comprising an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C;
  • a second mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide, wherein the constitutively active human STING polypeptide comprises mutation V155M,
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio of 5:1.
  • the concatemer of 4 KRAS activating oncogene mutation peptides comprises the amino acid sequence set forth in SEQ ID NO: 137.
  • the first mRNA encoding the concatemer of 4 KRAS activating oncogene mutation peptides comprises the nucleotide sequence set forth in SEQ ID NO: 169.
  • the constitutively active human STING polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.
  • the mRNA encoding the constitutively active human STING polypeptide comprises the nucleotide sequence shown in SEQ ID NO: 170.
  • the first mRNA comprises a 5′ UTR comprising the nucleotide sequence shown in SEQ ID NO: 176.
  • the second mRNA comprises a 5′ UTR comprising the nucleotide sequence shown in SEQ ID NO: 176.
  • the second mRNA encoding the constitutively active human STING polypeptide comprises a 3′ UTR having a miR-122 microRNA binding site.
  • the miR-122 microRNA binding site comprises the nucleotide sequence shown in SEQ ID NO: 175.
  • the first and second mRNAs each comprise a poly A tail.
  • the poly A tail comprises about 100 nucleotides.
  • the first and second mRNAs each comprise a 5′ Cap 1 structure. In some aspects, the first and second mRNAs each comprise at least one chemical modification. In some aspects, the chemical modification is N1-methylpseudouridine. In some aspects, the first mRNA is fully modified with N1-methylpseudouridine. In some aspects, the second mRNA is fully modified with N1-methylpseudouridine.
  • the disclosure provides a lipid nanoparticle comprising:
  • first and second mRNA are each fully modified with N1-methylpseudouridine
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio of 5:1.
  • the lipid nanoparticle comprises a molar ratio of about 20-60% ionizable amino lipid:5-25% phospholipid:25-55% sterol; and 0.5-15% PEG-modified lipid.
  • the ionizable amino lipid comprises a compound of any of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe).
  • the ionizable amino lipid comprises a compound of Formula (I).
  • the compound of Formula (I) is Compound 25.
  • the lipid nanoparticle comprises a molar ratio of about 50% Compound 25:about 10% DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG.
  • the disclosure provides pharmaceutical composition comprising the lipid nanoparticle, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is formulated for intramuscular delivery.
  • the disclosure provides a lipid nanoparticle, and an optional pharmaceutically acceptable carrier, or a pharmaceutical composition for use in treating or delaying progression of cancer in an individual, wherein the treatment comprises administration of the composition in combination with a second composition, wherein the second composition comprises a checkpoint inhibitor polypeptide and an optional pharmaceutically acceptable carrier.
  • the disclosure provides use of a lipid nanoparticle, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of cancer in an individual, wherein the medicament comprises the lipid nanoparticle and an optional pharmaceutically acceptable carrier and wherein the treatment comprises administration of the medicament in combination with a composition comprising a checkpoint inhibitor polypeptide and an optional pharmaceutically acceptable carrier.
  • the disclosure provides a kit comprising a container comprising a lipid nanoparticle, and an optional pharmaceutically acceptable carrier, or a pharmaceutical composition, and a package insert comprising instructions for administration of the lipid nanoparticle or pharmaceutical composition for treating or delaying progression of cancer in an individual.
  • the package insert further comprises instructions for administration of the lipid nanoparticle or pharmaceutical composition in combination with a composition comprising a checkpoint inhibitor polypeptide and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
  • the disclosure provides a kit comprising a medicament comprising a lipid nanoparticle, and an optional pharmaceutically acceptable carrier, or a pharmaceutical composition, and a package insert comprising instructions for administration of the medicament alone or in combination with a composition comprising a checkpoint inhibitor polypeptide and an optional pharmaceutically acceptable carrier for treating or delaying progression of cancer in an individual.
  • the kit further comprises a package insert comprising instructions for administration of the first medicament prior to, current with, or subsequent to administration of the second medicament for treating or delaying progression of cancer in an individual.
  • the disclosure provides a lipid nanoparticle, a composition, or the use thereof, or a kit comprising a lipid nanoparticle or a composition as described herein, wherein the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof.
  • the checkpoint inhibitor polypeptide is an antibody.
  • the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
  • the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab. In some asepcts, the checkpoint inhibitor polypeptide is an anti-PD1 antibody, wherein the anti-PD1 antibody is pembrolizumab.
  • the disclosure provides a method of reducing or decreasing a size of a tumor or inhibiting a tumor growth in a subject in need thereof comprising administering to the subject any of the foregoing or related lipid nanoparticles of the disclosure, or any of the foregoing or related compositions of the disclosure.
  • the disclosure provides a method inducing an anti-tumor response in a subject with cancer comprising administering to the subject any of the foregoing or related lipid nanoparticles of the disclosure, or any of the foregoing or related compositions of the disclosure.
  • the anti-tumor response comprises a T-cell response.
  • the T-cell response comprises CD8+ T cells.
  • the composition is administered by intramuscular injection.
  • the method further comprises administering a second composition comprising a checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
  • the checkpoint inhibitor polypeptide inhibits PD1, PD-L, CTLA4, or a combination thereof.
  • the checkpoint inhibitor polypeptide is an antibody.
  • the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
  • the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab. In some aspects, the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab. In some asepcts, the checkpoint inhibitor polypeptide is an anti-PD1 antibody, wherein the anti-PD1 antibody is pembrolizumab.
  • the composition comprising the checkpoint inhibitor polypeptide is administered by intravenous injection. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered once every 2 to 3 weeks. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered once every 2 weeks or once every 3 weeks. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered prior to, concurrent with, or subsequent to administration of the lipid nanoparticle or pharmaceutical composition thereof.
  • the subject has a histologically confirmed KRAS mutation selected from G12D, G12V, G13D or G12C.
  • the subject has metastatic colorectal cancer.
  • the subject has non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the subject has pancreatic cancer
  • the disclosure provides a method of reducing or decreasing a size of a tumor, inhibiting a tumor growth or inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an immunomodulatory therapeutic composition comprising: one or more first mRNA each comprising an open reading frame encoding a KRAS activating oncogene mutation peptide, and optionally one or more second mRNA each comprising an open reading frame encoding a constitutively active human STING polypeptide, and optionally wherein the first mRNA and second mRNA are at a mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1; and a pharmaceutically acceptable carrier, thereby reducing or decreasing a size of a tumor, inhibiting a tumor growth or inducing an anti-tumor response in the subject.
  • the composition comprises 1, 2, 3, or 4 mRNAs encoding 1, 2, 3, or 4 KRAS activating oncogene mutation peptides. In some aspects, the composition comprises 4 mRNAs encoding 4 KRAS activating oncogene mutation peptides. In some aspects, the 4 KRAS activating oncogene mutation peptides comprise G12D, G12V, G12C, and G13D.
  • the method comprises administering an immunomodulatory therapeutic composition comprising a first, second, third, fourth, and fifth mRNA, wherein
  • the first mRNA comprises an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12D;
  • the second mRNA comprises an open reading frame encoding a KRAS activating oncogene mutation peptide comprises G12V;
  • the third mRNA comprises an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G12C;
  • the fourth mRNA comprises an open reading frame encoding a KRAS activating oncogene mutation peptide comprising G13D;
  • the fifth mRNA comprises an open reading frame encoding a constitutively active human STING polypeptide
  • first, second, third, fourth and fifth mRNAs are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.
  • KRAS activating oncogene mutation peptides comprise the amino acid sequences set forth in SEQ ID NOs: 39-41 and 72.
  • the mRNA encoding the KRAS activating oncogene mutation peptide comprises the nucleotide sequences set forth in SEQ ID NOs: 126-128 and 132.
  • the method comprises administering an immunomodulatory therapeutic composition comprising an mRNA comprising an open reading frame encoding a concatemer of two or more KRAS activating oncogene mutation peptides.
  • the concatemer comprises G12D, G12V, G12C, and G13D.
  • the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C.
  • the concatemer comprises from N- to C-terminus G12C, G13D, G12V, and G12D.
  • the concatemer comprises an amino acid sequence selected from the group set forth in SEQ ID NOs: 42-47, 73 and 137.
  • the mRNA encoding the concatemer comprises the nucleotide sequence selected from the group set forth in SEQ ID NOs: 129-131, 133 and 138.
  • the disclosure provides a method of reducing or decreasing a size of a tumor, inhibiting a tumor growth or inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject a lipid nanoparticle comprising:
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1,
  • the lipid nanoparticle comprises
  • a second mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide, wherein the constitutively active human STING polypeptide comprises mutation V155M,
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.
  • the lipid nanoparticle comprises
  • a first mRNA comprises an open reading frame encoding a concatemer of 4 KRAS activating oncogene mutation peptides, wherein the concatemer comprises from N- to C-terminus G12D, G12V, G13D, and G12C;
  • a second mRNA comprising an open reading frame encoding a constitutively active human STING polypeptide, wherein the constitutively active human STING polypeptide comprises mutation V155M,
  • first mRNA and second mRNA are present at a KRAS:STING mass ratio selected from the group consisting of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.
  • the disclosure provides a method of reducing or decreasing a size of a tumor, inhibiting a tumor growth or inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject a lipid nanoparticle comprising:
  • the lipid nanoparticle comprises a molar ratio of about 50% Compound 25:about 10% DSPC:about 38.5% cholesterol; and about 1.5% PEG-DMG.
  • the lipid nanoparticle or composition is administered by intramuscular injection.
  • the anti-tumor response comprises a T-cell response, such as a CD8+ T cell response.
  • the disclosure provides a method of reducing or decreasing a size of a tumor, inhibiting a tumor growth or inducing an anti-tumor response in a subject in need thereof, comprising administering to the subject an immunomodulatory therapeutic composition or lipid nanoparticle of the disclosure in combination with (prior to, concurrent with or consecutively) a second composition comprising a checkpoint inhibitor polypeptide or polynucleotide encoding the same, and an optional pharmaceutically acceptable carrier.
  • the checkpoint inhibitor polypeptide inhibits PD1, PD-L1, CTLA4, or a combination thereof.
  • the checkpoint inhibitor polypeptide is an antibody.
  • the checkpoint inhibitor polypeptide is an antibody selected from an anti-CTLA4 antibody or antigen-binding fragment thereof that specifically binds CTLA4, an anti-PD1 antibody or antigen-binding fragment thereof that specifically binds PD1, an anti-PD-L1 antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
  • the checkpoint inhibitor polypeptide is an anti-PD-L1 antibody selected from atezolizumab, avelumab, or durvalumab.
  • the checkpoint inhibitor polypeptide is an anti-CTLA-4 antibody selected from tremelimumab or ipilimumab.
  • the checkpoint inhibitor polypeptide is an anti-PD1 antibody selected from nivolumab or pembrolizumab.
  • the composition comprising the checkpoint inhibitor polypeptide is administered by intravenous injection. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered once every 2 to 3 weeks. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered once every 2 weeks or once every 3 weeks. In some aspects, the composition comprising the checkpoint inhibitor polypeptide is administered prior to, concurrent with, or subsequent to administration of the lipid nanoparticle or composition.
  • the disclosure provides methods for treating subjects having a histologically confirmed KRAS mutation selected from G12D, G12V, G13D or G12C.
  • the subject has a histologically confirmed HLA subtype selected from HLA-A11 and/or HLA-C*08.
  • the tumor is metastatic colorectal cancer.
  • the tumor is non-small cell lung cancer (NSCLC).
  • the tumor is pancreatic cancer.
  • the subject is administered a chemotherapeutic agent prior to, concurrent with, or subsequent to administration of the lipid nanoparticle or composition.
  • FIG. 1 is a bar graph showing stimulation of IFN- ⁇ production in TF1a cells transfected with constitutively active STING mmRNA constructs.
  • FIG. 2 is a bar graph showing activation of an interferon-sensitive response element (ISRE) by constitutively active STING constructs.
  • STING variants 23a and 23b correspond to SEQ ID NO: 1
  • STING variant 42 corresponds to SEQ ID NO: 2
  • STING variants 19, 21a and 21b correspond to SEQ ID NO: 3
  • STING variant 41 corresponds to SEQ ID NO: 4
  • STING variant 43 corresponds to SEQ ID NO: 5
  • STING variant 45 corresponds to SEQ ID NO: 6
  • STING variant 46 corresponds to SEQ ID NO: 7
  • STING variant 47 corresponds to SEQ ID NO: 8
  • STING variant 56 corresponds to SEQ ID NO: 9
  • STING variant 57 corresponds to SEQ ID NO: 10.
  • FIGS. 3A-3B are bar graphs showing activation of an interferon-sensitive response element (ISRE) by constitutively active IRF3 constructs ( FIG. 3A ) or constitutively active IRF7 constructs ( FIG. 3B ).
  • IRF3 variants 1, 3 and 4 correspond to SEQ ID NO: 12 and IRF3 variants 2 and 5 correspond to SEQ ID NO: 11 (variants have different tags).
  • IRF7 variant 36 corresponds to SEQ ID NO: 18 and variant 31 is the murine version of SEQ ID NO: 18.
  • IRF7 variant 32 corresponds to SEQ ID NO: 17 and IRF7 variant 33 corresponds to SEQ ID NO: 14.
  • FIG. 4 is a bar graph showing activation of an NF ⁇ B-luciferase reporter gene by constitutively active cFLIP and IKK ⁇ mRNA constructs.
  • FIG. 5 is a graph showing activation of an NF ⁇ B-luciferase reporter gene by constitutively active RIPK1 mRNA constructs.
  • FIG. 6 is a bar graph showing TNF- ⁇ induction in SKOV3 cells transfected with DIABLO mmRNA constructs.
  • FIG. 7 is a bar graph showing interleukin 6 (IL-6) induction in SKOV3 cells transfected with DIABLO mmRNA constructs.
  • IL-6 interleukin 6
  • FIGS. 8A-8B are graphs showing MC38 antigen-specific responses by IFN- ⁇ intracellular staining (ICS) of day 21 ( FIG. 8A ) or day 35 ( FIG. 8B ) CD8 + spenocytes from mice immunized with MC38 neo-antigen vaccine construct (ADRvax) coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • ICS IFN- ⁇ intracellular staining
  • FIGS. 9A-9B are graphs showing the percentage of CD8b + cells among live CD45 + cells in spleen or PBMCs ( FIG. 9A ) or the percentage of CD62L lo cells among CD8b + cell in spleen or PBMCs ( FIG. 9B ) from mice immunized with MC38 neo-antigen vaccine construct (ADRvax) coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • ADRvax neo-antigen vaccine construct
  • FIG. 10 depicts NRAS and KRAS mutation frequency in colorectal cancer as identified using cBioPortal.
  • FIGS. 11A-11B are graphs showing intracellular staining (ICS) of CD8 + splenocytes from mice immunized with HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct on day 21 post first immunization.
  • FIG. 11A shows E7-specific responses for IFN- ⁇ ICS.
  • FIG. 11B shows E7-specific responses for TNF- ⁇ ICS.
  • FIGS. 12A-12B are graphs showing intracellular staining (ICS) of CD8 + splenocytes from mice immunized with HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • FIG. 12A shows E6-specific responses for IFN- ⁇ ICS.
  • FIG. 12B shows E6-specific responses for TNF- ⁇ ICS.
  • FIGS. 13A-13B are graphs showing E7-specific responses for IFN- ⁇ intracellular staining (ICS) of day 21 ( FIG. 13A ) or day 53 ( FIG. 13B ) CD8 + splenocytes from mice immunized with HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • ICS IFN- ⁇ intracellular staining
  • FIGS. 14A-14B are graphs showing the percentage of CD8b + cells among the live CD45 + cells for day 21 ( FIG. 14A ) or day 53 ( FIG. 14B ) spleen cells from mice immunized with HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • FIGS. 15A-15B are graphs showing E7-MHC1-tetramer staining of day 21 ( FIG. 15A ) or day 53 ( FIG. 15B ) CD8b + splenocytes from mice immunized with HPV E6/E7 vaccine constructs coformulated with either a STING, IRF3 or IRF7 immune potentiator mRNA construct.
  • FIGS. 16A-16D are graphs showing that the majority of E7-tetramer + CD8 + cells have an “effector memory” CD62L lo phenotype, with comparison of day 21 versus day 53 E7-tetramer + CD8 cells demonstrating that this “effector-memory” CD62L lo phenotype was maintained throughout the study.
  • FIGS. 16A (d21) and 16 B (d53) show increased % of CD8 with effector memory ‘CD62Llo phenotype.
  • FIGS. 16C and 16D show increased % of E7-tetramer+ CD8 are CD62Llo.
  • FIGS. 17A-17C are graphs showing tumor volume from mice vaccinated prophylactically as indicated with HPV E6/E7 construct together with a STING immune potentiator mRNA construct (alone or in combination with anti-CTLA-4 or anti-PD1 treatment), either prior to or at the time of challenge with a TC1 tumor that expresses HPV E7, showing inhibition of tumor growth by the HPV E6/E7+STING treatment.
  • Certain mice were treated on days ⁇ 14 and ⁇ 7 with soluble E6/E7+STING ( FIG. 17A ) or with intracellular E6/E7+STING ( FIG. 17B ), with tumor challenge on day 1.
  • Other mice were treated on days 1 and 8 with soluble E6/E7+STING ( FIG. 17C ), with tumor challenge on day 1.
  • FIGS. 18A-18I are graphs showing tumor volume from mice vaccinated therapeutically as indicated with HPV E6/E7 construct together with a STING immune potentiator mRNA construct ( FIG. 18A ), alone or in combination with anti-CTLA-4 ( FIG. 18B ) or anti-PD1 treatment ( FIG. 18C ), after challenge with a TC1 tumor that expresses HPV E7, showing inhibition of tumor growth by the HPV E6/E7+STING treatment.
  • FIGS. 18D-18I show control treatments.
  • FIG. 19 is a graph showing intracellular staining (ICS) of CD8 + splenocytes for IFN- ⁇ from mice immunized with an ADR vaccine construct coformulated with a STING immune potentiator at the indicated Ag:STING ratios on day 21 post first immunization.
  • CD8+ cells were restimulated with either the mutant ADR antigen composition (comprising three peptides) or the wild-type ADR composition (as a control).
  • FIG. 20 is a graph showing intracellular staining (ICS) of CD8 + splenocytes for TNF- ⁇ from mice immunized with an ADR vaccine construct coformulated with a STING immune potentiator at the indicated Ag:STING ratios on day 21 post first immunization.
  • CD8+ cells were restimulated with either the mutant ADR antigen composition (comprising three peptides) or the wild-type ADR composition (as a control).
  • FIGS. 21A-21C are graphs showing intracellular staining (ICS) of CD8 + splenocytes for IFN- ⁇ from mice immunized with an ADR vaccine construct coformulated with a STING immune potentiator at the indicated Ag:STING ratios on day 21 post first immunization.
  • CD8+ cells were restimulated with either a mutant or wild-type (as a control) peptide contained within the ADR antigen composition.
  • FIG. 21A shows responses to the Adpk1 peptide within the ADR composition.
  • FIG. 21B shows the response to the Reps1 peptide within the ADR composition.
  • FIG. 21C shows the response to the Dpagt1 peptide within the ADR composition.
  • FIG. 22 is a graph showing antigen-specific T cell responses to MHC class I epitopes within the CA-132 vaccine, as measured by ELISpot analysis for IFN- ⁇ , from mice treated with a coformulation of CA-132 and STING immune potentiator, at the indicated different Ag:STING ratios.
  • FIGS. 23A-23B show results for Ag:STING ratio studies from mice immunized with HPV E6/E7 vaccine construct coformulated with a STING immune potentiator mRNA construct.
  • FIG. 23A shows intracellular staining (ICS) of CD8+ splenocytes for IFN- ⁇ from mice immunized at the indicated Ag:STING ratios on day 21 post immunization.
  • FIG. 23B shows H2-Kb/E7 peptide-tetramer staining of day 21 CD8+ splenocytes from mice immunized at the indicated Ag:STING ratios.
  • FIGS. 24A-24C are bar graphs showing TNF ⁇ intracellular staining (ICS) results for CD8+ T cells from cynomolgus monkeys vaccinated with HPV vaccine+STING constructs, followed by ex vivo stimulation with either HPV16 E6 peptide pool ( FIG. 24A ), HPV16 E7 peptide pool ( FIG. 24B ) or medium (negative control) ( FIG. 24C ).
  • ICS TNF ⁇ intracellular staining
  • FIGS. 25A-25C are bar graphs showing IL-2 intracellular staining (ICS) results for CD8+ T cells from cynomolgus monkeys vaccinated with HPV vaccine+STING constructs, followed by ex vivo stimulation with either HPV16 E6 peptide pool ( FIG. 25A ), HPV16 E7 peptide pool ( FIG. 25B ) or medium (negative control) ( FIG. 25C ).
  • ICS IL-2 intracellular staining
  • FIG. 26 is a graph showing ELISA results for anti-E6 IgG in serum from cynomolgus monkeys vaccinated/immunized with HPV vaccine+STING constructs.
  • FIG. 27 is a graph showing ELISA results for anti-E7 IgG in serum from cynomolgus monkeys vaccinated/immunized with HPV vaccine+STING constructs.
  • FIG. 28 is a graph showing ELISA results for anti-E6 IgG in a two-fold dilution series of day 25 serum from cynomolgus monkeys treated with HPV vaccine+STING construct at a 1:10 STING:Ag ratio.
  • FIGS. 29A-29B are graphs showing calculated titer values of ELISA results for anti-E6 IgG ( FIG. 29A ) or anti-E7 IgG ( FIG. 29B ) in day 25 serum from cynomolgus monkeys treated with HPV vaccine+STING construct at the indicated STING:Ag ratios.
  • FIG. 30 is a graph showing the intracellular staining (ICS) results for CD8+ splenocytes for IFN ⁇ from mice immunized with mutant KRAS vaccine+STING construct followed by ex vivo stimulation with KRAS-G12V peptide.
  • FIG. 31 is a graph showing the intracellular staining (ICS) results for CD8+ splenocytes for IFN ⁇ from mice immunized with mutant KRAS vaccine+STING construct followed by ex vivo stimulation with KRAS-G12D peptide.
  • FIG. 32 is a graph showing the intracellular staining (ICS) results or CD8+ splenocytes for IFN ⁇ from mice immunized with mutant KRAS vaccine+STING construct followed by ex vivo co-culture with Cos7 cells virally transduced with HLA*A11 allele and pulsed with KRAS-G12V.
  • ICS intracellular staining
  • FIG. 33 is a graph showing the intracellular staining (ICS) results or CD8+ splenocytes for IFN-g from mice immunized with mutant KRAS vaccine+STING construct followed by ex vivo co-culture with Cos7 cells virally transduced with HLA*A11 allele and pulsed with KRAS-G12D.
  • ICS intracellular staining
  • FIG. 34 is a graph showing the intracellular staining (ICS) results or CD8+ splenocytes for IFN-g from mice immunized with an A11 viral epitope concatemer+STING construct followed by ex vivo stimulation with individual viral epitopes.
  • ICS intracellular staining
  • immunomodulatory therapeutic compositions including mRNA compositions and/or lipid nanoparticles comprising the same, comprising one or more RNAs (e.g., messenger RNAs (mRNAs)) that can safely direct the body's cellular machinery to produce a cancer protein or fragment thereof of interest, e.g., an activating oncogene mutation peptide.
  • RNAs e.g., messenger RNAs (mRNAs)
  • mRNAs messenger RNAs
  • the RNA is a modified RNA.
  • the immunomodulatory therapeutic compositions and lipid nanoparticles of the present disclosure may be used to induce a balanced immune response against cancers, comprising both cellular and humoral immunity, without risking the possibility of insertional mutagenesis, for example.
  • an immunomodulatory therapeutic composition including a lipid-based composition such as a lipid nanoparticles, comprising: one or more mRNA each having an open reading frame encoding an activating oncogene mutation peptide, and optionally one or more mRNA each having an open reading frame encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject, wherein the immune response comprises a cellular or humoral immune.
  • the disclosure provides an immunomodulatory therapeutic composition
  • an immunomodulatory therapeutic composition comprising four different activating oncogene mutation peptides (e.g., KRAS G12D, G12C, G12V and G13D), which is capable of treating patients having any one of colorectal cancer, pancreactic carcinoma, and non-small cell lung carcinoma.
  • the ability to target to four different mutations and three different cancers is a significant advantage of the compositions and methods provided herein.
  • an mRNA encoding a polypeptide that enhances an immune response to the activating oncogene mutation peptide in a subject is also referred to herein as “an immune potentiator mRNA” or “mRNA encoding an immune potentiator” or simply “immune potentiator.”
  • An enhanced immune response can be a cellular response, a humoral response or both.
  • a “cellular” immune response is intended to encompass immune responses that involve or are mediated by T cells, whereas a “humoral” immune response is intended to encompass immune responses that involve or are mediated by B cells.
  • An mRNA encoding an immune potentiator may enhance an immune response by, for example,
  • Type I interferon pathway signaling is intended to encompass activating one or more components of the Type I interferon signaling pathway (e.g., modifying phosphorylation, dimerization or the like of such components to thereby activate the pathway), stimulating transcription from an interferon-sensitive response element (ISRE) and/or stimulating production or secretion of Type I interferon (e.g., IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ and/or IFN- ⁇ ).
  • ISRE interferon-sensitive response element
  • stimulating NFkB pathway signaling is intended to encompass activating one or more components of the NFkB signaling pathway (e.g., modifying phosphorylation, dimerization or the like of such components to thereby activate the pathway), stimulating transcription from an NFkB site and/or stimulating production of a gene product whose expression is regulated by NFkB.
  • stimulating an inflammatory response is intended to encompass stimulating the production of inflammatory cytokines (including but not limited to Type I interferons, IL-6 and/or TNF ⁇ ).
  • stimulating dendritic cell development, activity or mobilization is intended to encompass directly or indirectly stimulating dendritic cell maturation, proliferation and/or functional activity.
  • compositions including mRNA compositions and/or lipid nanoparticles comprising the same, which include one or more mRNA constructs encoding a polypeptide that enhances immune responses to an activating oncogene mutation peptide (also referred to herein as “an antigen of interest”), referred to herein as immune potentiator mRNA or immune potentiator mRNAs, including chemically modified mRNAs (mmRNAs).
  • the immune potentiator mRNAs of the disclosure enhance immune responses by, for example, activating Type I interferon pathway signaling such that antigen-specific responses to an antigen of interest (i.e., activating oncogene mutation peptide(s)) are stimulated.
  • the immune potentiator mRNAs of the disclosure enhance immune responses to an exogenous antigen that is administered to the subject with the immune potentiator mRNA (e.g., an mRNA construct encoding activating oncogene mutation peptide(s) that is coformulated and coadministered with the immune potentiator mRNA or an mRNA construct encoding activating oncogene mutation peptide(s) that is formulated and administered separately from the immune potentiator mRNA).
  • an exogenous antigen that is administered to the subject with the immune potentiator mRNA
  • an immune potentiator mRNA enhances an immune response in a subject by stimulating, for example, cytokine production, T cells responses (e.g., antigen-specific CD8 + or CD4 + T cell responses) or B cell responses (e.g., antigen-specific antibody production) in the subject.
  • T cells responses e.g., antigen-specific CD8 + or CD4 + T cell responses
  • B cell responses e.g., antigen-specific antibody production
  • compositions including mRNA compositions and lipid nanoparticles, comprising one or more mRNA constructs (e.g., one or more mmRNA constructs), wherein the one or more mRNA constructs encode an activating oncogene mutation peptide(s) and, in the same or a separate mRNA construct, encode a polypeptide that enhances an immune response to the antigen of interest.
  • the disclosure provides nanoparticles, e.g., lipid nanoparticles, which include an immune potentiator mRNA that enhances an immune response, alone or in combination with mRNAs that encode activating oncogene mutation peptide(s).
  • the disclosure also provides pharmaceutical compositions comprising any of the mRNAs as described herein or nanoparticles, e.g., lipid nanoparticles comprising any of the mRNAs as described herein.
  • the disclosure provides methods for enhancing an immune response to an activating oncogene mutation peptide(s) by administering to a subject one or more mRNAs encoding activating oncogene mutation peptide(s) and a mRNA encoding a polypeptide that enhances an immune response to the peptide(s) of interest, or lipid nanoparticle thereof, or pharmaceutical composition thereof, such that an immune response to the activating oncogene mutation peptide(s) is enhanced in the subject.
  • the methods of enhancing an immune response can be used, for example, to stimulate an immunogenic response to a tumor in a subject.
  • the immune potentiators mRNAs of the disclosure are useful in combination with any type of antigen for which enhancement of an immune response is desired, including with mRNA sequences encoding at least one antigen of interest (on either the same or a separate mRNA construct) to enhance immune responses against the antigen of interest, such as a tumor antigen.
  • the immune potentiator mRNAs of the disclosure enhance, for example, mRNA vaccine responses, thereby acting as genetic adjuvants.
  • the antigen(s) of interest is a tumor antigen.
  • the tumor antigen comprises a tumor neoepitope, e.g., mutant peptide from a tumor antigen.
  • the tumor antigen is a Ras antigen.
  • a comprehensive survey of Ras mutations in cancer has been described in the art (Prior, I. A. et al. (2012) Cancer Res. 72:2457-2467). Accordingly, a Ras amino acid sequence comprising at least one mutation associated with cancer can be used as an antigen of interest.
  • the tumor antigen is a mutant KRAS antigen. Mutant KRAS antigens have been implicated in acquired resistance to certain therapeutic agents (see e.g., Misale, S. et al. (2012) Nature 486:532-536; Diaz, L. A. et al. (2012) Nature 486:537-540).
  • RNA immunomodulatory therapeutic compositions including mRNA compositions
  • the therapeutic efficacy of these RNA compositions has not yet been fully established.
  • the inventors have discovered a class of formulations for delivering mRNA immunomodulatory therapeutic compositions that results in significantly enhanced, and in many respects synergistic, immune responses including enhanced T cell responses.
  • KRAS is the most frequently mutated oncogene in human cancer ( ⁇ 15%). Such KRAS mutations are mostly conserved in a few “hotspots” and activate the oncogene.
  • the immunomodulatory therapeutic compositions of the invention include activating oncogene mutation peptides, such as KRAS mutation peptides.
  • oncogene mutation peptides such as KRAS mutation peptides.
  • Prior research has shown limited ability to raise T cells specific to the oncogenic mutation. Much of this research was done in the context of the most common HLA allele (A2, which occurs in ⁇ 50% of Caucasians). More recent work has explored the generation of specific T cells against point mutations in the context of less common HLA alleles (A11, C8). These findings have significant implications for the treatment of cancer.
  • Oncogenic mutations are common in many cancers. The ability to target these mutations and generate T cells that are sufficient to kill tumors has broad applicability to cancer therapy.
  • the invention involves, in some aspects, the surprising finding that activating oncogenic mutation antigens delivered in vivo in the form of an mRNA significantly enhances the generation of T cell effector and memory responses.
  • HLA class I molecules are highly polymorphic trans-membrane glycoproteins composed of two polypeptide chains (heavy chain and light chain). Human leukocyte antigen, the major histocompatibility complex in humans, is specific to each individual and has hereditary features.
  • the class I heavy chains are encoded by three genes: HLA-A, HLA-B and HLA-C.
  • HLA class I molecules are important for establishing an immune response by presenting endogenous antigens to T lymphocytes, which initiates a chain of immune reactions that lead to tumor cell elimination by cytotoxic T cells. Altered levels of production of HLA class I antigens is a widespread phenomenon in malignancies and is accompanied by significant inhibition of anti-tumor T cell function.
  • the generation of cancer antigens that elicit a desired immune response (e.g. T-cell responses) against targeted polypeptide sequences in immunomodulatory therapeutic development remains a challenging task.
  • the invention involves technology to overcome hurdles associated with such development. Through the use of the technology of the invention, it is possible to elicit a desired immune response by selecting appropriate activating oncogene mutation peptides and formulating the mRNA encoding peptides for effective delivery in vivo.
  • the immunomodulatory therapeutic compositions provide unique therapeutic alternatives to peptide based or DNA vaccines.
  • the mRNA containing immunomodulatory therapeutic composition When the mRNA containing immunomodulatory therapeutic composition is delivered to a cell, the mRNA will be translated into a polypeptide by the intracellular machinery which can then process the polypeptide into sensitive fragments capable of being presented on MDC and stimulating an immune response against the tumor.
  • the immunomodulatory therapeutic compositions described herein include at least one ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one cancer antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to cancer).
  • the antigenic peptide includes an activating oncogenic mutation.
  • the composition is multiple epitopes of a mixture of activating oncogenic mutations. Many activating oncogenic mutations are known in the art.
  • KRAS gene Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
  • KRAS KRAS relays external signals to the cell nucleus and contributes to regulation of cell division.
  • Activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states.
  • KRAS activation leads to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors.
  • KRAS amino acid sequence is provided below (UniProtKB P01116). KRAS mutations are common in many cancers, and G12 is the site of most common KRAS mutations.
  • N-RAS proteins are highly prevalent in certain types of cancers and are useful as cancer vaccines. For instance, 29% of Cutaneous Melanoma involves a RAS mutation, of which 94% are of N-RAS origin. This represents about 2,500 new US cases/year of the most aggressive form of melanoma accounting for the majority of melanoma deaths. (Channing Der, Are A11 RAS Proteins Created Equal in Cancer?, Sep. 22, 2014, cancer.gov). There are 30,280 news cases of multiple myeloma annually, of which 26% are NRAS*. This represents ⁇ 6,100 new NRAS* cases per year. Thus, the N-Ras vaccines described herein are useful in some embodiments in the treatment of melanoma and multiple myeloma as well as other malignancies that harbor NRAS mutations.
  • the present invention provides mRNA encoding peptide sequences resulting from certain activating mutations in one or more oncogenes, not limited to missense SNVs and often resulting in alternative splicing, for use as targets for therapeutic vaccination.
  • the activating oncogene mutation is a KRAS mutation.
  • the KRAS mutation is a G12 mutation.
  • the G12 KRAS mutation is selected from a G12D, G12V, G12S, G12C, G12A, and a G12R KRAS mutation, e.g., the G12 KRAS mutation is selected from a G12D, G12V, and a G12S KRAS mutation.
  • the G12 KRAS mutation is selected from a G12D, G12V, and a G12C KRAS mutation.
  • the KRAS mutation is a G13 mutation, e.g., the G13 KRAS mutation is a G13D KRAS mutation.
  • the activating oncogene mutation is a H-RAS or N-RAS mutation.
  • one or more mRNAs encode a mutant KRAS peptide(s) comprising an amino acid sequence having one or more mutations selected from G12D, G12V, G13D and G12C, and combinations thereof.
  • mutant KRAS antigens include those comprising one or more of the amino acid sequences shown in SEQ ID NOs: 36-41 and 72, 125.
  • CD8+ T cells specific for the G12D or G12V mutations can be restricted by HLA-A*02:01, A*03:01; -A*11:01, -B*35:01, -Cw*08:02, and potentially others.
  • a KRAS mutation is selected for inclusion in an immunomodulatory therapeutic composition for a subject having T cells that are restricted by HLA-A*02:01, A*03:01; -A*11:01, -B*35:01, or -Cw*08:02.
  • the subject has T cells that are HLA-A*02:01 restricted.
  • the mutant KRAS antigen is one or more mutant KRAS 15-mer peptides comprising a mutation selected from G12D, G12V, G13D and G12C, non-limiting examples of which are shown in SEQ ID NO: 36-38 and 125.
  • the mutant KRAS antigen is one or more mutant KRAS 25-mer peptides comprising a mutation selected from G12D, G12V, G13D and G12C, non-limiting examples of which are shown in SEQ ID NO: 39-41 and 72.
  • the mutant KRAS antigen is one or more mutant KRAS 3 ⁇ 15mer peptides (3 copies of the 15-mer peptide) comprising a mutation selected from G12D, G12V, G13D and G12C, non-limiting examples of which are shown in SEQ ID NO: 42-44 and 183.
  • the mutant KRAS antigen is one or more mutant KRAS 3 ⁇ 25mer peptides (three copies of the 25-mer peptide) comprising a mutation selected from G12D, G12V, G13D and G12C, non-limiting examples of which are shown in SEQ ID NO: 45-47 and 73.
  • the mutant KRAS antigen is a 100-mer concatemer peptide of the 25-mer peptides containing the G12D, G12V, G13D and G12C mutations (i.e., a 100-mer concatemer of SEQ ID NOs: 39, 40, 41 and 72).
  • the mutant KRAS antigen comprises an mRNA construct encoding SEQ ID NOs: 39, 40, 41 and 72.
  • Non-limiting examples of nucleotide sequences encoding a concatemer of peptides containing G12D, G12V, G13D and G12C mutations include SEQ ID NO: 138, SEQ ID NO: 167 and SEQ ID NO: 169. Further description of mutant KRAS antigens, amino acid sequences thereof, and mRNA sequences encoding therefor, are disclosed in U.S. Application Ser. No. 62/453,465, the entire contents of which is expressly incorporated herein by reference.
  • Some embodiments of the present disclosure provide immunomodulatory therapeutic compositions that include an mRNA having an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides.
  • at least two of the peptide epitopes are separated from one another by a single Glycine.
  • the concatemer comprises 3-10 activating oncogene mutation peptides.
  • all of the peptide epitopes are separated from one another by a single Glycine.
  • at least two of the peptide epitopes are linked directly to one another without a linker.
  • a tumor antigen is encoded by an mRNA construct that also comprises an immune potentiator (i.e., also encodes a polypeptide that enhances an immune response against the tumor antigen).
  • an immune potentiator i.e., also encodes a polypeptide that enhances an immune response against the tumor antigen.
  • Non-limiting examples of such constructs include the KRAS-STING constructs encoding one of the amino acid sequences shown in SEQ ID NOs: 48-71.
  • nucleotide sequences encoding the KRAS-STING constructs are shown in SEQ ID NOs: 160-163 and 221-224.
  • the disclosure provides an immunomodulatory therapeutic composition, comprising: an mRNA having an open reading frame encoding a concatemer of two or more activating oncogene mutation peptides, wherein the concatemer comprises KRAS activating oncogene mutation peptides G12D, G12V, G12C, and G13D; and one or more mRNA each having an open reading frame encoding a polypeptide that enhances an immune response to the KRAS activating oncogene mutation peptides in a subject, such as a STING immune potentiator mRNA.
  • Such an immunomodulatory composition targets somatic point mutations of KRAS, which constitute not only Meinish-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-A serine-associated neotruncal event (an early event and therefore present in many tumor cells).
  • HPV E6 and E7 are viral oncogenic proteins whose expression is vital for the transformed phenotype, like mutant KRAS. Accordingly, HPV E6 and E7 are suitable model antigens because, similar to mutant KRAS, they are oncogenic drivers.
  • each antigen contains a single missense mutation relative to its wild-type counterpart which is likely to be more challenging to recognize as “non-self” by the immune system than a viral antigen and (2) they are concatemerized.
  • the immunomodulatory therapeutic compositions of the disclosure may include one or more cancer antigens.
  • the immunomodulatory therapeutic composition is composed of 2 or more, 3 or more, 4 or more, 5 or more 6 or more 7 or more, 8 or more, 9 or more antigens, e.g., activating oncogene mutation peptides.
  • the immunomodulatory therapeutic composition is composed of 1000 or less, 900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 or less, 30 or less, 20 or less or 100 or less cancer antigens, e.g., activating oncogene mutation peptides.
  • the immunomodulatory therapeutic composition has 3-10, 3-100, 5-100, 10-100, 15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100, 60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300, 100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 cancer antigens, e.g., activating oncogene mutation peptides.
  • activating oncogene mutation peptides e.g., activating oncogene mutation peptides.
  • An epitope also known as an antigenic determinant, as used herein is a portion of an antigen that is recognized by the immune system in the appropriate context, specifically by antibodies, B cells, or T cells.
  • Epitopes include B cell epitopes and T cell epitopes.
  • B-cell epitopes are peptide sequences which are required for recognition by specific antibody producing B-cells.
  • B cell epitopes refer to a specific region of the antigen that is recognized by an antibody.
  • the portion of an antibody that binds to the epitope is called a paratope.
  • An epitope may be a conformational epitope or a linear epitope, based on the structure and interaction with the paratope.
  • a linear, or continuous, epitope is defined by the primary amino acid sequence of a particular region of a protein.
  • the sequences that interact with the antibody are situated next to each other sequentially on the protein, and the epitope can usually be mimicked by a single peptide.
  • Conformational epitopes are epitopes that are defined by the conformational structure of the native protein. These epitopes may be continuous or discontinuous, i.e. components of the epitope can be situated on disparate parts of the protein, which are brought close to each other in the folded native protein structure.
  • T-cell epitopes are peptide sequences which, in association with proteins on APC, are required for recognition by specific T-cells.
  • T cell epitopes are processed intracellularly and presented on the surface of APCs, where they are bound to MHC molecules including MHC class II and MHC class I.
  • the peptide epitope may be any length that is reasonable for an epitope. In some embodiments the peptide epitope is 9-30 amino acids.
  • the length is 9-22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24, 9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21, 10-20, 11-22, 22-21, 11-20, 12-22, 12-21, 12-20, 13-22, 13-21, 13-20, 14-19, 15-18, or 16-17 amino acids.
  • the immunomodulatory therapeutic composition may include a recall antigen, also sometimes referred to as a memory antigen.
  • a recall antigen is an antigen that has previously been encountered by an individual and for which there are pre-existent memory lymphocytes.
  • the recall antigen may be an infectious disease antigen that the individual has likely encountered such as an influenza antigen. The recall antigen helps promote a more robust immune response.
  • the therapeutic mRNA can be delivered alone or in combination with other cancer therapeutics such as checkpoint inhibitors to provide a significantly enhanced immune response against tumors.
  • the checkpoint inhibitors can enhance the effects of the mRNA encoding activating oncogenic peptides by eliminating some of the obstacles to promoting an immune response, thus allowing the activated T cells to efficiently promote an immune response against the tumor.
  • the mRNA may be delivered to the subject in the form of carrier such as a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • a number of LNPs are known in the art. For instance some LNPs such as those which have been used previously to deliver siRNA various in animal models as well as in humans have been observed to cause an undesirable inflammatory response associated with a transient IgM response, typically leading to a reduction in antigen production and a compromised immune response.
  • lipid nanoparticle-mRNA immunomodulatory therapeutic compositions are provided herein that generate T cell responses sufficient for therapeutic methods rather than promoting transient IgM responses.
  • the LNPs described herein are not liposomes.
  • a liposome as used herein is a lipid based structure having a simple lipid bilayer shell with a nucleic acid payload in the core.
  • An mRNA construct encoding an antigen(s) of interest typically comprises, in addition to the antigen-encoding sequences, other structural properties as described herein for mRNA constructs (e.g., modified nucleobases, 5′ cap, 5′ UTR, 3′ UTR, miR binding site(s), polyA tail, as described herein). Suitable mRNA construct components are as described herein.
  • the cancer antigens can be personalized cancer antigens.
  • Personalized immunomodulatory therapeutic compositions may include RNA encoding for one or more known cancer antigens specific for the tumor or cancer antigens specific for each subject, referred to as neoepitopes or subject specific epitopes or antigens.
  • a “subject specific cancer antigen” is an antigen that has been identified as being expressed in a tumor of a particular patient. The subject specific cancer antigen may or may not be typically present in tumor samples generally.
  • Neoepitopes Tumor associated antigens that are not expressed or rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced in comparison to that in cancerous cells and that induce an immune response induced upon vaccination, are referred to as neoepitopes.
  • Neoepitopes like tumor associated antigens, are completely foreign to the body and thus would not produce an immune response against healthy tissue or be masked by the protective components of the immune system.
  • personalized immunomodulatory therapeutic compositions based on neoepitopes are desirable because such vaccine formulations will maximize specificity against a patient's specific tumor.
  • Mutation-derived neoepitopes can arise from point mutations, non-synonymous mutations leading to different amino acids in the protein; read-through mutations in which a stop codon is modified or deleted, leading to translation of a longer protein with a novel tumor-specific sequence at the C-terminus; splice site mutations that lead to the inclusion of an intron in the mature mRNA and thus a unique tumor-specific protein sequence; chromosomal rearrangements that give rise to a chimeric protein with tumor-specific sequences at the junction of 2 proteins (i.e., gene fusion); frameshift mutations or deletions that lead to a new open reading frame with a novel tumor-specific protein sequence; and translocations.
  • the immunomodulatory therapeutic compositions include at least 1 cancer antigens including mutations selected from the group consisting of frame-shift mutations and recombinations or any of the other mutations described herein.
  • Methods for generating personalized immunomodulatory therapeutic compositions generally involve identification of mutations, e.g., using deep nucleic acid or protein sequencing techniques, identification of neoepitopes, e.g., using application of validated peptide-MHC binding prediction algorithms or other analytical techniques to generate a set of candidate T cell epitopes that may bind to patient HLA alleles and are based on mutations present in tumors, optional demonstration of antigen-specific T cells against selected neoepitopes or demonstration that a candidate neoepitope is bound to HLA proteins on the tumor surface and development of the vaccine.
  • the immunomodulatory therapeutic compositions of the invention may include multiple copies of a single neoepitope, multiple different neoepitopes based on a single type of mutation, i.e. point mutation, multiple different neoepitopes based on a variety of mutation types, neoepitopes and other antigens, such as tumor associated antigens or recall antigens.
  • Examples of techniques for identifying mutations include but are not limited to dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system as well as various DNA “chip” technologies i.e. Affymetrix SNP chips, and methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle amplification.
  • DASH dynamic allele-specific hybridization
  • MADGE microplate array diagonal gel electrophoresis
  • pyrosequencing oligonucleotide-specific ligation
  • TaqMan system as well as various DNA “chip” technologies i.e. Affymetrix SNP chips, and methods based on the generation of small signal molecules by invasive cleavage followed by mass spectrometry or immobilized padlock probes and rolling-circle amplification.
  • the deep nucleic acid or protein sequencing techniques are known in the art. Any type of sequence analysis method can be used. Nucleic acid sequencing may be performed on whole tumor genomes, tumor exomes (protein-encoding DNA), tumor transcriptomes, or exosomes. Real-time single molecule sequencing-by-synthesis technologies rely on the detection of fluorescent nucleotides as they are incorporated into a nascent strand of DNA that is complementary to the template being sequenced. Other rapid high throughput sequencing methods also exist. Protein sequencing may be performed on tumor proteomes. Additionally, protein mass spectrometry may be used to identify or validate the presence of mutated peptides bound to MHC proteins on tumor cells.
  • Peptides can be acid-eluted from tumor cells or from HLA molecules that are immunoprecipitated from tumor cells, and then identified using mass spectrometry.
  • the results of the sequencing may be compared with known control sets or with sequencing analysis performed on normal tissue of the patient.
  • the present invention relates to methods for identifying and/or detecting neoepitopes of an antigen, such as T-cell epitopes.
  • the invention provides methods of identifying and/or detecting tumor specific neoepitopes that are useful in inducing a tumor specific immune response in a subject.
  • these neoepitopes bind to class I HLA proteins with a greater affinity than the wild-type peptide and/or are capable of activating anti-tumor CD8 T-cells. Identical mutations in any particular gene are rarely found across tumors.
  • Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells.
  • the proteins of MHC class I are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells, and are then presented to cytotoxic T-lymphocytes (CTLs).
  • CTLs cytotoxic T-lymphocytes
  • T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules of MHC class I.
  • Each cytotoxic T-lymphocyte expresses a unique T-cell receptor which is capable of binding specific MHC/peptide complexes.
  • T-cell epitopes i.e. peptide sequences
  • MHC molecules of class I or class II in the form of a peptide-presenting complex
  • T-cell receptors of T-lymphocytes examples include for instance: Lonza Epibase, SYFPEITHI (Rammensee et al., Immunogenetics, 50 (1999), 213-219) and HLA_BIND (Parker et al., J. Immunol., 152 (1994), 163-175).
  • neoepitopes Once putative neoepitopes are selected, they can be further tested using in vitro and/or in vivo assays. Conventional in vitro lab assays, such as Elispot assays may be used with an isolate from each patient, to refine the list of neoepitopes selected based on the algorithm's predictions. Neoepitope vaccines, methods of use thereof and methods of preparing are all described in PCT/US2016/044918 which is incorporated herein by reference in its entirety.
  • the tumor antigen is an endogenous tumor antigen, such as a tumor antigen that is released upon destruction of tumor cells in situ. It has been established in the art that natural mechanisms exist that results in cell death in vivo leading to release of intracellular components such that an immune response may be stimulated against the intracellular components. Such mechanisms are referred to herein as immunogenic cell death and include necroptosis and pyroptosis. Accordingly, in one embodiment, an immune potentiator mRNA construct of the disclosure is administered to a tumor-bearing subject under conditions in which endogenous immunogenic cell death is occurring such that one or more endogenous tumor antigens are released, to thereby enhance an immune response against the tumor antigens.
  • the immune potentiator mRNA construct is administered to a tumor-bearing subject together with a second mRNA construct encoding an “executioner mRNA construct”, which stimulates immunogenic cell death of tumor cells in the subject.
  • executioner mRNA constructs include those encoding MLKL, RIPK3, RIPK1, DIABLO, FADD, GSDMD, caspase-4, caspase-5, caspase-11, Pyrin, NLRP3 and ASC/PYCARD.
  • Executioner mRNA constructs, and their use in combination with an immune potentiator mRNA construct are described in further detail in U.S. Application Ser. No. 62/412,933, the entire contents of which is expressly incorporated herein by reference.
  • the activating oncogene mutation peptides selected for inclusion in the immunomodulatory therapeutic composition typically will be high affinity binding peptides.
  • the activating oncogene mutation peptide binds an HLA protein with greater affinity than a wild-type peptide.
  • the activating oncogene mutation peptides has an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less in some embodiments.
  • peptides with predicted IC50 ⁇ 50 nM are generally considered medium to high affinity binding peptides and will be selected for testing their affinity empirically using biochemical assays of HLA-binding.
  • subject specific activating oncogene mutation peptides may be identified in a sample of a patient.
  • the sample may be a tissue sample or a tumor sample.
  • a sample of one or more tumor cells may be examined for the presence of subject specific activating oncogene mutations.
  • the tumor sample may be examined using whole genome, exome or transcriptome analysis in order to identify the subject specific activating oncogene mutations.
  • the subject specific activating oncogene mutation peptides may be identified in an exosome of the subject.
  • the activating oncogene mutation peptides are identified in an exosome of the subject, such peptides are said to be representative of exosome peptides of the subject.
  • Exosomes are small microvesicles shed by cells, typically having a diameter of approximately 30-100 nm. Exosomes are classically formed from the inward invagination and pinching off of the late endosomal membrane, resulting in the formation of a multivesicular body (MVB) laden with small lipid bilayer vesicles, each of which contains a sample of the parent cell's cytoplasm. Fusion of the MVB with the cell membrane results in the release of these exosomes from the cell, and their delivery into the blood, urine, cerebrospinal fluid, or other bodily fluids. Exosomes can be recovered from any of these biological fluids for further analysis.
  • MVB multivesicular body
  • Nucleic acids within exosomes have a role as biomarkers for tumor antigens.
  • An advantage of analyzing exosomes in order to identify subject specific cancer antigens, is that the method circumvents the need for biopsies. This can be particularly advantageous when the patient needs to have several rounds of therapy including identification of cancer antigens, and vaccination.
  • a number of methods of isolating exosomes from a biological sample have been described in the art. For example, the following methods can be used: differential centrifugation, low speed centrifugation, anion exchange and/or gel permeation chromatography, sucrose density gradients or organelle electrophoresis, magnetic activated cell sorting (MACS), nanomembrane ultrafiltration concentration, Percoll gradient isolation and using microfluidic devices. Exemplary methods are described in US Patent Publication No. 2014/0212871 for instance.
  • One aspect of the disclosure pertains to mRNAs that encode a polypeptide that stimulates or enhances an immune response against one or more antigens of interest (activating oncogene mutation peptide(s)).
  • mRNAs that enhance immune responses to an antigen(s) of interest are referred to herein as immune potentiator mRNA constructs or immune potentiator mRNAs, including chemically modified mRNAs (mmRNAs).
  • the disclosure provides an mRNA encoding a polypeptide that stimulates or enhances an immune response in a subject in need thereof (e.g., potentiates an immune response in the subject) by, for example, inducing adaptive immunity (e.g., by stimulating Type I interferon production), stimulating an inflammatory response, stimulating NFkB signaling and/or stimulating dendritic cell (DC) development, activity or mobilization in the subject.
  • administration of an immune potentiator mRNA to a subject in need thereof enhances cellular immunity (e.g., T cell-mediated immunity), humoral immunity (e.g., B cell-mediated immunity) or both cellular and humoral immunity in the subject.
  • administering stimulates cytokine production (e.g., inflammatory cytokine production), stimulates antigen-specific CD8 + effector cell responses, stimulates antigen-specific CD4 + helper cell responses, increases the effector memory CD62L lo T cell population, stimulates B cell activity or stimulates antigen-specific antibody production, including combinations of the foregoing responses.
  • cytokine production e.g., inflammatory cytokine production
  • stimulates antigen-specific CD8 + effector cell responses stimulates antigen-specific CD4 + helper cell responses
  • increases the effector memory CD62L lo T cell population stimulates B cell activity or stimulates antigen-specific antibody production, including combinations of the foregoing responses.
  • administration of an immune potentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD8 + effector cell responses.
  • administration of an immune potentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production), and stimulates antigen-specific CD4 + helper cell responses.
  • administration of an immune potentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production), and increases the effector memory CD62L lo T cell population.
  • administration of an immune potentiator mRNA stimulates cytokine production (e.g., inflammatory cytokine production), and stimulates B cell activity or stimulates antigen-specific antibody production.
  • the disclosure provides an immune potentiator mRNA encoding a polypeptide that stimulates or enhances an immune response against an antigen of interest by simulating or enhancing Type I interferon pathway signaling, thereby stimulating or enhancing Type I interferon (IFN) production.
  • IFN Type I interferon
  • host cell DNA for example derived from damaged or dying cells
  • Type I IFN signaling pathway plays a role in the development of adaptive anti-tumor immunity.
  • many pathogens and cancer cells have evolved mechanisms to reduce or inhibit Type I interferon responses.
  • activation (including stimulation and/or enhancement) of the Type I IFN signaling pathway in a subject in need thereof by providing an immune potentiator mRNA of the disclosure to the subject, stimulates or enhances an immune response in the subject in a wide variety of clinical situations, including treatment of cancer and pathogenic infections, as well as in potentiating vaccine responses to provide protective immunity.
  • Type I interferons are pro-inflammatory cytokines that are rapidly produced in multiple different cell types, typically upon viral infection, and known to have a wide variety of effects.
  • the canonical consequences of type I IFN production in vivo is the activation of antimicrobial cellular programs and the development of innate and adaptive immune responses.
  • Type I IFN induces a cell-intrinsic antimicrobial state in infected and neighboring cells that limits the spread of infectious agents, particularly viral pathogens.
  • Type I IFN also modulates innate immune cell activation (e.g., maturation of dendritic cells) to promote antigen presentation and nature killer cell functions.
  • Type I IFN also promotes the development of high-affinity antigen-specific T and B cell responses and immunological memory (Ivashkiv and Donlin (2014) Nat Rev Immunol 14(1):36-49).
  • Type I IFN activates dendritic cells (DCs) and promotes their T cell stimulatory capacity through autocrine signaling (Montoya et al., (2002) Blood 99:3263-3271).
  • Type I IFN exposure facilitates maturation of DCs via increasing the expression of chemokine receptors and adhesion molecules (e.g., to promote DC migration into draining lymph nodes), co-stimulatory molecules, and MHC class I and class II antigen presentation.
  • DCs that mature following type I IFN exposure can effectively prime protective T cell responses (Wijesundara et al., (2014) Front Immunol 29(412) and references therein).
  • Type I IFN can either promote or inhibit T cell activation, proliferation, differentiation and survival depending largely on the timing of type I IFN signaling relative to T cell receptor signaling (Crouse et al., (2015) Nat Rev Immunol 15:231-242).
  • MHC-I expression is upregulated in response to type I IFN in multiple cell types (Lindahl et al., (1976), J Infect Dis 133(Suppl):A66-A68; Lindahl et al., (1976) Proc Natl Acad Sci USA 17:1284-1287) which is a requirement for optimal T cell stimulation, differentiation, expansion and cytolytic activity.
  • Type I IFN can exert potent co-stimulatory effects on CD8 T cells, enhancing CD8 T cell proliferation and differentiation (Curtsinger et al., (2005) J Immunol 174:4465-4469; Kolumam et al., (2005) J Exp Med 202:637-650).
  • type I IFN signaling has both positive and negative effects on B cell responses depending on the timing and context of exposure (Braun et al., (2002) Int Immunol 14(4):411-419; Lin et al, (1998) 187(1):79-87).
  • the survival and maturation of immature B cells can be inhibited by type I IFN signaling.
  • type I IFN exposure has been shown to promote B cell activation, antibody production and isotype switch following viral infection or following experimental immunization (Le Bon et al., (2006) J Immunol 176:4:2074-2078; Swanson et al., (2010) J Exp Med 207:1485-1500).
  • Type I IFN pathway signaling A number of components involved in Type I IFN pathway signaling have been established, including STING, Interferon Regulatory Factors, such as IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9, TBK1, IKKi, MyD88 and TRAM. Additional components involved in Type I IFN pathway signaling include TRAF3, TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI and IFI16.
  • STING Interferon Regulatory Factors
  • IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9 TBK1, IKKi, MyD88 and TRAM.
  • Additional components involved in Type IFN pathway signaling include TRAF3, TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI
  • an immune potentiator mRNA encodes any of the foregoing components involved in Type I IFN pathway signaling.
  • STING STimulator of INterferon Genes; also known as transmembrane protein 173 (TMEM173), mediator of IRF3 activation (MITA), methionine-proline-tyrosine-serine (MPYS), and ER IFN stimulator (ERIS)
  • TMEM173 transmembrane protein 173
  • MIAA mediator of IRF3 activation
  • MPYS methionine-proline-tyrosine-serine
  • ERIS ER IFN stimulator
  • ER endoplasmic reticulum
  • STING functions as a signaling adaptor linking the cytosolic detection of DNA to the TBK1/IRF3/Type I IFN signaling axis.
  • the signaling adaptor functions of STING are activated through the direct sensing of cyclic dinucleotides (CDNs).
  • CDNs include cyclic di-GMP (guanosine 5′-monophosphate), cyclic di-AMP (adenosine 5′-monophosphate) and cyclic GMP-AMP (cGAMP).
  • CDNs are now known to constitute a class of pathogen-associated molecular pattern molecules (PAMPs) that activate the TBK1/IRF3/type I IFN signaling axis via direct interaction with STING.
  • PAMPs pathogen-associated molecular pattern molecules
  • STING is capable of sensing aberrant DNA species and/or CDNs in the cytosol of the cell, including CDNs derived from bacteria, and/or from the host protein cyclic GMP-AMP synthase (cGAS).
  • cGAS host protein cyclic GMP-AMP synthase
  • the cGAS protein is a DNA sensor that produces cGAMP in response to detection of DNA in the cytosol (Burdette et al., (2011) Nature 478:515-518; Sun et al., (2013) Science 339:786-791; Diner et al., (2013) Cell Rep 3:1355-1361; Ablasser et al., (2013) Nature 498:380-384).
  • TANK-binding kinase 1 (TBK1) (Ouyang et al., (2012) Immunity 36(6):1073-1086).
  • TNK1 TANK-binding kinase 1
  • This complex translocates to the perinuclear Golgi, resulting in delivery of TBK1 to endolysosomal compartments where it phosphorylates IRF3 and NF- ⁇ B transcription factors (Zhong et al., (2008) Immunity 29:538-550).
  • Mutant STING proteins resulting from polymorphisms mapped to the human TMEM173 gene have been described exhibiting a gain-of function or constitutively active phenotype. When expressed in vitro, mutant STING alleles were shown to potently stimulate induction of type I IFN (Liu et al., (2014) N Engl J Med 371:507-518; Jeremiah et al., (2014) J Clin Invest 124:5516-5520; Dobbs et al., (2015) Cell Host Microbe 18(2):157-168; Tang & Wang, (2015) PLoS ONE 10(3):e0120090; Melki et al., (2017) J Allergy Clin Immunol In Press; Konig et al., (2017) Ann Rheum Dis 76(2):468-472; Burdette et al. (2011) Nature 478:515-518).
  • mRNAs e.g., mmRNAs
  • mmRNAs encoding constitutively active forms of STING, including mutant human STING isoforms for use as immune potentiators as described herein.
  • mmRNAs encoding constitutively active forms of STING, including mutant human STING isoforms are set forth in the Sequence Listing herein.
  • the amino acid residue numbering for mutant human STING polypeptides used herein corresponds to that used for the 379 amino acid residue wild type human STING (isoform 1) available in the art as Genbank Accession Number NP_938023.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 155, in particular an amino acid substitution, such as a V155M mutation.
  • the mRNA e.g., mmRNAs
  • the STING V155M mutant is encoded by a nucleotide sequence shown in SEQ ID NO: 139, SEQ ID NO: 168 or SEQ ID NO: 170.
  • the mRNA (e.g., mmRNAs) comprises a 3′ UTR sequence as shown in SEQ ID NO: 149, which includes an miR122 binding site.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 284, such as an amino acid substitution.
  • residue 284 substitutions include R284T, R284M and R284K.
  • the mutant human STING protein has as a R284T mutation, for example has the amino acid sequence set forth in SEQ ID NO: 2 or is encoded by an the nucleotide sequence shown in SEQ ID NO: 140 or 201.
  • the mutant human STING protein has a R284M mutation, for example has the amino acid sequence as set forth in SEQ ID NO: 3 or is encoded by the nucleotide sequence shown in SEQ ID NO: 141 or 202.
  • the mutant human STING protein has a R284K mutation, for example has the amino acid sequence as set forth in SEQ ID NO: 4 or 164, or is encoded by the nucleotide sequence shown in SEQ ID NO: 142, 165, 203 or 225.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 154, such as an amino acid substitution, such as a N154S mutation.
  • the mutant human STING protein has a N154S mutation, for example has the amino acid sequence as set forth in SEQ ID NO: 5 or is encoded by the nucleotide sequence shown in SEQ ID NO: 143 or 204.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 147, such as an amino acid substitution, such as a V147L mutation.
  • the mutant human STING protein having a V147L mutation has the amino acid sequence as set forth in SEQ ID NO: 6 or is encoded by the nucleotide sequence shown in SEQ ID NO: 144 or 205.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 315, such as an amino acid substitution, such as a E315Q mutation.
  • the mutant human STING protein having a E315Q mutation has the amino acid sequence as set forth in SEQ ID NO: 7 or is encoded by the nucleotide sequence shown in SEQ ID NO: 145 or 206.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a mutation at amino acid residue 375, such as an amino acid substitution, such as a R375A mutation.
  • the mutant human STING protein having a R375A mutation has the amino acid sequence as set forth in SEQ ID NO: 8 or is encoded by the nucleotide sequence shown in SEQ ID NO: 146 or 207.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a one or more or a combination of two, three, four or more of the foregoing mutations. Accordingly, in one aspect the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having one or more mutations selected from the group consisting of: V147L, N154S, V155M, R284T, R284M, R284K, E315Q and R375A, and combinations thereof.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a combination of mutations selected from the group consisting of: V155M and R284T; V155M and R284M; V155M and R284K; V155M and V147L; V155M and N154S; V155M and E315Q; and V155M and R375A.
  • a mRNA e.g., mmRNA
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a V155M and one, two, three or more of the following mutations: R284T; R284M; R284K; V147L; N154S; E315Q; and R375A.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L and N154S mutations.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S mutations, and, optionally, a mutation at amino acid 284.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S mutations, and a mutation at amino acid 284 selected from R284T, R284M and R284K.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S, and R284T mutations.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S, and R284M mutations.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having V155M, V147L, N154S, and R284K mutations.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein having a combination of mutations at amino acid residue 147, 154, 155 and, optionally, 284, in particular amino acid substitutions, such as a V147L, N154S, V155M and, optionally, R284M.
  • the mutant human STING protein has V147N, N154S and V155M mutations, such as the amino acid sequence as set forth in SEQ ID NO: 9 or encoded by the nucleotide sequence shown in SEQ ID NO: 147.
  • the mutant human STING protein has R284M, V147N, N154S and V155M mutations, such as the amino acid sequence as set forth in SEQ ID NO: 10 or encoded by the nucleotide sequence shown in SEQ ID NO: 148 or 209.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human STING protein that is a constitutively active truncated form of the full-length 379 amino acid wild type protein, such as a constitutively active human STING polypeptide consisting of amino acids 137-379.
  • a mRNA e.g., mmRNA
  • a mutant human STING protein that is a constitutively active truncated form of the full-length 379 amino acid wild type protein, such as a constitutively active human STING polypeptide consisting of amino acids 137-379.
  • the present disclosure provides mRNA (including mmRNA) encoding Interferon Regulatory Factors, such as IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9 as immune potentiators.
  • IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9 Interferon Regulatory Factors
  • the IRF transcription factor family is involved in the regulation of gene expression leading to the production of type I interferons (IFNs) during innate immune responses.
  • IFNs type I interferons
  • IFNs type I interferons
  • DBDs N-terminal binding domains
  • IRF1, IRF3, IRF5, and IRF7 have been specifically implicated as positive regulators of type I IFN gene transcription (Honda et al., (2006) Immunity 25(3):349-360).
  • IRF1 was the first family member discovered to activate type I IFN gene promoters (Miyamoto et al., (1988) Cell 54:903-913). Although studies show that IRF1 participates in type I IFN gene expression, normal induction of type IFN was observed in virus-infected IRF1 ⁇ / ⁇ murine fibroblasts, suggesting dispensability (Matsuyama et al., (1993) Cell 75:83-97).
  • IRF5 was also shown to be dispensable for type I IFN induction by viruses or TLR agonists (Takaoka et al., (2005) Nature 434:243-249).
  • the disclosure provides mRNA encoding constitutively active forms of human IRF1, IRF3, IRF5, IRF7, IRF8, and IRF9 as immune potentiators.
  • the disclosure provides mRNA encoding constitutively active forms of human IRF3 and/or IRF7.
  • IRF-3 plays a critical role in the early induction of type I IFNs.
  • the IRF3 transcription factor is constitutively expressed and shuttles between the nucleus and cytoplasm of cells in latent form, with a predominantly cytosolic localization prior to phosphorylation (Hiscott (2007) J Biol Chem 282(21):15325-15329; Kumar et al., (2000) Mol Cell Biol 20(11):4159-4168).
  • IRF3 Upon phosphorylation of serine residues at the C-terminus by TBK-1 (TANK binding kinase 1; also known as T2K and NAK) and/or IKK ⁇ (inducible I ⁇ B kinase; also known as IKKi), IRF3 translocates from the cytoplasm into the nucleus (Fitzgerald et al., (2003) Nat Immuno 4(5):491-496; Sharma et al., (2003) Science 300:1148-1151; Hemmi et al., (2004) J Exp Med 199:1641-1650). The transcriptional activity of IRF3 is mediated by these phosphorylation and translocation events.
  • TBK-1 TANK binding kinase 1
  • IKK ⁇ inducible I ⁇ B kinase
  • a model for IRF3 activation proposes that C-terminal phosphorylation induces a conformational change in IRF3 that promotes homo- and/or heterodimerization (e.g. with IRF7; see Honda et al., (2006) Immunity 25(3):346-360), nuclear localization, and association with the transcriptional co-activators CBP and/or p300 (Lin et al., (1999) Mol Cell Biol 19(4):2465-2474).
  • IRF3 While inactive IRF3 constitutively shuttles into and out of the nucleus, phosphorylated IRF3 proteins remain associated with CBP and/or p300, are retained in the nucleus, and induce transcription of IFN and other genes (Kumar et al., (2000) Mol Cell Biol 20(11):4159-4168).
  • IRF7 In contrast to IRF3, IRF7 exhibits a low expression level in most cells, but is strongly induced by type I IFN-mediated signaling, supporting the notion that IRF3 is primarily responsible for the early induction of IFN genes and that IRF7 is involved in the late induction phase (Sato et al., (2000) Immunity 13(4):539-548).
  • Ligand-binding to the type I IFN receptor results in the activation of a heterotrimeric transcriptional activator, termed IFN-stimulated gene factor 3 (ISGF3), which consists of IRF9, STAT1, and STAT2, and is responsible for the induction of the IRF7 gene (Marie et al., (1998) EMBO J 17(22):6660-6669).
  • IGF3 IFN-stimulated gene factor 3
  • IRF7 can partition between cytoplasm and nucleus after serine phosphorylation of its C-terminal region, allowing its dimerization and nuclear translocation. IRF7 forms a homodimer or a heterodimer with IRF3, and each of these different dimers differentially acts on the type I IFN gene family members. IRF3 is more potent in activating the IFN- ⁇ gene than the IFN- ⁇ genes, whereas IRF7 efficiently activates both IFN- ⁇ and IFN- ⁇ genes (Marie et al., (1998) EMBO J 17(22):6660-6669).
  • mRNAs e.g., mmRNAs
  • mRNAs encoding constitutively active forms of IRF3 and IRF7 including mutant human IRF3 and mutant human IRF7 isoforms for use as immune potentiators as described herein.
  • mRNAs e.g., mmRNAs
  • constitutively active forms of IRF3 and IRF7, including mutant human IRF3 and IRF7 isoforms are set forth in the Sequence Listing herein.
  • the amino acid residue numbering for mutant human IRF3 polypeptides used herein corresponds to that used for the 427 amino acid residue wild type human IRF3 (isoform 1) available in the art as Genbank Accession Number NP_001562.
  • the amino acid residue numbering for mutant human IRF7 polypeptides used herein corresponds to that used for the 503 amino acid residue wild type human IRF7 (isoform a) available in the art as Genbank Accession Number NP_001563.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human IRF3 protein that is constitutively active, e.g., having a mutation at amino acid residue 396, such as an amino acid substitution, such as a S396D mutation, for example as set forth in the amino acid sequence of SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 151 or 212.
  • a mRNA e.g., mmRNA
  • a mutant human IRF3 protein that is constitutively active e.g., having a mutation at amino acid residue 396, such as an amino acid substitution, such as a S396D mutation, for example as set forth in the amino acid sequence of SEQ ID NO: 12 or encoded by the nucleotide sequence shown in SEQ ID NO: 151 or 212.
  • the mRNA (e.g., mmRNA) construct encodes a constitutively active mouse IRF3 polypeptide comprising an S396D mutation, for example as set forth in the amino acid sequence of SEQ ID NO: 11 or encoded by the nucleotide sequence shown in SEQ ID NO: 150 or 211.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human IRF7 protein that is constitutively active.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IR7 protein comprising one or more point mutations (amino acid substitutions compared to wild-type).
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IR7 protein comprising a truncated form of the protein (amino acid deletions compared to wild-type).
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IR7 protein comprising a truncated form of the protein that also includes one or more point mutations (a combination of amino acid deletions and amino acid substitutions compared to wild-type).
  • a mRNA e.g., mmRNA
  • a constitutively active IR7 protein comprising a truncated form of the protein that also includes one or more point mutations (a combination of amino acid deletions and amino acid substitutions compared to wild-type).
  • the wild-type amino acid sequence of human IRF7 (isoform a) is set forth in SEQ ID NO: 13, encoded by the nucleotide sequence shown in SEQ ID NO: 152 or 213.
  • a series of constitutively active forms of human IRF7 were prepared comprising point mutations, deletions, or both, as compared to the wild-type sequence.
  • the disclosure provides an immune potentiator mRNA construct encoding a constitutively active IRF7 polypeptide comprising one or more of the following mutations: S475D, S476D, S477D, S479D, L480D, S483D and S487D, and combinations thereof.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IRF7 polypeptide comprising mutations S477D and S479D, as set forth in the amino acid sequence of SEQ ID NO: 14, encoded by the nucleotide sequence shown in SEQ ID NO: 153 or 214.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IRF7 polypeptide comprising mutations S475D, S477D and L480D, as set forth in the amino acid sequence of SEQ ID NO: 15, encoded by the nucleotide sequence shown in SEQ ID NO: 154 or 215.
  • the disclosure provides a mRNA (e.g., mmRNAs) encoding a constitutively active IRF7 polypeptide comprising mutations S475D, S476D, S477D, S479D, S483D and S487D, as set forth in the amino acid sequence of SEQ ID NO: 16, encoded by the nucleotide sequence shown in SEQ ID NO: 155 or 216.
  • a mRNA e.g., mmRNAs
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IRF7 polypeptide comprising a deletion of amino acid residues 247-467 (i.e., comprising amino acid residues 1-246 and 468-503), as set forth in the amino acid sequence of SEQ ID NO: 17, encoded by the nucleotide sequence shown in SEQ ID NO: 156 or 217.
  • a mRNA e.g., mmRNA
  • a constitutively active IRF7 polypeptide comprising a deletion of amino acid residues 247-467 (i.e., comprising amino acid residues 1-246 and 468-503), as set forth in the amino acid sequence of SEQ ID NO: 17, encoded by the nucleotide sequence shown in SEQ ID NO: 156 or 217.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active IRF7 polypeptide comprising a deletion of amino acid residues 247-467 (i.e., comprising amino acid residues 1-246 and 468-503) and further comprising mutations S475D, S476D, S477D, S479D, S483D and S487D, as set forth in the amino acid sequence of SEQ ID NO: 18, encoded by the nucleotide sequence shown in SEQ ID NO: 157 or 218.
  • a mRNA e.g., mmRNA
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a truncated IRF7 inactive “null” polypeptide construct comprising a deletion of residues 152-246 (i.e., comprising amino acid residues 1-151 and 247-503), as set forth in the amino acid sequence of SEQ ID NO: 19, encoded by the nucleotide sequence shown in SEQ ID NO: 158 or 219 (used, for example, for control purposes).
  • a mRNA e.g., mmRNA
  • a truncated IRF7 inactive “null” polypeptide construct comprising a deletion of residues 152-246 (i.e., comprising amino acid residues 1-151 and 247-503), as set forth in the amino acid sequence of SEQ ID NO: 19, encoded by the nucleotide sequence shown in SEQ ID NO: 158 or 219 (used, for example, for control purposes).
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a truncated IRF7 inactive “null” polypeptide construct comprising a deletion of residues 1-151 (i.e., comprising amino acid residues 152-503), as set forth in the amino acid sequence of SEQ ID NO: 20, encoded by the nucleotide sequence shown in SEQ ID NO: 159 or 220 (used, for example, for control purposes).
  • a mRNA e.g., mmRNA
  • a truncated IRF7 inactive “null” polypeptide construct comprising a deletion of residues 1-151 (i.e., comprising amino acid residues 152-503), as set forth in the amino acid sequence of SEQ ID NO: 20, encoded by the nucleotide sequence shown in SEQ ID NO: 159 or 220 (used, for example, for control purposes).
  • the disclosure provides mRNA constructs encoding additional components of the Type I IFN signaling pathway that can be use as immune potentiators to enhance immune responses through activation of the Type I IFN signaling pathway.
  • the immune potentiator mRNA construct encodes a MyD88 protein.
  • MyD88 is known in the art to signal upstream of IRF7.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a constitutively active MyD88 protein, such as mutant MyD88 protein having one or more point mutations.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mutant human or mouse MyD88 protein having a L265P substitutions, as set forth in SEQ ID NOs: 75 and 76, respectively.
  • an immune potentiator mRNA construct encodes a TRAM (TICAM2) protein.
  • TRAM is known in the art to signal upstream of IRF3.
  • the disclosure encompasses a mRNA (e.g., mmRNA) encoding a constitutively active TRAM protein, such as mutant TRAM protein having one or more point mutations.
  • the disclosure encompasses a wild-type TRAM protein that is overexpressed.
  • the disclosure provides a mRNA (e.g., mmRNA) encoding a mouse TRAM protein as shown in SEQ ID NO: 77.
  • the disclosure provides an immune potentiator mRNA construct encoding a TANK-binding kinase 1 (TBK1) or an inducible I ⁇ B kinase (IKKi, also known as IKK ⁇ ), including constitutively active forms of TBK1 or IKKi, as immune potentiators.
  • TBK1 and IKKi have been demonstrated to be components of the virus-activated kinase that phosphorylates IRF3 and IRF7, thus acting upstream from IRF3 and IRF7 in the Type I IFN signaling pathway (Sharma, S. et al. (2003) Science 300:1148-1151).
  • TBK1 and IKKi are involved in the phosphorylation and activation of transcription factors (e.g. IRF3/7 & NF- ⁇ B) that induce expression of type I IFN genes as well as IFN-inducible genes (Fitzgerald, K. A. et al., (2003) Nat Immunol 4(5):491-496).
  • transcription factors e.g. IRF3/7 & NF- ⁇ B
  • the disclosure provides an immune potentiator mRNA construct that encodes a TBK1 protein, including a constitutively active form of TBK1, including mutant human TBK1 isoforms.
  • an immune potentiator mRNA construct encodes a IKKi protein, including a constitutively active form of IKKi, including mutant human IKKi isoforms.
  • the disclosure provides immune potentiator mRNA constructs that enhance an immune response by stimulating an inflammatory response.
  • agents that stimulate an inflammatory response include STAT1, STAT2, STAT4 and STAT6.
  • the disclosure provides an immune potentiator mRNA construct encoding one or a combination of these inflammation-inducing proteins, including a constitutively active form.
  • mRNAs e.g., mmRNAs
  • mRNAs e.g., mmRNAs
  • mutant human STAT6 isoforms for use as immune potentiators as described herein.
  • mRNAs e.g., mmRNAs
  • encoding constitutively active forms of STAT6, including mutant human STAT6 isoforms are set forth in the Sequence Listing herein.
  • the amino acid residue numbering for mutant human STAT6 polypeptides used herein corresponds to that used for the 847 amino acid residue wild type human STAT6 (isoform 1) available in the art as Genbank Accession Number NP_001171550.1.
  • the disclosure provides a mRNA construct encoding a constitutively active human STAT6 construct comprising one or more amino acid mutations selected from the group consisting of S407D, V547A, T548A, Y641F, and combinations thereof.
  • the mRNA construct encodes a constitutively active human STAT6 construct comprising V547A and T548A mutations, such as the sequence shown in SEQ ID NO: 78.
  • the mRNA construct encodes a constitutively active human STAT6 construct comprising a S407D mutation, such as the sequence shown in SEQ ID NO: 79.
  • the mRNA construct encodes a constitutively active human STAT6 construct comprising S407D, V547A and T548A mutations, such as the sequence shown in SEQ ID NO: 80.
  • the mRNA construct encodes a constitutively active human STAT6 construct comprising V547A, T548A and Y641F mutations, such as the sequence shown in SEQ ID NO: 81.
  • the disclosure provides immune potentiator mRNA constructs that enhance an immune response by stimulating an NFkB signaling, which is known to be involved in stimulation of immune responses.
  • proteins that stimulate NFkB signaling include c-FLIP, IKK ⁇ , RIPK1, Btk and TAK-TAB1.
  • an immune potentiator mRNA construct of the present disclosure can encode any of these NFkB pathway-inducing proteins, for example in a constitutively active form.
  • the disclosure provides an immune potentiator mRNA construct that activates NF ⁇ B signaling encodes a c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) protein (also known in the art as CASP8 and FADD-like apoptosis regulator), including a constitutively active c-FLIP.
  • c-FLIP cellular caspase 8 (FLICE)-like inhibitory protein) protein
  • CASP8 and FADD-like apoptosis regulator also known in the art as CASP8 and FADD-like apoptosis regulator
  • mutant human c-FLIP isoforms
  • the amino acid residue numbering for mutant human c-FLIP polypeptides used herein corresponds to that used for the 480 amino acid residue wild type human c-FLIP (isoform 1) available in the art as Genbank Accession Number NP_003870.
  • the mRNA encodes a c-FLIP long (L) isoform comprising two DED domains, a p20 domain and a p12 domain, such as having the sequence shown in SEQ ID NO: 82.
  • the mRNA encodes a c-FLIP short (S) isoform, encoding amino acids 1-227, comprising two DED domains, such as having the sequence shown in SEQ ID NO: 83.
  • the mRNA encodes a c-FLIP p22 cleavage product, encoding amino acids 1-198, such as having the sequence shown in SEQ ID NO: 84.
  • the mRNA encodes a c-FLIP p43 cleavage product, encoding amino acids 1-376, such as having the sequence shown in SEQ ID NO: 85.
  • the mRNA encodes a c-FLIP p12 cleavage product, encoding amino acids 377-480, such as having the sequence shown in SEQ ID NO: 86.
  • an immune potentiator mRNA construct that activates NF ⁇ B signaling encodes a constitutively active IKK ⁇ mRNA construct or a constitutively active IKK ⁇ mRNA construct.
  • the constitutively active human IKK ⁇ polypeptide comprises S177E and S181E mutations, such as the sequence shown in SEQ ID NO: 87.
  • the constitutively active human IKK ⁇ polypeptide comprises S177A and S181A mutations, such as the sequence shown in SEQ ID NO: 88.
  • the mRNA construct encodes a constitutively active mouse IKK ⁇ polypeptide.
  • the constitutively active mouse IKK ⁇ polypeptide comprises S177E and S181E mutations, such as the sequence shown in SEQ ID NO: 148.
  • the constitutively active mouse IKK ⁇ polypeptide comprises S177A and S181A mutations, such as the sequence shown in SEQ ID NO: 89.
  • the mRNA construct encodes a constitutively active human or mouse IKK ⁇ polypeptide comprising a PEST mutation, such as having a sequence as shown in SEQ ID NOs: 91-92 (human) or 95-96 (mouse).
  • the mRNA construct encodes a constitutively active human or mouse IKK ⁇ polypeptide comprising a PEST mutation, such as having the sequence shown in SEQ ID NOs: 93-94 (human) or 97-98 (mouse).
  • the disclosure provides an immune potentiator mRNA construct that activates NF ⁇ B signaling encoding a receptor-interacting protein kinase 1 (RIPK1) protein.
  • RIPK1 receptor-interacting protein kinase 1
  • the mRNA construct encodes RIPK1 amino acids 1-555 of a human or mouse RIPK1 polypeptide as well as an IZ domain, such as having the sequence shown in SEQ ID N: 99 (human) or 102 (mouse). In one embodiment, the mRNA construct encodes RIPK1 amino acids 1-555 of a human or mouse RIPK1 polypeptide as well as EE and DM domains, such as having the sequence shown in SEQ ID NO: 100 (human) or 103 (mouse).
  • the mRNA construct encodes RIPK1 amino acids 1-555 of a human or mouse RIPK1 polypeptide as well as RR and DM domains, such as having the sequence shown in SEQ ID NO: 101 (human) or 104 (mouse).
  • an immune potentiator mRNA construct that activates NF ⁇ B signaling encodes a Btk polypeptide, such as a mutant Btk polypeptide such as a Btk(E41K) polypeptide (e.g., encoding an ORF amino acid sequence shown in SEQ ID NO: 114)
  • an immune potentiator mRNA construct that activates NF ⁇ B signaling encodes a TAK-TAB1 protein, such as a constitutively active TAK-TAB1.
  • an immune potentiator mRNA construct encodes a human TAK-TAB1 protein, such as having the sequence shown in SEQ ID NO: 105.
  • an immune potentiator mRNA construct encodes direct IAP binding protein with low pI (DIABLO) (also known as SMAC/DIABLO).
  • DIABLO constructs induce release of cytokines.
  • the disclosure provides a mRNA construct encoding a wild-type human DIABLO Isoform 1 sequence, such as having the sequence shown in SEQ ID NO: 106 (corresponding to the 239 amino acid human DIABLO isoform 1 precursor disclosed in the art as Genbank Accession No. NP_063940.1).
  • the mRNA construct encodes a human DIABLO Isoform 1 sequence comprising an S126L mutation, such as having the sequence shown in SEQ ID NO: 107.
  • the mRNA construct encodes amino acids 56-239 of human DIABLO Isoform 1, such as having the sequence shown in SEQ ID N: 108.
  • the mRNA construct encodes amino acids 56-239 of human DIABLO Isoform 1 and comprises an S126L mutation, such as having the sequence shown in SEQ ID NO: 109.
  • the mRNA construct encodes a wild-type human DIABLO Isoform 3 sequence, such as having the sequence shown in SEQ ID NO: 110 (corresponding to the 195 amino acid human DIABLO isoform 3 disclosed in the art as Genbank Accession No. NP_001265271.1).
  • the mRNA construct encodes a human DIABLO Isoform 3 sequence comprising an S82L mutation, such as having the sequence shown in SEQ ID NO: 110.
  • the mRNA construct encodes amino acids 56-195 of human DIABLO Isoform 3, such as having the sequence shown in SEQ ID NO: 111.
  • the mRNA construct encodes amino acids 56-195 of human DIABLO Isoform 3 and comprises an S82L mutation, such as having the sequence shown in SEQ ID NO: 112.
  • the immune potentiator mRNA construct encodes a SOC3 polypeptide (e.g., encoding an ORF amino acid sequence shown in SEQ ID NO: 115) or encodes a self-activating caspase-1 polypeptide (e.g, encoding any of the ORF amino acid sequences shown in SEQ ID NOs: 116-119), which can promote cleavage of pro-IL1 ⁇ (and pro-IL18 to their respective mature forms.
  • a SOC3 polypeptide e.g., encoding an ORF amino acid sequence shown in SEQ ID NO: 115
  • a self-activating caspase-1 polypeptide e.g, encoding any of the ORF amino acid sequences shown in SEQ ID NOs: 116-119
  • an immune potentiator mRNA construct encodes a protein that modulates dendritic cell (DC) activity, such as stimulating DC production, activity or mobilization.
  • DC dendritic cell
  • a non-limiting example of a protein that stimulates DC mobilization is FLT3. Accordingly, in one embodiment, the immune potentiator mRNA construct encodes a FLT3 protein.
  • An immune potentiator mRNA construct typically comprises, in addition to the polypeptide-encoding sequences, other structural properties as described herein for mRNA constructs (e.g., modified nucleobases, 5′ cap, 5′ UTR, 3′ UTR, miR binding site(s), polyA tail, as described herein). Suitable mRNA construct components are as described herein.
  • the disclosure provides a composition
  • at least one messenger RNA e.g., modified mRNA (mmRNA)
  • mmRNA modified mRNA
  • the disclosure provides a composition
  • at least one messenger RNA e.g., modified mRNA (mmRNA)
  • RNA e.g., modified mRNA (mmRNA)
  • at least one antigen of interest an activating oncogene mutation peptide(s)
  • a polypeptide that enhances an immune response against the at least one antigen of interest (an activating oncogene mutation peptide(s)) when the at least on mRNA is administered to a subject
  • said mRNA comprises one or more modified nucleobases.
  • compositions comprising an immune potentiator mRNA and an mRNA encoding an antigen of interest (an activating oncogene mutation peptide(s)), wherein a single mRNA construct can encode both the antigen(s) or interest and the polypeptide that enhances an immune response to the antigen(s) or, alternatively, the composition can comprise two or more separate mRNA constructs, a first mRNA and a second mRNA (or third or fourth mRNA), wherein the first mRNA encodes the at least one antigen of interest and the second mRNA encodes the polypeptide that enhances an immune response to the antigen(s) (i.e., the second mRNA comprises the immune potentiator).
  • the first mRNA and the second mRNAs can be coformulated together (e.g., prior to coadministration), such as coformulated in the same lipid nanoparticle.
  • the sequences encoding the polypeptide can be positioned on the mRNA construct either upstream or downstream of the sequences encoding the antigen of interest.
  • mRNA constructs encoding both an antigen and an immunostimulatory polypeptide include those encoding at least one mutant KRAS antigen and a constitutively active STING polypeptide, e.g., encoding an amino acid sequence shown in any one of SEQ ID NOs: 48-71.
  • the constitutively active STING polypeptide is located at the N-terminal end of the construct (i.e., upstream of the antigen-encoding sequences), as shown in SEQ ID NOs: 48-57. In another embodiment, the constitutively active STING polypeptide is located at the C-terminal end of the construct (i.e., downstream of the antigen-encoding sequences), as shown in SEQ ID NOs: 58-71.
  • mRNAs encoding antigens of interest e.g., mRNA vaccines
  • an immune potentiator mRNA of the disclosure are described in further detail below.
  • An mRNA may be a naturally or non-naturally occurring mRNA.
  • An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides, as described below, in which case it may be referred to as a “modified mRNA” or “mmRNA.”
  • nucleoside is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
  • nucleotide is defined as a nucleoside including a phosphate group.
  • An mRNA may include a 5′ untranslated region (5′-UTR), a 3′ untranslated region (3′-UTR), and/or a coding region (e.g., an open reading frame).
  • An exemplary 5′ UTR for use in the constructs is shown in SEQ ID NO: 21.
  • An exemplary 3′ UTR for use in the constructs is shown in SEQ ID NO: 22.
  • An exemplary 3′ UTR comprising miR-122 and miR-142.3p binding sites for use in the constructs is shown in SEQ ID NO: 23.
  • An mRNA may include any suitable number of base pairs, including tens (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100), hundreds (e.g., 200, 300, 400, 500, 600, 700, 800, or 900) or thousands (e.g., 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) of base pairs.
  • Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified.
  • an mRNA as described herein may include a 5′ cap structure, a chain terminating nucleotide, optionally a Kozak sequence (also known as a Kozak consensus sequence), a stem loop, a polyA sequence, and/or a polyadenylation signal.
  • a Kozak sequence also known as a Kozak consensus sequence
  • a 5′ cap structure or cap species is a compound including two nucleoside moieties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA).
  • a cap species may include one or more modified nucleosides and/or linker moieties.
  • a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5′ positions, e.g., m 7 G(5′)ppp(5′)G, commonly written as m 7 GpppG.
  • a cap species may also be an anti-reverse cap analog.
  • a non-limiting list of possible cap species includes m 7 GpppG, m 7 Gpppm 7 G, m 7 3′dGpppG, m 2 7,O3′ GpppG, m 2 7,O3′ GppppG, m 2 7,O2′ GppppG, m 7 Gpppm 7 G, m 7 3′dGpppG, m 2 7,O3′ GpppG, m 2 7,O3′ GppppG, and m 2 7,O2′ GppppG.
  • An mRNA may instead or additionally include a chain terminating nucleoside.
  • a chain terminating nucleoside may include those nucleosides deoxygenated at the 2′ and/or 3′ positions of their sugar group.
  • Such species may include 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytosine, 3′-deoxyguanosine, 3′-deoxythymine, and 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, and 2′,3′-dideoxythymine.
  • incorporation of a chain terminating nucleotide into an mRNA may result in stabilization of the mRNA, as described, for example, in International Patent Publication No. WO 2013/103659.
  • An mRNA may instead or additionally include a stem loop, such as a histone stem loop.
  • a stem loop may include 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs.
  • a stem loop may include 4, 5, 6, 7, or 8 nucleotide base pairs.
  • a stem loop may be located in any region of an mRNA.
  • a stem loop may be located in, before, or after an untranslated region (a 5′ untranslated region or a 3′ untranslated region), a coding region, or a polyA sequence or tail.
  • a stem loop may affect one or more function(s) of an mRNA, such as initiation of translation, translation efficiency, and/or transcriptional termination.
  • An mRNA may instead or additionally include a polyA sequence and/or polyadenylation signal.
  • a polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof.
  • a polyA sequence may be a tail located adjacent to a 3′ untranslated region of an mRNA.
  • a polyA sequence may affect the nuclear export, translation, and/or stability of an mRNA.
  • An mRNA may instead or additionally include a microRNA binding site.
  • an mRNA is a bicistronic mRNA comprising a first coding region and a second coding region with an intervening sequence comprising an internal ribosome entry site (IRES) sequence that allows for internal translation initiation between the first and second coding regions, or with an intervening sequence encoding a self-cleaving peptide, such as a 2A peptide.
  • IRES sequences and 2A peptides are typically used to enhance expression of multiple proteins from the same vector.
  • a variety of IRES sequences are known and available in the art and may be used, including, e.g., the encephalomyocarditis virus IRES.
  • the polynucleotides of the present disclosure may include a sequence encoding a self-cleaving peptide.
  • the self-cleaving peptide may be, but is not limited to, a 2A peptide.
  • a variety of 2A peptides are known and available in the art and may be used, including e.g., the foot and mouth disease virus (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, the Thosea asigna virus 2A peptide, and the porcine teschovirus-1 2A peptide.
  • FMDV foot and mouth disease virus
  • 2A peptides are used by several viruses to generate two proteins from one transcript by ribosome-skipping, such that a normal peptide bond is impaired at the 2A peptide sequence, resulting in two discontinuous proteins being produced from one translation event.
  • the 2A peptide may have the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 24), fragments or variants thereof.
  • the 2A peptide cleaves between the last glycine and last proline.
  • the polynucleotides of the present disclosure may include a polynucleotide sequence encoding the 2A peptide having the protein sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:24) fragments or variants thereof.
  • a polynucleotide sequence encoding the 2A peptide is: GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAG AACCCTGGACCT (SEQ ID NO: 25).
  • a 2A peptide is encoded by the following sequence: 5′-TCCGGACTCAGATCCGGGGATCTCAAAATTGTCGCTCCTGTCAAACAAACTCTTA ACTTTGATTTACTCAAACTGGCTGGGGATGTAGAAAGCAATCCAGGTCCACTC-3′(SEQ ID NO: 26).
  • 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 regions of two or more polypeptides of interest.
  • the sequence encoding the F2A peptide may be between a first coding region A and a second coding region B (A-F2Apep-B).
  • the presence of the F2A peptide results in the cleavage of the one long protein between the glycine and the proline at the end of the F2A peptide sequence (NPGP is cleaved to result in NPG and P) thus creating separate protein A (with 21 amino acids of the F2A peptide attached, ending with NPG) and separate protein B (with 1 amino acid, P, of the F2A peptide attached).
  • Protein A and protein B may be the same or different peptides or polypeptides of interest.
  • protein A is a polypeptide that induces immunogenic cell death and protein B is another polypeptide that stimulates an inflammatory and/or immune response and/or regulates immune responsiveness (as described further below).
  • an mRNA of the disclosure 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.
  • Exemplary 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-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-pseudoisocy
  • the modified nucleobase is a modified adenine.
  • exemplary nucleobases and nucleosides having a modified adenine include ⁇ -thio-adenosine, 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),
  • the modified nucleobase is a modified guanine.
  • exemplary nucleobases and nucleosides having a modified guanine include ⁇ -thio-guanosine, 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-a
  • an mRNA of the disclosure 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 disclosure 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 N1-methylpseudouridine (m 1 ⁇ ) and the mRNA of the disclosure is fully modified with N1-methylpseudouridine (m 1 ⁇ ).
  • N1-methylpseudouridine (m 1 ⁇ ) represents from 75-100% of the uracils in the mRNA.
  • N1-methylpseudouridine (m 1 ⁇ ) represents 100% of the uracils in the mRNA.
  • 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.
  • an mRNA of the disclosure 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 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).
  • an mRNA of the disclosure 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 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-guano sine (preQ 1 ), 7-methyl-guanosine (m 7 G), 1-methyl-guanosine (m 1 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
  • an mRNA of the disclosure 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 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 disclosure 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 mRNA comprises pseudouridine ( ⁇ ). In some embodiments, 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 disclosure is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification.
  • an mRNA can be uniformly modified with N1-methylpseudouridine (m 1 ⁇ ) or 5-methyl-cytidine (m 5 C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are replaced with N1-methylpseudouridine (m 1 ⁇ ) or 5-methyl-cytidine (m 5 C).
  • mRNAs of the disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
  • an mRNA of the disclosure may be modified in 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 disclosure include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.
  • the mmRNAs of the disclosure 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 nucleosides and modified nucleoside combinations are provided below in Table 1 and Table 2. These combinations of modified nucleotides can be used to form the mmRNAs of the disclosure.
  • the modified nucleosides may be partially or completely substituted for the natural nucleotides of the mRNAs of the disclosure.
  • the natural nucleotide uridine may be substituted with a modified nucleoside described herein.
  • the natural nucleoside 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% of the natural uridines) with at least one of the modified nucleoside disclosed herein.
  • polynucleotides of the disclosure may be synthesized to comprise the combinations or single modifications of Table 1 or Table 2.
  • 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 mRNAs of the present disclosure, or regions thereof, may be codon optimized. Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in 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 proteins trafficking sequences, remove/add post translation modification sites in encoded proteins (e.g., glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization methods are known in the art and may be useful for a variety of purposes: matching codon frequencies in 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 imp
  • Codon optimization tools, algorithms and services are known in the art; non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, Calif.) and/or proprietary methods.
  • the mRNA sequence is optimized using optimization algorithms, e.g., to optimize expression in mammalian cells or enhance mRNA stability.
  • the present disclosure includes polynucleotides having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein.
  • mRNAs of the present disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In one embodiment, mRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT/US2013/30062, the contents of which are incorporated herein by reference in their entirety. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.
  • Non-natural modified nucleobases may be introduced into polynucleotides, e.g., mRNA, during synthesis or post-synthesis.
  • modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugar.
  • the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain; with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT application No. PCT/US2012/058519. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
  • Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc.
  • Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990).
  • Polynucleotides of the disclosure can include regulatory elements, for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • regulatory elements for example, microRNA (miRNA) binding sites, transcription factor binding sites, structured mRNA sequences and/or motifs, artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid binding molecules, and combinations thereof.
  • polynucleotides including such regulatory elements are referred to as including “sensor sequences.”
  • sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • ORF open reading frame
  • miRNA binding site(s) provides for regulation of polynucleotides of the disclosure, and in turn, of the polypeptides encoded therefrom, based on tissue-specific and/or cell-type specific expression of naturally-occurring miRNAs.
  • a miRNA e.g., a natural-occurring miRNA
  • a miRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature miRNA.
  • a miRNA seed can comprise positions 2-8 or 2-7 of the mature miRNA.
  • a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1.
  • a miRNA seed can comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature miRNA), wherein the seed-complementary site in the corresponding miRNA binding site is flanked by an adenosine (A) opposed to miRNA position 1. See, for example, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul.
  • RNA profiling of the target cells or tissues can be conducted to determine the presence or absence of miRNA in the cells or tissues.
  • a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a polynucleotide of the disclosure comprises one or more microRNA binding sites, microRNA target sequences, microRNA complementary sequences, or microRNA seed complementary sequences.
  • sequences can correspond to, e.g., have complementarity to, any known microRNA such as those taught in US Publication US2005/0261218 and US Publication US2005/0059005, the contents of each of which are incorporated herein by reference in their entirety.
  • microRNA binding site refers to a sequence within a polynucleotide, e.g., within a DNA or within an RNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficient complementarity to all or a region of a miRNA to interact with, associate with or bind to the miRNA.
  • a polynucleotide of the disclosure comprising an ORF encoding a polypeptide of interest and further comprises one or more miRNA binding site(s).
  • a 5′UTR and/or 3′UTR of the polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of a polynucleotide, e.g., miRNA-mediated translational repression or degradation of the polynucleotide.
  • a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the polynucleotide, e.g., miRNA-guided RNA-induced silencing complex (RISC)-mediated cleavage of mRNA.
  • miRNA-guided RNA-induced silencing complex RISC
  • the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence.
  • a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence.
  • Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA is preferred when the desired regulation is mRNA degradation.
  • a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with a miRNA seed sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA seed sequence. In some embodiments, a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with an miRNA sequence. In some embodiments, the miRNA binding site includes a sequence that has complete complementarity with a miRNA sequence. In some embodiments, a miRNA binding site has complete complementarity with a miRNA sequence but for 1, 2, or 3 nucleotide substitutions, terminal additions, and/or truncations.
  • the miRNA binding site is the same length as the corresponding miRNA. In other embodiments, the miRNA binding site is one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve nucleotide(s) shorter than the corresponding miRNA at the 5′ terminus, the 3′ terminus, or both. In still other embodiments, the microRNA binding site is two nucleotides shorter than the corresponding microRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA binding sites that are shorter than the corresponding miRNAs are still capable of degrading the mRNA incorporating one or more of the miRNA binding sites or preventing the mRNA from translation.
  • the miRNA binding site binds the corresponding mature miRNA that is part of an active RISC containing Dicer. In another embodiment, binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA containing the miRNA binding site or prevents the mRNA from being translated. In some embodiments, the miRNA binding site has sufficient complementarity to miRNA so that a RISC complex comprising the miRNA cleaves the polynucleotide comprising the miRNA binding site. In other embodiments, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA induces instability in the polynucleotide comprising the miRNA binding site. In another embodiment, the miRNA binding site has imperfect complementarity so that a RISC complex comprising the miRNA represses transcription of the polynucleotide comprising the miRNA binding site.
  • the miRNA binding site has one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) from the corresponding miRNA.
  • the miRNA binding site has at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one contiguous nucleotides complementary to at least about ten, at least about eleven, at least about twelve, at least about thirteen, at least about fourteen, at least about fifteen, at least about sixteen, at least about seventeen, at least about eighteen, at least about nineteen, at least about twenty, or at least about twenty-one, respectively, contiguous nucleotides of the corresponding miRNA.
  • the polynucleotide By engineering one or more miRNA binding sites into a polynucleotide of the disclosure, the polynucleotide can be targeted for degradation or reduced translation, provided the miRNA in question is available. This can reduce off-target effects upon delivery of the polynucleotide. For example, if a polynucleotide of the disclosure is not intended to be delivered to a tissue or cell but ends up is said tissue or cell, then a miRNA abundant in the tissue or cell can inhibit the expression of the gene of interest if one or multiple binding sites of the miRNA are engineered into the 5′UTR and/or 3′UTR of the polynucleotide.
  • miRNA binding sites can be removed from polynucleotide sequences in which they naturally occur in order to increase protein expression in specific tissues.
  • a binding site for a specific miRNA can be removed from a polynucleotide to improve protein expression in tissues or cells containing the miRNA.
  • a polynucleotide of the disclosure can include at least one miRNA-binding site in the 5′UTR and/or 3′UTR in order to regulate cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • a polynucleotide of the disclosure can include two, three, four, five, six, seven, eight, nine, ten, or more miRNA-binding sites in the 5′-UTR and/or 3′-UTR in order to regulate cytotoxic or cytoprotective mRNA therapeutics to specific cells such as, but not limited to, normal and/or cancerous cells.
  • Regulation of expression in multiple tissues can be accomplished through introduction or removal of one or more miRNA binding sites, e.g., one or more distinct miRNA binding sites.
  • the decision whether to remove or insert a miRNA binding site can be made based on miRNA expression patterns and/or their profilings in tissues and/or cells in development and/or disease. Identification of miRNAs, miRNA binding sites, and their expression patterns and role in biology have been reported (e.g., Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20.
  • miRNAs and miRNA binding sites can correspond to any known sequence, including non-limiting examples described in U.S. Publication Nos. 2014/0200261, 2005/0261218, and 2005/0059005, each of which are incorporated herein by reference in their entirety.
  • tissues where miRNA are known to regulate mRNA, and thereby protein expression include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).
  • 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
  • miRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killer cells, etc.
  • APCs antigen presenting cells
  • Immune cell specific miRNAs are involved in immunogenicity, autoimmunity, the immune response to infection, inflammation, as well as unwanted immune response after gene therapy and tissue/organ transplantation. Immune cell specific miRNAs also regulate many aspects of development, proliferation, differentiation and apoptosis of hematopoietic cells (immune cells).
  • miR-142 and miR-146 are exclusively expressed in immune cells, particularly abundant in myeloid dendritic cells. It has been demonstrated that the immune response to a polynucleotide can be shut-off by adding miR-142 binding sites to the 3′-UTR of the polynucleotide, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; Brown B D, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
  • An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
  • Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of a polynucleotide of the disclosure can selectively repress gene expression in antigen presenting cells through miR-142 mediated degradation, limiting antigen presentation in antigen presenting cells (e.g., dendritic cells) and thereby preventing antigen-mediated immune response after the delivery of the polynucleotide.
  • the polynucleotide is then stably expressed in target tissues or cells without triggering cytotoxic elimination.
  • binding sites for miRNAs that are known to be expressed in immune cells can be engineered into a polynucleotide of the disclosure to suppress the expression of the polynucleotide in antigen presenting cells through miRNA mediated RNA degradation, subduing the antigen-mediated immune response. Expression of the polynucleotide is maintained in non-immune cells where the immune cell specific miRNAs are not expressed.
  • any miR-122 binding site can be removed and a miR-142 (and/or mirR-146) binding site can be engineered into the 5′UTR and/or 3′UTR of a polynucleotide of the disclosure.
  • a polynucleotide of the disclosure can include a further negative regulatory element in the 5′UTR and/or 3′UTR, either alone or in combination with miR-142 and/or miR-146 binding sites.
  • the further negative regulatory element is a Constitutive Decay Element (CDE).
  • Immune cell specific miRNAs include, but are not limited to, hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p, hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p, hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p, hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p, miR-142-3p, miR-142-5p, miR-
  • novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11, 288, the content of each of which is incorporated herein by reference in its entirety.)
  • miRNAs that are known to be expressed in the liver include, but are not limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p, miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.
  • liver specific miRNA binding sites from any liver specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the liver.
  • Liver specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the lung include, but are not limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, and miR-381-5p.
  • miRNA binding sites from any lung specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the lung.
  • Lung specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the heart include, but are not limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p, miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p, miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p, miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p.
  • miRNA binding sites from any heart specific microRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the heart.
  • Heart specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the nervous system include, but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p, miR-30
  • miRNAs enriched in the nervous system further include those specifically expressed in neurons, including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326, miR-328, miR-922 and those specifically expressed in glial cells, including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657.
  • miRNA binding sites from any CNS specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the nervous system.
  • Nervous system specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the pancreas include, but are not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944.
  • pancreas specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the pancreas.
  • Pancreas specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g. APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the kidney include, but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.
  • kidney specific miRNA binding sites from any kidney specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the kidney.
  • Kidney specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs that are known to be expressed in the muscle include, but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p.
  • miRNA binding sites from any muscle specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the muscle.
  • Muscle specific miRNA binding sites can be engineered alone or further in combination with immune cell (e.g., APC) miRNA binding sites in a polynucleotide of the disclosure.
  • miRNAs are also differentially expressed in different types of cells, such as, but not limited to, endothelial cells, epithelial cells, and adipocytes.
  • miRNAs that are known to be expressed in endothelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p, miR-296-5p
  • miRNA binding sites from any endothelial cell specific miRNA can be introduced to or removed from a polynucleotide of the disclosure to regulate expression of the polynucleotide in the endothelial cells.
  • miRNAs that are known to be expressed in epithelial cells include, but are not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific in respiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b, miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5p specific in renal epithelial cells, and miR-762 specific in corneal epithelial cells. miRNA binding sites from any epithelial cell
  • a large group of miRNAs are enriched in embryonic stem cells, controlling stem cell self-renewal as well as the development and/or differentiation of various cell lineages, such as neural cells, cardiac, hematopoietic cells, skin cells, osteogenic cells and muscle cells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764; Vidigal J A and Ventura A, Semin Cancer Biol.
  • miRNAs abundant in embryonic stem cells include, but are not limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p, miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246, miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p, miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p, miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p, miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p, miR-369-3p
  • the binding sites of embryonic stem cell specific miRNAs can be included in or removed from the 3′UTR of a polynucleotide of the disclosure to modulate the development and/or differentiation of embryonic stem cells, to inhibit the senescence of stem cells in a degenerative condition (e.g. degenerative diseases), or to stimulate the senescence and apoptosis of stem cells in a disease condition (e.g. cancer stem cells).
  • a degenerative condition e.g. degenerative diseases
  • apoptosis of stem cells e.g. cancer stem cells
  • miRNAs are abnormally over-expressed in certain cancer cells and others are under-expressed.
  • miRNAs are differentially expressed in cancer cells (WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224); pancreatic cancers and diseases (US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S. Pat. No.
  • miRNA binding sites for miRNAs that are over-expressed in certain cancer and/or tumor cells can be removed from the 3′UTR of a polynucleotide of the disclosure, restoring the expression suppressed by the over-expressed miRNAs in cancer cells, thus ameliorating the corresponsive biological function, for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death.
  • the corresponsive biological function for instance, transcription stimulation and/or repression, cell cycle arrest, apoptosis and cell death.
  • Normal cells and tissues, wherein miRNAs expression is not up-regulated, will remain unaffected.
  • miRNA can also regulate complex biological processes such as angiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176).
  • angiogenesis e.g., miR-132
  • miRNA binding sites that are involved in such processes can be removed or introduced, in order to tailor the expression of the polynucleotides to biologically relevant cell types or relevant biological processes.
  • the polynucleotides of the disclosure are defined as auxotrophic polynucleotides.
  • the therapeutic window and/or differential expression (e.g., tissue-specific expression) of a polypeptide of the disclosure may be altered by incorporation of a miRNA binding site into an mRNA encoding the polypeptide.
  • an mRNA may include one or more miRNA binding sites that are bound by miRNAs that have higher expression in one tissue type as compared to another.
  • an mRNA may include one or more miRNA binding sites that are bound by miRNAs that have lower expression in a cancer cell as compared to a non-cancerous cell of the same tissue of origin. When present in a cancer cell that expresses low levels of such an miRNA, the polypeptide encoded by the mRNA typically will show increased expression.
  • Liver cancer cells e.g., hepatocellular carcinoma cells
  • liver cancer cells typically express low levels of miR-122 as compared to normal liver cells. Therefore, an mRNA encoding a polypeptide that includes at least one miR-122 binding site (e.g., in the 3′-UTR of the mRNA) will typically express comparatively low levels of the polypeptide in normal liver cells and comparatively high levels of the polypeptide in liver cancer cells.
  • the mRNA includes at least one miR-122 binding site, at least two miR-122 binding sites, at least three miR-122 binding sites, at least four miR-122 binding sites, or at least five miR-122 binding sites.
  • the miRNA binding site binds miR-122 or is complementary to miR-122.
  • the miRNA binding site binds to miR-122-3p or miR-122-5p.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 175, wherein the miRNA binding site binds to miR-122.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 173, wherein the miRNA binding site binds to miR-122. These sequences are shown below in Table 3.
  • a polynucleotide of the disclosure comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from Table 3, including one or more copies of any one or more of the miRNA binding site sequences.
  • a polynucleotide of the disclosure further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, or more of the same or different miRNA binding sites selected from Table 3, including any combination thereof.
  • the miRNA binding site binds to miR-142 or is complementary to miR-142.
  • the miR-142 comprises SEQ ID NO: 27.
  • the miRNA binding site binds to miR-142-3p or miR-142-5p.
  • the miR-142-3p binding site comprises SEQ ID NO: 29.
  • the miR-142-5p binding site comprises SEQ ID NO: 31.
  • the miRNA binding site comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 29 or SEQ ID NO: 31
  • a miRNA binding site is inserted in the polynucleotide of the disclosure in any position of the polynucleotide (e.g., the 5′UTR and/or 3′UTR).
  • the 5′UTR comprises a miRNA binding site.
  • the 3′UTR comprises a miRNA binding site.
  • the 5′UTR and the 3′UTR comprise a miRNA binding site.
  • the insertion site in the polynucleotide can be anywhere in the polynucleotide as long as the insertion of the miRNA binding site in the polynucleotide does not interfere with the translation of a functional polypeptide in the absence of the corresponding miRNA; and in the presence of the miRNA, the insertion of the miRNA binding site in the polynucleotide and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the polynucleotide or preventing the translation of the polynucleotide.
  • a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the disclosure comprising the ORF. In some embodiments, a miRNA binding site is inserted in at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucle
  • a miRNA binding site is inserted in about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 90 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 70 nucleotides, about 50 nucleotides to about 60 nucleotides, about 45 nucleotides to about 65 nucleotides downstream from the stop codon of an ORF in a polynucleotide of the disclosure.
  • miRNA gene regulation can be influenced by the sequence surrounding the miRNA such as, but not limited to, the species of the surrounding sequence, the type of sequence (e.g., heterologous, homologous, exogenous, endogenous, or artificial), regulatory elements in the surrounding sequence and/or structural elements in the surrounding sequence.
  • the miRNA can be influenced by the 5′UTR and/or 3′UTR.
  • a non-human 3′UTR can increase the regulatory effect of the miRNA sequence on the expression of a polypeptide of interest compared to a human 3′UTR of the same sequence type.
  • other regulatory elements and/or structural elements of the 5′UTR can influence miRNA mediated gene regulation.
  • a regulatory element and/or structural element is a structured IRES (Internal Ribosome Entry Site) in the 5′UTR, which is necessary for the binding of translational elongation factors to initiate protein translation. EIF4A2 binding to this secondarily structured element in the 5′-UTR is necessary for miRNA mediated gene expression (Meijer H A et al., Science, 2013, 340, 82-85, incorporated herein by reference in its entirety).
  • the polynucleotides of the disclosure can further include this structured 5′UTR in order to enhance microRNA mediated gene regulation.
  • At least one miRNA binding site can be engineered into the 3′UTR of a polynucleotide of the disclosure.
  • at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more miRNA binding sites can be engineered into a 3′UTR of a polynucleotide of the disclosure.
  • 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineered into the 3′UTR of a polynucleotide of the disclosure.
  • miRNA binding sites incorporated into a polynucleotide of the disclosure can be the same or can be different miRNA sites.
  • a combination of different miRNA binding sites incorporated into a polynucleotide of the disclosure can include combinations in which more than one copy of any of the different miRNA sites are incorporated.
  • miRNA binding sites incorporated into a polynucleotide of the disclosure can target the same or different tissues in the body.
  • tissue-, cell-type-, or disease-specific miRNA binding sites in the 3′-UTR of a polynucleotide of the disclosure can be reduced.
  • specific cell types e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR, about halfway between the 5′ terminus and 3′ terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in a polynucleotide of the disclosure.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.
  • a miRNA binding site can be engineered near the 3′ terminus of the 3′UTR and about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.
  • a miRNA binding site can be engineered near the 5′ terminus of the 3′UTR and near the 3′ terminus of the 3′UTR.
  • a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA binding sites.
  • the miRNA binding sites can be complementary to a miRNA, miRNA seed sequence, and/or miRNA sequences flanking the seed sequence.
  • a polynucleotide of the disclosure can be engineered to include more than one miRNA site expressed in different tissues or different cell types of a subject.
  • a polynucleotide of the disclosure can be engineered to include miR-192 and miR-122 to regulate expression of the polynucleotide in the liver and kidneys of a subject.
  • a polynucleotide of the disclosure can be engineered to include more than one miRNA site for the same tissue.
  • the therapeutic window and or differential expression associated with the polypeptide encoded by a polynucleotide of the disclosure can be altered with a miRNA binding site.
  • a polynucleotide encoding a polypeptide that provides a death signal can be designed to be more highly expressed in cancer cells by virtue of the miRNA signature of those cells.
  • the polynucleotide encoding the binding site for that miRNA (or miRNAs) would be more highly expressed.
  • the polypeptide that provides a death signal triggers or induces cell death in the cancer cell.
  • Neighboring noncancer cells harboring a higher expression of the same miRNA would be less affected by the encoded death signal as the polynucleotide would be expressed at a lower level due to the effects of the miRNA binding to the binding site or “sensor” encoded in the 3′UTR.
  • cell survival or cytoprotective signals can be delivered to tissues containing cancer and non-cancerous cells where a miRNA has a higher expression in the cancer cells—the result being a lower survival signal to the cancer cell and a larger survival signal to the normal cell.
  • Multiple polynucleotides can be designed and administered having different signals based on the use of miRNA binding sites as described herein.
  • the expression of a polynucleotide of the disclosure can be controlled by incorporating at least one sensor sequence in the polynucleotide and formulating the polynucleotide for administration.
  • a polynucleotide of the disclosure can be targeted to a tissue or cell by incorporating a miRNA binding site and formulating the polynucleotide in a lipid nanoparticle comprising a cationic lipid, including any of the lipids described herein.
  • a polynucleotide of the disclosure can be engineered for more targeted expression in specific tissues, cell types, or biological conditions based on the expression patterns of miRNAs in the different tissues, cell types, or biological conditions. Through introduction of tissue-specific miRNA binding sites, a polynucleotide of the disclosure can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
  • a polynucleotide of the disclosure can be designed to incorporate miRNA binding sites that either have 100% identity to known miRNA seed sequences or have less than 100% identity to miRNA seed sequences.
  • a polynucleotide of the disclosure can be designed to incorporate miRNA binding sites that have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to known miRNA seed sequences.
  • the miRNA seed sequence can be partially mutated to decrease miRNA binding affinity and as such result in reduced downmodulation of the polynucleotide.
  • the degree of match or mis-match between the miRNA binding site and the miRNA seed can act as a rheostat to more finely tune the ability of the miRNA to modulate protein expression.
  • mutation in the non-seed region of a miRNA binding site can also impact the ability of a miRNA to modulate protein expression.
  • a miRNA sequence can be incorporated into the loop of a stem loop.
  • a miRNA seed sequence can be incorporated in the loop of a stem loop and a miRNA binding site can be incorporated into the 5′ or 3′ stem of the stem loop.
  • a translation enhancer element can be incorporated on the 5′end of the stem of a stem loop and a miRNA seed can be incorporated into the stem of the stem loop.
  • a TEE can be incorporated on the 5′ end of the stem of a stem loop, a miRNA seed can be incorporated into the stem of the stem loop and a miRNA binding site can be incorporated into the 3′ end of the stem or the sequence after the stem loop.
  • the miRNA seed and the miRNA binding site can be for the same and/or different miRNA sequences.
  • the incorporation of a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation.
  • a miRNA sequence and/or a TEE sequence changes the shape of the stem loop region which can increase and/or decrease translation.
  • the 5′-UTR of a polynucleotide of the disclosure can comprise at least one miRNA sequence.
  • the miRNA sequence can be, but is not limited to, a 19 or 22 nucleotide sequence and/or a miRNA sequence without the seed.
  • the miRNA sequence in the 5′UTR can be used to stabilize a polynucleotide of the disclosure described herein.
  • a miRNA sequence in the 5′UTR of a polynucleotide of the disclosure can be used to decrease the accessibility of the site of translation initiation such as, but not limited to a start codon.
  • a site of translation initiation such as, but not limited to a start codon.
  • LNA antisense locked nucleic acid
  • EJCs exon-junction complexes
  • a polynucleotide of the disclosure can comprise a miRNA sequence, instead of the LNA or EJC sequence described by Matsuda et al, near the site of translation initiation in order to decrease the accessibility to the site of translation initiation.
  • the site of translation initiation can be prior to, after or within the miRNA sequence.
  • the site of translation initiation can be located within a miRNA sequence such as a seed sequence or binding site.
  • the site of translation initiation can be located within a miR-122 sequence such as the seed sequence or the mir-122 binding site.
  • a polynucleotide of the disclosure can include at least one miRNA in order to dampen the antigen presentation by antigen presenting cells.
  • the miRNA can be the complete miRNA sequence, the miRNA seed sequence, the miRNA sequence without the seed, or a combination thereof.
  • a miRNA incorporated into a polynucleotide of the disclosure can be specific to the hematopoietic system.
  • a miRNA incorporated into a polynucleotide of the disclosure to dampen antigen presentation is miR-142-3p.
  • a polynucleotide of the disclosure can include at least one miRNA in order to dampen expression of the encoded polypeptide in a tissue or cell of interest.
  • a polynucleotide of the disclosure can include at least one miR-122 binding site in order to dampen expression of an encoded polypeptide of interest in the liver.
  • a polynucleotide of the disclosure can include at least one miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3p binding site without the seed, miR-142-5p binding site, miR-142-5p seed sequence, miR-142-5p binding site without the seed, miR-146 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.
  • a polynucleotide of the disclosure can comprise at least one miRNA binding site in the 3′UTR in order to selectively degrade mRNA therapeutics in the immune cells to subdue unwanted immunogenic reactions caused by therapeutic delivery.
  • the miRNA binding site can make a polynucleotide of the disclosure more unstable in antigen presenting cells.
  • these miRNAs include mir-142-5p, mir-142-3p, mir-146a-5p, and mir-146-3p.
  • a polynucleotide of the disclosure comprises at least one miRNA sequence in a region of the polynucleotide that can interact with a RNA binding protein.
  • the polynucleotide of the disclosure e.g., a RNA, e.g., a mRNA
  • a RNA e.g., a mRNA
  • a sequence-optimized nucleotide sequence e.g., an ORF
  • a miRNA binding site e.g., a miRNA binding site that binds to miR-142.
  • the polynucleotide of the disclosure comprises a uracil-modified sequence encoding a polypeptide disclosed herein and a miRNA binding site disclosed herein, e.g., a miRNA binding site that binds to miR-142 or miR-122.
  • the uracil-modified sequence encoding a polypeptide comprises at least one chemically modified nucleobase, e.g., 5-methoxyuracil.
  • at least 95% of a type of nucleobase (e.g., uracil) in a uracil-modified sequence encoding a polypeptide of the disclosure are modified nucleobases.
  • uricil in a uracil-modified sequence encoding a polypeptide is 5-methoxyuridine.
  • the polynucleotide comprising a nucleotide sequence encoding a polypeptide disclosed herein and a miRNA binding site is formulated with a delivery agent, e.g., a compound having the Formula (I), e.g., any of Compounds 1-147.
  • the present disclosure provides synthetic polynucleotides comprising a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity.
  • a modification e.g., an RNA element
  • the disclosure provides a polynucleotide comprising a 5′ untranslated region (UTR), an initiation codon, a full open reading frame encoding a polypeptide, a 3′ UTR, and at least one modification, wherein the at least one modification provides a desired translational regulatory activity, for example, a modification that promotes and/or enhances the translational fidelity of mRNA translation.
  • the desired translational regulatory activity is a cis-acting regulatory activity.
  • the desired translational regulatory activity is an increase in the residence time of the 43S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some embodiments, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome.
  • the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the initiation of polypeptide synthesis at any codon within the mRNA other than the initiation codon. In some embodiments, the desired translational regulatory activity is inhibition or reduction of the amount of polypeptide translated from any open reading frame within the mRNA other than the full open reading frame. In some embodiments, the desired translational regulatory activity is inhibition or reduction in the production of aberrant translation products. In some embodiments, the desired translational regulatory activity is a combination of one or more of the foregoing translational regulatory activities.
  • the present disclosure provides a polynucleotide, e.g., an mRNA, comprising an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity as described herein.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that promotes and/or enhances the translational fidelity of mRNA translation.
  • the mRNA comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that provides a desired translational regulatory activity, such as inhibiting and/or reducing leaky scanning.
  • the disclosure provides an mRNA that comprises an RNA element that comprises a sequence and/or an RNA secondary structure(s) that inhibits and/or reduces leaky scanning thereby promoting the translational fidelity of the mRNA.
  • the RNA element comprises natural and/or modified nucleotides. In some embodiments, the RNA element comprises of a sequence of linked nucleotides, or derivatives or analogs thereof, that provides a desired translational regulatory activity as described herein. In some embodiments, the RNA element comprises a sequence of linked nucleotides, or derivatives or analogs thereof, that forms or folds into a stable RNA secondary structure, wherein the RNA secondary structure provides a desired translational regulatory activity as described herein.
  • RNA elements can be identified and/or characterized based on the primary sequence of the element (e.g., GC-rich element), by RNA secondary structure formed by the element (e.g.
  • RNA molecules e.g., located within the 5′ UTR of an mRNA
  • translational enhancer element e.g., translational enhancer element
  • the disclosure provides an mRNA having one or more structural modifications that inhibits leaky scanning and/or promotes the translational fidelity of mRNA translation, wherein at least one of the structural modifications is a GC-rich RNA element.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR of the mRNA.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′ UTR of the mRNA. In another embodiment, the GC-rich RNA element is located 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about 3 nucleotides, derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60% cytosine, 40-50% cytosine, or 30-40% cytosine.
  • the disclosure provides a GC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is about 80% cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, or about 30% cytosine.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR of the mRNA, wherein the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′ UTR of the mRNA, and wherein the GC-rich RNA element comprises a sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides, or derivatives or analogs thereof, linked in any order, wherein the sequence composition is >50% cytosine.
  • at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in
  • the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a sequence of linked nucleotides, or derivatives or analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR of the mRNA, wherein the GC-rich RNA element comprises any one of the sequences set forth in Table 4.
  • the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V1 [CCCCGGCGCC] (SEQ ID NO: 179) as set forth in Table 4, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence V1 as set forth in Table 4 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence V1 as set forth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA. In other embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V2 [CCCCGGC] as set forth in Table 4, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence V2 as set forth in Table 4 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence V2 as set forth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence V2 as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence EK [GCCGCC] as set forth in Table 4, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence EK as set forth in Table 4 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence EK as set forth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the GC-rich element comprises the sequence EK as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA.
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence V1[CCCCGGCGCC] (SEQ ID NO: 179) as set forth in Table 4, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5′ UTR of the mRNA, wherein the 5′ UTR comprises the following sequence shown in Table 4:
  • the GC-rich element comprises the sequence V1 as set forth in Table 4 located immediately adjacent to and upstream of the Kozak consensus sequence in the 5′ UTR sequence shown in Table 4. In some embodiments, the GC-rich element comprises the sequence V1 as set forth in Table 4 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA, wherein the 5′ UTR comprises the following sequence shown in Table 4:
  • the GC-rich element comprises the sequence V1 as set forth in Table 4 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence in the 5′ UTR of the mRNA, wherein the 5′ UTR comprises the following sequence shown in Table 4:
  • the 5′ UTR comprises the following sequence set forth in Table 4:
  • the 5′ UTR comprises the following sequence set forth in Table 4:
  • the disclosure provides a modified mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising a stable RNA secondary structure comprising a sequence of nucleotides, or derivatives or analogs thereof, linked in an order which forms a hairpin or a stem-loop.
  • the stable RNA secondary structure is upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 30, about 25, about 20, about 15, about 10, or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 20, about 15, about 10 or about 5 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure is located about 5, about 4, about 3, about 2, about 1 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located about 15-30, about 15-20, about 15-25, about 10-15, or about 5-10 nucleotides upstream of the Kozak consensus sequence. In another embodiment, the stable RNA secondary structure is located 12-15 nucleotides upstream of the Kozak consensus sequence.
  • the stable RNA secondary structure has a deltaG of about ⁇ 30 kcal/mol, about ⁇ 20 to ⁇ 30 kcal/mol, about ⁇ 20 kcal/mol, about ⁇ 10 to ⁇ 20 kcal/mol, about ⁇ 10 kcal/mol, about ⁇ 5 to ⁇ 10 kcal/mol.
  • the modification is operably linked to an open reading frame encoding a polypeptide and wherein the modification and the open reading frame are heterologous.
  • sequence of the GC-rich RNA element is comprised exclusively of guanine (G) and cytosine (C) nucleobases.
  • RNA elements that provide a desired translational regulatory activity as described herein can be identified and characterized using known techniques, such as ribosome profiling.
  • Ribosome profiling is a technique that allows the determination of the positions of PICs and/or ribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science 324(5924):218-23, incorporated herein by reference). The technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion. Protection results in the generation of a 30-bp fragment of RNA termed a ‘footprint’.
  • RNA footprints can be analyzed by methods known in the art (e.g., RNA-seq).
  • the footprint is roughly centered on the A-site of the ribosome. If the PIC or ribosome dwells at a particular position or location along an mRNA, footprints generated at these position would be relatively common. Studies have shown that more footprints are generated at positions where the PIC and/or ribosome exhibits decreased processivity and fewer footprints where the PIC and/or ribosome exhibits increased processivity (Gardin et al., (2014) eLife 3:e03735).
  • residence time or the time of occupancy of a the PIC or ribosome at a discrete position or location along an polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.
  • RNA preparations produced using traditional IVT processes have properties that enable the production of qualitatively and quantitatively superior compositions.
  • RNA produced using traditional IVT methods is qualitatively and quantitatively distinct from the RNA preparations produced by the modified IVT processes. For instance, the purified RNA preparations are less immunogenic in comparison to RNA preparations made using traditional IVT. Additionally, increased protein expression levels with higher purity are produced from the purified RNA preparations.
  • RNA polymerase equimolar quantities of nucleotide triphosphates, including GTP, ATP, CTP, and UTP in a transcription buffer.
  • An RNA transcript having a 5′ terminal guanosine triphosphate is produced from this reaction.
  • These reactions also result in the production of a number of impurities such as double stranded and single stranded RNAs which are immunostimulatory and may have an additive impact.
  • the purity methods described herein prevent formation of reverse complements and thus prevent the innate immune recognition of both species.
  • the modified IVT methods result in the production of RNA having significantly reduced T cell activity than an RNA preparation made using prior art methods with equimolar NTPs.
  • the modified IVT methods involve the manipulation of one or more of the reaction parameters in the IVT reaction to produce a RNA preparation of highly functional RNA without one or more of the undesirable contaminants produced using the prior art processes.
  • One parameter in the IVT reaction that may be manipulated is the relative amount of a nucleotide or nucleotide analog in comparison to one or more other nucleotides or nucleotide analogs in the reaction mixture (e.g., disparate nucleotide amounts or concentration).
  • the IVT reaction may include an excess of a nucleotides, e.g., nucleotide monophosphate, nucleotide diphosphate or nucleotide triphosphate and/or an excess of nucleotide analogs and/or nucleoside analogs.
  • a nucleotides e.g., nucleotide monophosphate, nucleotide diphosphate or nucleotide triphosphate and/or an excess of nucleotide analogs and/or nucleoside analogs.
  • Nucleotide analogs are compounds that have the general structure of a nucleotide or are structurally similar to a nucleotide or portion thereof.
  • nucleotide analogs are nucleotides which contain, for example, an analogue of the nucleic acid portion, sugar portion and/or phosphate groups of the nucleotide.
  • Nucleotides include, for instance, nucleotide monophosphates, nucleotide diphosphates, and nucleotide triphosphates.
  • a nucleotide analog, as used herein is structurally similar to a nucleotide or portion thereof but does not have the typical nucleotide structure (nucleobase-ribose-phosphate).
  • Nucleoside analogs are compounds that have the general structure of a nucleoside or are structurally similar to a nucleoside or portion thereof.
  • nucleoside analogs are nucleosides which contain, for example, an analogue of the nucleic acid and/or sugar portion of the nucleoside.
  • nucleotide analogs useful in the methods are structurally similar to nucleotides or portions thereof but, for example, are not polymerizable by T7.
  • Nucleotide/nucleoside analogs as used herein include for instance, antiviral nucleotide analogs, phosphate analogs (soluble or immobilized, hydrolyzable or non-hydrolyzable), dinucleotide, trinucleotide, tetranucleotide, e.g., a cap analog, or a precursor/substrate for enzymatic capping (vaccinia, or ligase), a nucleotide labelled with a functional group to facilitate ligation/conjugation of cap or 5′ moiety (IRES), a nucleotide labelled with a 5′ PO4 to facilitate ligation of cap or 5
  • the IVT reaction typically includes the following: an RNA polymerase, e.g., a T7 RNA polymerase at a final concentration of, e.g., 1000-12000 U/mL, e.g., 7000 U/mL; the DNA template at a final concentration of, e.g., 10-70 nM, e.g., 40 nM; nucleotides (NTPs) at a final concentration of e.g., 0.5-10 mM, e.g., 7.5 mM each; magnesium at a final concentration of, e.g., 12-60 mM, e.g., magnesium acetate at 40 mM; a buffer such as, e.g., HEPES or Tris at a pH of, e.g., 7-8.5, e.g.
  • an RNA polymerase e.g., a T7 RNA polymerase at a final concentration of, e.g., 1000-12000
  • an RNase inhibitor is included in the IVT reaction to ensure no RNase induced degradation during the transcription reaction.
  • murine RNase inhibitor can be utilized at a final concentration of 1000 U/mL.
  • a pyrophosphatase is included in the IVT reaction to cleave the inorganic pyrophosphate generated following each nucleotide incorporation into two units of inorganic phosphate. This ensures that magnesium remains in solution and does not precipitate as magnesium pyrophosphate.
  • an E. coli inorganic pyrophosphatase can be utilized at a final concentration of 1 U/mL.
  • the modified method may also be produced by forming a reaction mixture comprising a DNA template, and one or more NTPs such as ATP, CTP, UTP, GTP (or corresponding analog of aforementioned components) and a buffer. The reaction is then incubated under conditions such that the RNA is transcribed.
  • the modified methods utilize the presence of an excess amount of one or more nucleotides and/or nucleotide analogs that can have significant impact on the end product. These methods involve a modification in the amount (e.g., molar amount or quantity) of nucleotides and/or nucleotide analogs in the reaction mixture.
  • one or more nucleotides and/or one or more nucleotide analogs may be added in excess to the reaction mixture.
  • An excess of nucleotides and/or nucleotide analogs is any amount greater than the amount of one or more of the other nucleotides such as NTPs in the reaction mixture.
  • an excess of a nucleotide and/or nucleotide analog may be a greater amount than the amount of each or at least one of the other individual NTPs in the reaction mixture or may refer to an amount greater than equimolar amounts of the other NTPs.
  • the NTP may be present in a higher concentration than all three of the other NTPs included in the reaction mixture.
  • the other three NTPs may be in an equimolar concentration to one another.
  • one or more of the three other NTPs may be in a different concentration than one or more of the other NTPs.
  • the IVT reaction may include an equimolar amount of nucleotide triphosphate relative to at least one of the other nucleotide triphosphates.
  • reaction mixture comprising a DNA template and NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP) and optionally guanosine diphosphate (GDP), and (eg. buffer containing T7 co-factor eg. magnesium).
  • NTPs including adenosine triphosphate (ATP), cytidine triphosphate (CTP), uridine triphosphate (UTP), guanosine triphosphate (GTP) and optionally guanosine diphosphate (GDP), and (eg. buffer containing T7 co-factor eg. magnesium).
  • the concentration of at least one of GTP, CTP, ATP, and UTP is at least 2 ⁇ greater than the concentration of any one or more of ATP, CTP or UTP or the reaction further comprises a nucleotide analog and wherein the concentration of the nucleotide analog is at least 2 ⁇ greater than the concentration of any one or more of ATP, CTP or UTP.
  • the ratio of concentration of GTP to the concentration of any one ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:1.
  • the ratio of concentration of GTP to concentration of ATP, CTP and UTP is, in some embodiments 2:1, 4:1 and 4:1, respectively.
  • the ratio of concentration of GTP to concentration of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.
  • the reaction mixture may comprise GTP and GDP and wherein the ratio of concentration of GTP plus GDP to the concentration of any one of ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:1 In some embodiments the ratio of concentration of GTP plus GDP to concentration of ATP, CTP and UTP is 3:1, 6:1 and 6:1, respectively.
  • the method involves incubating the reaction mixture under conditions such that the RNA is transcribed, wherein the effective concentration of phosphate in the reaction is at least 150 mM phosphate, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM, at least 210 mM or at least 220 mM.
  • the effective concentration of phosphate in the reaction may be 180 mM.
  • the effective concentration of phosphate in the reaction in some embodiments is 195 mM. In other embodiments the effective concentration of phosphate in the reaction is 225 mM.
  • the RNA is produced by a process or is preparable by a process comprising wherein a buffer magnesium-containing buffer is used when forming the reaction mixture comprising a DNA template and ATP, CTP, UTP, GTP.
  • the magnesium-containing buffer comprises Mg2+ and wherein the molar ratio of concentration of ATP plus CTP plus UTP pus GTP to concentration of Mg2+ is at least 1.0, at least 1.25, at least 1.5, at least 1.75, at least 1.85, at least 3 or higher.
  • the molar ratio of concentration of ATP plus CTP plus UTP pus GTP to concentration of Mg2+ may be 1.5.
  • the molar ratio of concentration of ATP plus CTP plus UTP pus GTP to concentration of Mg2+ in some embodiments is 1.88.
  • the molar ratio of concentration of ATP plus CTP plus UTP pus GTP to concentration of Mg2+ in some embodiments is 3.
  • the composition is produced by a process which does not comprise an dsRNase (e.g., RNaseII) treatment step.
  • the composition is produced by a process which does not comprise a reverse phase (RP) chromatography purification step.
  • the composition is produced by a process which does not comprise a high-performance liquid chromatography (HPLC) purification step.
  • dsRNase e.g., RNaseII
  • RP reverse phase
  • HPLC high-performance liquid chromatography
  • the ratio of concentration of GTP to the concentration of any one ATP, CTP or UTP is at least 2:1, at least 3:1, at least 4:1, at least 5:1 or at least 6:1 to produce the RNA.
  • the purity of the products may be assessed using known analytical methods and assays.
  • the amount of reverse complement transcription product or cytokine-inducing RNA contaminant may be determined by high-performance liquid chromatography (such as reverse-phase chromatography, size-exclusion chromatography), Bioanalyzer chip-based electrophoresis system, ELISA, flow cytometry, acrylamide gel, a reconstitution or surrogate type assay.
  • the assays may be performed with or without nuclease treatment (P1, RNase III, RNase H etc.) of the RNA preparation. Electrophoretic/chromatographic/mass spec analysis of nuclease digestion products may also be performed.
  • the purified RNA preparations comprise contaminant transcripts that have a length less than a full length transcript, such as for instance at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides less than the full length.
  • Contaminant transcripts can include reverse or forward transcription products (transcripts) that have a length less than a full length transcript, such as for instance at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides less than the full length.
  • Exemplary forward transcripts include, for instance, abortive transcripts.
  • the composition comprises a tri-phosphate poly-U reverse complement of less than 30 nucleotides.
  • the composition comprises a tri-phosphate poly-U reverse complement of any length hybridized to a full length transcript.
  • the composition comprises a single stranded tri-phosphate forward transcript.
  • the composition comprises a single stranded RNA having a terminal tri-phosphate-G.
  • the composition comprises single or double stranded RNA of less than 12 nucleotides or base pairs (including forward or reverse complement transcripts).
  • the composition may include less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of any one of or combination of these less than full length transcripts.
  • mRNAs of the disclosure may be formulated in nanoparticles or other delivery vehicles, e.g., to protect them from degradation when delivered to a subject.
  • Illustrative nanoparticles are described in Panyam, J. & Labhasetwar, V. Adv. Drug Deliv. Rev. 55, 329-347 (2003) and Peer, D. et al. Nature Nanotech. 2, 751-760 (2007).
  • an mRNA of the disclosure is encapsulated within a nanoparticle.
  • a nanoparticle is a particle having at least one dimension (e.g., a diameter) less than or equal to 1000 nM, less than or equal to 500 nM or less than or equal to 100 nM.
  • a nanoparticle includes a lipid.
  • Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any of a number of lipids may be present, including cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, PEGylated lipids, and/or structural lipids. Such lipids can be used alone or in combination.
  • a lipid nanoparticle comprises one or more mRNAs described herein.
  • the lipid nanoparticle formulations of the mRNAs described herein may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • cationic and/or ionizable lipids include, but are not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-
  • lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL).
  • LIPOFECTIN® including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECTAMINE® including DOSPA and DOPE, available from GIBCO/BRL
  • KL10, KL22, and KL25 are described, for example, in U.S. Pat. No. 8,691,750, which is incorporated herein by reference in its entirety.
  • the lipid is DLin-MC3-DMA or DLin-KC2-DMA.
  • Anionic lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids.
  • Neutral lipids suitable for use in lipid nanoparticles of the disclosure include, but are not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Lipids having a variety of acyl chain groups of varying chain length and degree of saturation are available or may be isolated or synthesized by well-known techniques. Additionally, lipids having mixtures of saturated and unsaturated fatty acid chains can be used. In some embodiments, the neutral lipids used in the disclosure are DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine. In some embodiments, the neutral lipid may be composed of sphingomyelin, dihydrosphingomyeline, or phospholipids with other head groups, such as serine and inositol.
  • amphipathic lipids are included in nanoparticles of the disclosure.
  • Exemplary amphipathic lipids suitable for use in nanoparticles of the disclosure include, but are not limited to, sphingolipids, phospholipids, and aminolipids.
  • a phospholipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl
  • phosphorus-lacking compounds such as sphingolipids, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, may also be used. Additionally, such amphipathic lipids can be readily mixed with other lipids, such as triglycerides and sterols.
  • the lipid component of a nanoparticle of the disclosure may include one or more PEGylated lipids.
  • a PEGylated lipid (also known as a PEG lipid or a PEG-modified lipid) is a lipid modified with polyethylene glycol.
  • the lipid component may include one or more PEGylated lipids.
  • a PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.
  • a PEGylated lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.
  • a lipid nanoparticle of the disclosure may include one or more structural lipids.
  • Exemplary, non-limiting structural lipids that may be present in the lipid nanoparticles of the disclosure include cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, or alpha-tocopherol.
  • one or more mRNA of the disclosure may be formulated in a lipid nanoparticle having a diameter from about 1 nm to about 900 nm, e.g., about 1 nm to about 100 nm, about 1 nm to about 200 nm, about 1 nm to about 300 nm, about 1 nm to about 400 nm, about 1 nm to about 500 nm, about 1 nm to about 600 nm, about 1 nm to about 700 nm, about 1 nm to 800 nm, about 1 nm to about 900 nm.
  • the nanoparticle may have a diameter from about 10 nm to about 300 nm, about 20 nm to about 200 nm, about 30 nm to about 100 nm, or about 40 nm to about 80 nm. In some embodiments, the nanoparticle may have a diameter from about 30 nm to about 300 nm, about 40 nm to about 200 nm, about 50 nm to about 150 nm, about 70 to about 110 nm, or about 80 nm to about 120 nm.
  • an mRNA may be formulated in a lipid nanoparticle having a diameter from about 10 to about 100 nm including ranges in between such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 nm
  • an mRNA may be formulated in a lipid nanoparticle having a diameter from about 30 nm to about 300 nm, about 40 nm to about 200 nm, about 50 nm to about 150 nm, about 70 to about 110 nm, or about 80 nm to about 120 nm including ranges in between.
  • a 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, or greater than 950 nm.
  • the particle size of the lipid nanoparticle may be increased and/or decreased.
  • the change in particle size may be able to help counter a biological reaction such as, but not limited to, inflammation, or may increase the biological effect of the mRNA delivered to a patient or subject.
  • a nanoparticle e.g., a lipid nanoparticle
  • a targeting moiety that is specific to a cell type and/or tissue type.
  • a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety.
  • a nanoparticle comprises one or more mRNA described herein and a targeting moiety.
  • targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, or F(ab′)2 fragments), single domain antibodies, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)).
  • the targeting moiety may be a polypeptide.
  • the targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof.
  • a targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor.
  • a variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J. Liposome Res. 12:1-3, 2002.
  • a lipid nanoparticle may include a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains (see, e.g., Allen et al., Biochimica et Biophysica Acta 1237: 99-108, 1995; DeFrees et al., Journal of the American Chemistry Society 118: 6101-6104, 1996; Blume et al., Biochimica et Biophysica Acta 1149: 180-184, 1993; Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; U.S. Pat. No.
  • PEG polyethylene glycol
  • a targeting moiety for targeting the lipid nanoparticle is linked to the polar head group of lipids forming the nanoparticle.
  • the targeting moiety is attached to the distal ends of the PEG chains forming the hydrophilic polymer coating (see, e.g., Klibanov et al., Journal of Liposome Research 2: 321-334, 1992; Kirpotin et al., FEBS Letters 388: 115-118, 1996).
  • Standard methods for coupling the targeting moiety or moieties may be used.
  • phosphatidylethanolamine which can be activated for attachment of targeting moieties
  • derivatized lipophilic compounds such as lipid-derivatized bleomycin
  • Antibody-targeted liposomes can be constructed using, for instance, liposomes that incorporate protein A (see, e.g., Renneisen et al., J. Bio. Chem., 265:16337-16342, 1990 and Leonetti et al., Proc. Natl. Acad. Sci . (USA), 87:2448-2451, 1990).
  • Other examples of antibody conjugation are disclosed in U.S. Pat. No. 6,027,726.
  • targeting moieties can also include other polypeptides that are specific to cellular components, including antigens associated with neoplasms or tumors.
  • Polypeptides used as targeting moieties can be attached to the liposomes via covalent bonds (see, for example Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)).
  • Other targeting methods include the biotin-avidin system.
  • a lipid nanoparticle of the disclosure includes a targeting moiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatic tumor cells).
  • a targeting moiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes, colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem
  • the targeting moiety targets the lipid nanoparticle to a hepatocyte. In other embodiments, the targeting moiety targets the lipid nanoparticle to a colon cell. In some embodiments, the targeting moiety targets the lipid nanoparticle to a liver cancer cell (e.g., a hepatocellular carcinoma cell) or a colorectal cancer cell (e.g., a primary tumor or a metastasis).
  • a liver cancer cell e.g., a hepatocellular carcinoma cell
  • a colorectal cancer cell e.g., a primary tumor or a metastasis
  • lipid nanoparticles are provided.
  • a lipid nanoparticle comprises lipids including an ionizable lipid, a structural lipid, a phospholipid, and one or more mRNAs.
  • Each of the LNPs described herein may be used as a formulation for the mRNA described herein.
  • a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, a PEG-modified lipid and one or more mRNAs.
  • the LNP comprises an ionizable lipid, a PEG-modified lipid, a sterol and a phospholipid.
  • the LNP has a molar ratio of about 20-60% ionizable lipid:about 5-25% phospholipid:about 25-55% sterol; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% cholesterol and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about 10% phospholipid.
  • the ionizable lipid is an ionizable amino or cationic lipid and the neutral lipid is a phospholipid, and the sterol is a cholesterol.
  • the LNP has a molar ratio of 50:38.5:10:1.5 of ionizable lipid:cholesterol:DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine):PEG-DMG.
  • the present disclosure provides pharmaceutical compositions with advantageous properties.
  • the lipids described herein e.g. those having any of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId), (IIe), (III), (IV), (V), or (VI) may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or organs.
  • the lipids described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • a reference lipid e.g., MC3, KC2, or DLinDMA
  • the present application provides pharmaceutical compositions comprising:
  • the delivery agent comprises a lipid compound having the Formula (I)
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —N(R)R 8 , —O(CH 2 ) n OR,
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or stereoisomers thereof.
  • a subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , and —C(R)N(R)OR, and each n is independently
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • alkyl and alkenyl groups may be linear or branched.
  • a subset of compounds of Formula (I) includes those in which when R 4 is —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, or —CQ(R) 2 , then (i) Q is not —N(R) 2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN(
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN(
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • Another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • R 8 is selected from the group consisting of C 3-6 carbocycle and heterocycle
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1-6 alkyl, —OR, —S(O) 2 R, —S(O) 2 N(R) 2 , C 2-6 alkenyl, C 3-6 carbocycle and heterocycle;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • Another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, —CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —CRN
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 2-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is —(CH 2 ) n Q or —(CH 2 ) n CHQR, where Q is —N(R) 2 , and n is selected from 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 2-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is —(CH 2 ) n Q or —(CH 2 ) n CHQR, where Q is —N(R) 2 , and n is selected from 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • Another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-30 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is —N(R) 2 , and n is selected from 1, 2, 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, —S—S—, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • Another subset of compounds of Formula (I) includes those in which
  • R 1 is selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R*YR′′, —YR′′, and —R′′M′R′;
  • R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle;
  • R 4 is selected from the group consisting of —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is —N(R) 2 , and n is selected from 1, 2, 3, 4, and 5;
  • each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R 6 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • R 7 is selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H;
  • each R′ is independently selected from the group consisting of C 1-18 alkyl, C 2-18 alkenyl, —R*YR′′, —YR′′, and H;
  • each R′′ is independently selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 1-12 alkenyl;
  • each Y is independently a C 3-6 carbocycle
  • each X is independently selected from the group consisting of F, Cl, Br, and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,
  • a subset of compounds of Formula (I) includes those of Formula (IA):
  • M 1 is a bond or M′;
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • a subset of compounds of Formula (I) includes those of Formula (IA), or a salt or stereoisomer thereof,
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which Q is OH, —NHC(S)N(R) 2 , or —NHC(O)N(R) 2 ;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • a subset of compounds of Formula (I) includes those of Formula (II):
  • M 1 is a bond or M′
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • a subset of compounds of Formula (I) includes those of Formula (II), or a salt or stereoisomer thereof, wherein
  • 1 is selected from 1, 2, 3, 4, and 5;
  • M 1 is a bond or M′
  • R 4 is unsubstituted C 1-3 alkyl, or —(CH 2 ) n Q, in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R) 2 , or —NHC(O)N(R) 2 ;
  • M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, an aryl group, and a heteroaryl group; and
  • R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C 2-14 alkenyl.
  • the compound of formula (I) is of the formula (IIa),
  • the compound of formula (I) is of the formula (IIb),
  • the compound of formula (I) is of the formula (IIc),
  • the compound of formula (I) is of the formula (IIe):
  • the compound of formula (IIa), (IIb), (IIc), or (IIe) comprises an R 4 which is selected from —(CH 2 ) n Q and —(CH 2 ) n CHQR, wherein Q, R and n are as defined above.
  • Q is selected from the group consisting of —OR, —OH, —O(CH 2 ) n N(R) 2 , —OC(O)R, —CX 3 , —CN, —N(R)C(O)R, —N(H)C(O)R, —N(R)S(O) 2 R, —N(H)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(H)C(O)N(R) 2 , —N(H)C(O)N(H)(R), —N(R)C(S)N(R) 2 , —N(H)C(S)N(R) 2 , —N(H)C(S)N(H)(R), and a heterocycle, wherein R is as defined above.
  • n is 1 or 2.
  • Q is OH, —NHC(S)N(R)
  • the compound of formula (I) is of the formula (IId),
  • R 2 and R 3 are independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl, n is selected from 2, 3, and 4, and R′, R′′, R 5 , R 6 and m are as defined above.
  • R 2 is C 8 alkyl. In some aspects of the compound of formula (IId), R 3 is C 5 -C 9 alkyl. In some aspects of the compound of formula (IId), m is 5, 7, or 9. In some aspects of the compound of formula (IId), each R 5 is H. In some aspects of the compound of formula (IId), each R 6 is H.
  • the present application provides a lipid composition (e.g., a lipid nanoparticle (LNP)) comprising: (1) a compound having the formula (I); (2) optionally a helper lipid (e.g. a phospholipid); (3) optionally a structural lipid (e.g. a sterol); and (4) optionally a lipid conjugate (e.g. a PEG-lipid).
  • the lipid composition e.g., LNP
  • the lipid composition further comprises a polynucleotide encoding a polypeptide of interest, e.g., a polynucleotide encapsulated therein.
  • alkyl or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms).
  • C 1-14 alkyl means a linear or branched, saturated hydrocarbon including 1-14 carbon atoms.
  • An alkyl group can be optionally substituted.
  • alkenyl or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond.
  • C 2-14 alkenyl means a linear or branched hydrocarbon including 2-14 carbon atoms and at least one double bond.
  • An alkenyl group can include one, two, three, four, or more double bonds.
  • C 18 alkenyl can include one or more double bonds.
  • a C 18 alkenyl group including two double bonds can be a linoleyl group.
  • An alkenyl group can be optionally substituted.
  • “carbocycle” or “carbocyclic group” means a mono- or multi-cyclic system including one or more rings of carbon atoms. Rings can be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen membered rings.
  • C 3-6 carbocycle means a carbocycle including a single ring having 3-6 carbon atoms.
  • Carbocycles can include one or more double bonds and can be aromatic (e.g., aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. Carbocycles can be optionally substituted.
  • heterocycle or “heterocyclic group” means a mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom.
  • Heteroatoms can be, for example, nitrogen, oxygen, or sulfur atoms. Rings can be three, four, five, six, seven, eight, nine, ten, eleven, or twelve membered rings.
  • Heterocycles can include one or more double bonds and can be aromatic (e.g., heteroaryl groups).
  • heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl groups. Heterocycles can be optionally substituted.
  • a “biodegradable group” is a group that can facilitate faster metabolism of a lipid in a subject.
  • a biodegradable group can be, but is not limited to, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group.
  • an “aryl group” is a carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups.
  • heteroaryl group is a heterocyclic group including one or more aromatic rings.
  • heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted.
  • M and M′ can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole, and thiazole. In the formulas herein, M and M′ can be independently selected from the list of biodegradable groups above.
  • Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groups can be optionally substituted unless otherwise specified.
  • Optional substituents can be selected from the group consisting of, but are not limited to, a halogen atom (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., a hydroxyl, —OH), an ester (e.g., —C(O)OR or —OC(O)R), an aldehyde (e.g., —C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C ⁇ O), an acyl halide (e.g., —C(O)X, in which X is a halide selected from bromide, fluoride, chloride,
  • R is an alkyl or alkenyl group, as defined herein.
  • the substituent groups themselves can be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein.
  • a C 1-6 alkyl group can be further substituted with one, two, three, four, five, or six substituents as described herein.
  • the compounds of any one of formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId), and (IIe) include one or more of the following features when applicable.
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, 5- to 14-membered aromatic or non-aromatic heterocycle having one or more heteroatoms selected from N, O, S, and P, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —N(R) 2 , —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —C(R)N(R) 2 C(O
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —C(R)N(R) 2 C(O)
  • R 4 is selected from the group consisting of a C 3-6 carbocycle, —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, —OR, —O(CH 2 ) n N(R) 2 , —C(O)OR, —OC(O)R, —CX 3 , —CX 2 H, —CXH 2 , —CN, —C(O)N(R) 2 , —N(R)C(O)R, —N(R)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(R)C(S)N(R) 2 , —C(R)N(R) 2 C(O
  • R 4 is unsubstituted C 1-4 alkyl, e.g., unsubstituted methyl.
  • the disclosure provides a compound having the Formula (I), wherein R 4 is —(CH 2 ) n Q or —(CH 2 ) n CHQR, where Q is —N(R) 2 , and n is selected from 3, 4, and 5.
  • the disclosure provides a compound having the Formula (I), wherein R 4 is selected from the group consisting of —(CH 2 ) n Q, —(CH 2 ) n CHQR, —CHQR, and —CQ(R) 2 , where Q is —N(R) 2 , and n is selected from 1, 2, 3, 4, and 5.
  • the disclosure provides a compound having the Formula (I), wherein R 2 and R 3 are independently selected from the group consisting of C 2-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle, and R 4 is —(CH 2 ) n Q or —(CH 2 ) n CHQR, where Q is —N(R) 2 , and n is selected from 3, 4, and 5.
  • R 2 and R 3 are independently selected from the group consisting of C 2-14 alkyl, C 2-14 alkenyl, —R*YR′′, —YR′′, and —R*OR′′, or R 2 and R 3 , together with the atom to which they are attached, form a heterocycle or carbocycle.
  • R 1 is selected from the group consisting of C 5-20 alkyl and C 5-20 alkenyl.
  • R 1 is selected from the group consisting of —R*YR′′, —YR′′, and —R′′M′R′.
  • R 1 is selected from —R*YR′′ and —YR′′.
  • Y is a cyclopropyl group.
  • R* is C 8 alkyl or C 8 alkenyl.
  • R′′ is C 3-12 alkyl.
  • R′′ can be C 3 alkyl.
  • R′′ can be C 4-8 alkyl (e.g., C 4 , C 5 , C 6 , C 7 , or C 8 alkyl).
  • R 1 is C 5-20 alkyl. In some embodiments, R 1 is C 6 alkyl. In some embodiments, R 1 is C 8 alkyl. In other embodiments, R 1 is C 9 alkyl. In certain embodiments, R 1 is C 14 alkyl. In other embodiments, R 1 is C 18 alkyl.
  • R 1 is C 5-20 alkenyl. In certain embodiments, R 1 is C 18 alkenyl.
  • R 1 is linoleyl
  • R 1 is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, or heptadeca-9-yl).
  • R 1 is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl, or heptadeca-9-yl).
  • R 1 is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl,
  • R 1 is unsubstituted C 5-20 alkyl or C 5-20 alkenyl.
  • R′ is substituted C 5-20 alkyl or C 5-20 alkenyl (e.g., substituted with a C 3-6 carbocycle such as 1-cyclopropylnonyl).
  • R 1 is —R′′M′R′.
  • R′ is selected from —R*YR′′ and —YR′′.
  • Y is C 3-8 cycloalkyl.
  • Y is C 6-10 aryl.
  • Y is a cyclopropyl group.
  • Y is a cyclohexyl group.
  • R* is C 1 alkyl.
  • R′′ is selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl.
  • R′′ adjacent to Y is C 1 alkyl.
  • R′′ adjacent to Y is C 4-9 alkyl (e.g., C 4 , C 5 , C 6 , C 7 or C 8 or C 9 alkyl).
  • R′ is selected from C 4 alkyl and C 4 alkenyl. In certain embodiments, R′ is selected from C 5 alkyl and C 5 alkenyl. In some embodiments, R′ is selected from C 6 alkyl and C 6 alkenyl. In some embodiments, R′ is selected from C 7 alkyl and C 7 alkenyl. In some embodiments, R′ is selected from C 9 alkyl and C 9 alkenyl.
  • R′ is selected from C 11 alkyl and C 11 alkenyl. In other embodiments, R′ is selected from C 12 alkyl, C 12 alkenyl, C 13 alkyl, C 13 alkenyl, C 14 alkyl, C 14 alkenyl, C 15 alkyl, C 15 alkenyl, C 16 alkyl, C 16 alkenyl, C 17 alkyl, C 17 alkenyl, C 18 alkyl, and C 18 alkenyl.
  • R′ is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl or heptadeca-9-yl).
  • R′ is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, tetradecan-6-yl, 2-methylundecan-3-yl, 2-methyldecan-2-yl, 3-methylundecan-3-yl, 4-methyldodecan-4-yl or heptadeca-9-yl).
  • R′ is branched (e.g., decan-2-yl, undecan-3-yl, dodecan-4-yl, tridecan-5-yl, t
  • R′ is unsubstituted C 1-18 alkyl.
  • R′ is substituted C 1-8 i alkyl (e.g., C 1-15 alkyl substituted with a C 3-6 carbocycle such as 1-cyclopropylnonyl).
  • R′′ is selected from the group consisting of C 3-14 alkyl and C 3-14 alkenyl. In some embodiments, R′′ is C 3 alkyl, C 4 alkyl, C 5 alkyl, C 6 alkyl, C 7 alkyl, or C 8 alkyl. In some embodiments, R′′ is C 9 alkyl, C 10 alkyl, C 11 alkyl, C 12 alkyl, C 13 alkyl, or C 14 alkyl.
  • M′ is —C(O)O—. In some embodiments, M′ is —OC(O)—.
  • M′ is an aryl group or heteroaryl group.
  • M′ can be selected from the group consisting of phenyl, oxazole, and thiazole.
  • M is —C(O)O—. In some embodiments, M is —OC(O)—. In some embodiments, M is —C(O)N(R′)—. In some embodiments, M is —P(O)(OR′)O—.
  • M is an aryl group or heteroaryl group.
  • M can be selected from the group consisting of phenyl, oxazole, and thiazole.
  • M is the same as M′. In other embodiments, M is different from M′.
  • each R 5 is H. In certain such embodiments, each R 6 is also H.
  • R 7 is H. In other embodiments, R 7 is C 1-3 alkyl (e.g., methyl, ethyl, propyl, or i-propyl).
  • R 2 and R 3 are independently C 5-14 alkyl or C 5-14 alkenyl.
  • R 2 and R 3 are the same. In some embodiments, R 2 and R 3 are C 8 alkyl. In certain embodiments, R 2 and R 3 are C 2 alkyl. In other embodiments, R 2 and R 3 are C 3 alkyl. In some embodiments, R 2 and R 3 are C 4 alkyl. In certain embodiments, R 2 and R 3 are C 5 alkyl. In other embodiments, R 2 and R 3 are C 6 alkyl. In some embodiments, R 2 and R 3 are C 7 alkyl.
  • R 2 and R 3 are different. In certain embodiments, R 2 is C 8 alkyl.
  • R 3 is C 1-7 (e.g., C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , or C 7 alkyl) or C 9 alkyl.
  • R 7 and R 3 are H.
  • R 2 is H.
  • m is 5, 7, or 9.
  • R 4 is selected from —(CH 2 ) n Q and —(CH 2 ) n CHQR.
  • Q is selected from the group consisting of —OR, —OH, —O(CH 2 ) n N(R) 2 , —OC(O)R, —CX 3 , —CN, —N(R)C(O)R, —N(H)C(O)R, —N(R)S(O) 2 R, —N(H)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(H)C(O)N(R) 2 , —N(H)C(O)N(H)(R), —N(R)C(S)N(R) 2 , —N(H)C(S)N(R) 2 , —N(H)C(S)N(H)(R), —C(R)N(R) 2 C(O)OR, a carbocycle, and a heterocycle.
  • Q is —OH
  • Q is a substituted or unsubstituted 5- to 10-membered heteroaryl, e.g., Q is an imidazole, a pyrimidine, a purine, 2-amino-1,9-dihydro-6H-purin-6-one-9-yl (or guanin-9-yl), adenin-9-yl, cytosin-1-yl, or uracil-1-yl.
  • Q is a substituted 5- to 14-membered heterocycloalkyl, e.g., substituted with one or more substituents selected from oxo ( ⁇ O), OH, amino, and C 1-3 alkyl.
  • Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, or isoindolin-2-yl-1,3-dione.
  • Q is an unsubstituted or substituted C 6-10 aryl (such as phenyl) or C 3-6 cycloalkyl.
  • n is 1. In other embodiments, n is 2. In further embodiments, n is 3. In certain other embodiments, n is 4.
  • R 4 can be —(CH 2 ) 2 OH.
  • R 4 can be —(CH 2 ) 3 OH.
  • R 4 can be —(CH 2 ) 4 OH.
  • R 4 can be benzyl.
  • R 4 can be 4-methoxybenzyl.
  • R 4 is a C 3-6 carbocycle. In some embodiments, R 4 is a C 3-6 cycloalkyl.
  • R 4 can be cyclohexyl optionally substituted with e.g., OH, halo, C 1-6 alkyl, etc.
  • R 4 can be 2-hydroxycyclohexyl.
  • R is H.
  • R is unsubstituted C 1-3 alkyl or unsubstituted C 2-3 alkenyl.
  • R 4 can be —CH 2 CH(OH)CH 3 or —CH 2 CH(OH)CH 2 CH 3 .
  • R is substituted C 1-3 alkyl, e.g., CH 2 OH.
  • R 4 can be —CH 2 CH(OH)CH 2 OH.
  • R 2 and R 3 together with the atom to which they are attached, form a heterocycle or carbocycle. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form a 5- to 14-membered aromatic or non-aromatic heterocycle having one or more heteroatoms selected from N, O, S, and P. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form an optionally substituted C 3-20 carbocycle (e.g., C 3-18 carbocycle, C 3-15 carbocycle, C 3-12 carbocycle, or C 3-10 carbocycle), either aromatic or non-aromatic.
  • C 3-20 carbocycle e.g., C 3-18 carbocycle, C 3-15 carbocycle, C 3-12 carbocycle, or C 3-10 carbocycle
  • R 2 and R 3 together with the atom to which they are attached, form a C 3-6 carbocycle.
  • R 2 and R 3 together with the atom to which they are attached, form a C 6 carbocycle, such as a cyclohexyl or phenyl group.
  • the heterocycle or C 3-6 carbocycle is substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms).
  • R 2 and R 3 together with the atom to which they are attached, can form a cyclohexyl or phenyl group bearing one or more C 5 alkyl substitutions.
  • the heterocycle or C 3-6 carbocycle formed by R 2 and R 3 is substituted with a carbocycle groups.
  • R 2 and R 3 together with the atom to which they are attached, can form a cyclohexyl or phenyl group that is substituted with cyclohexyl.
  • R 2 and R 3 together with the atom to which they are attached, form a C 7-15 carbocycle, such as a cycloheptyl, cyclopentadecanyl, or naphthyl group.
  • R 4 is selected from —(CH 2 ) n Q and —(CH 2 ) n CHQR.
  • Q is selected from the group consisting of —OR, —OH, —O(CH 2 ) n N(R) 2 , —OC(O)R, —CX 3 , —CN, —N(R)C(O)R, —N(H)C(O)R, —N(R)S(O) 2 R, —N(H)S(O) 2 R, —N(H)S(O) 2 R, —N(R)C(O)N(R) 2 , —N(H)C(O)N(R) 2 , —N(H)C(O)N(H)(R), —N(R)C(S)N(R) 2 , —N(H)C(S)N(R) 2 , —N(H)C(S)N(H)(R), and a heterocycle
  • R 2 and R 3 together with the atom to which they are attached, form a heterocycle or carbocycle.
  • R 2 and R 3 together with the atom to which they are attached, form a C 3-6 carbocycle, such as a phenyl group.
  • the heterocycle or C 3-6 carbocycle is substituted with one or more alkyl groups (e.g., at the same ring atom or at adjacent or non-adjacent ring atoms).
  • R 2 and R 3 together with the atom to which they are attached, can form a phenyl group bearing one or more C 5 alkyl substitutions.
  • the pharmaceutical compositions of the present disclosure is selected from the group consisting of:
  • the compound of Formula (I) is selected from the group consisting of Compound 1-Compound 147, or salt or stereoisomers thereof.
  • ionizable lipids including a central piperazine moiety are provided.
  • the lipids described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents to mammalian cells or organs.
  • the lipids described herein have little or no immunogenicity.
  • the lipid compounds disclosed herein have a lower immunogenicity as compared to a reference lipid (e.g., MC3, KC2, or DLinDMA).
  • a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index as compared to a corresponding formulation which comprises a reference lipid (e.g., MC3, KC2, or DLinDMA) and the same therapeutic or prophylactic agent.
  • a reference lipid e.g., MC3, KC2, or DLinDMA
  • the delivery agent comprises a lipid compound having the formula (III)
  • t 1 or 2;
  • a 1 and A 2 are each independently selected from CH or N;
  • Z is CH 2 or absent wherein when Z is CH 2 , the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from the group consisting of C 5-20 alkyl, C 5-20 alkenyl, —R′′MR′, —R*YR′′, —YR′′, and —R*OR′′;
  • each M is independently selected from the group consisting of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O) 2 —, an aryl group, and a heteroaryl group;
  • X 1 , X 2 , and X 3 are independently selected from the group consisting of a bond, —CH 2 —, —(CH 2 ) 2 —, —CHR—, —CHY—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)—CH 2 —, —CH 2 —C(O)—, —C(O)O—CH 2 —, —OC(O)—CH 2 —, —CH 2 —C(O)O—, —CH 2 —OC(O)—, —CH(OH)—, —C(S)—, and —CH(SH—;
  • each Y is independently a C 3-6 carbocycle
  • each R* is independently selected from the group consisting of C 1-12 alkyl and C 2-12 alkenyl;
  • each R is independently selected from the group consisting of C 1-3 alkyl and a C 3-6 carbocycle;
  • each R′ is independently selected from the group consisting of C 1-12 alkyl, C 2-12 alkenyl, and H;
  • each R′′ is independently selected from the group consisting of C 3-12 alkyl and C 3-12 alkenyl

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US10881730B2 (en) 2021-01-05
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US20190175727A1 (en) 2019-06-13
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US20210128721A1 (en) 2021-05-06
WO2018144775A1 (en) 2018-08-09
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