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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/208—IL-12
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2086—IL-13 to IL-16
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
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  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
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  • 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
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
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  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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  • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5443—IL-15
• • 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
  • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Cancer is a disease characterized by uncontrolled cell division and growth within the body. In the United States, roughly a third of all women and half of all men will experience cancer in their lifetime. Cancers can generally be divided into two categories, solid tumors and disseminated cancers. Each type requires different considerations for developing effective therapeutic approaches.
• Disseminated cancers such as myeloid malignancies
• MDS myelodysplastic syndrome
• AML acute myeloid leukemia
• Treatment options for disseminated cancers such as myeloid malignancies, including AML, are limited, with conventional approaches such as chemotherapy and/or immunomodulatory cytokines or antibodies not being very effective in AML.
• interleukin-2 treatment alone was found not to be effective for remission maintenance therapy in AML patients (Buyse, M. et al. (2000) Blood 117:26).
• the treatment of solid tumors includes surgery, chemotherapy and/or radiotherapy. In surgery, most of the tumor or even the invaded organ is excised.
• Chemotherapy includes the use of drugs to destroy cancer cells. Some cancers are curable by chemotherapy while others are not. Chemotherapeutic drugs can affect not only cancer cells but also other rapidly dividing normal cells such as those in the gastrointestinal tract, bone marrow, hair follicles, and reproductive system which result in several side effects.
• Radiotherapy includes the use of x-rays to treat cancers. Some are curable by radiotherapy while others are not. With the host of undesired consequences brought about by standard treatments such as chemotherapy and radiotherapy used today, genetic therapy and immunotherapy approaches provide a more targeted approach to disease diagnosis, treatment and management. Therefore, there is a need for improved therapeutic approaches to treat cancer, including solid tumors and disseminated cancers, e.g., myeloid malignancies such as AML.
• the present disclosure relates to methods and compositions for treating cancer in a subject.
• the disclosure is based, at least in part, upon the discovery that administration of a combination of mRNAs encoding one or more cell-associated cytokines (e.g., IL-12 and IL- 15) and cell-associated costimulatory molecules (e.g., OX40L) induce T cell activation, NK cell activation or both T cell and NK cell activation, resulting in anti-tumor efficacy in solid tumors and disseminated cancers, such as myeloid malignancies (e.g., AML).
• cell-associated cytokines e.g., IL-12 and IL- 15
• cell-associated costimulatory molecules e.g., OX40L
• anti-tumor efficacy is enhanced by administration of a combination of mRNAs encoding two cell-associated cytokines (e.g., IL-12 and IL-15) with an mRNA encoding a costimulatory molecule (e.g., OX40L).
• mRNAs encoding two cell-associated cytokines (e.g., IL-12 and IL-15) with an mRNA encoding a costimulatory molecule (e.g., OX40L).
• the combination of mRNAs provides various signals to effectively induce T cell activation, NK cell activation or both T cell and NK cell activation.
• IL-15 is a unique cytokine that primarily exists bound to its high affinity receptor, IL-l5Ra.
• IL-15/IL- l5Ra complexes are shuttled to the cell surface to stimulate opposing cells through the b/g C receptor complex.
• mRNA encoding IL-15 and IL-l5Ra results in trans-presentation, thereby stimulating opposing cells having the b/g C receptor complex.
• a combination of mRNAs encoding one or more cell-associated cytokines and a costimulatory molecule provide enhanced anti-tumor efficacy for solid tumors and disseminated cancers, including myeloid malignancies, relative to a soluble, or secreted form of the same cytokine(s) due to their ability to form a stronger cancer cell:immune cell synapse, and to provide enhanced, prolonged or continuous activation of the cells with which they interact (e.g., T cells and NK cells).
• T cell activation requires three signals: signal 1 provided by interaction of MHC with a peptide; signal 2 provided by costimulatory molecules, such as OX40L; and signal 3 provided by immune potentiating molecules, including cytokines, such as IL-12 and IL-15. To establish anti-tumor immunity driven by T cells, all three signals are required. However, the tumor
• microenvironment often fails to provide the necessary signals to activate T cells and potentiate an anti-tumor immune response.
• costimulatory molecule such as human OX40L
• an mRNA encoding one or more cell-associated immune potentiating molecules e.g., mRNA encoding one or more cell-associated cytokines providing signal 3 (e.g., human IL-12, human IL-l5/IL-l5Ra), to a T cell in a tumor microenvironment by expression of the mRNAs by a cell (e.g., a leukemic cell or antigen presenting cell, such as a dendritic cell), T cells are activated to induce an anti tumor immune response. Further, by restricting exposure of the cytokines and costimulatory molecules to the cells that express the mRNA, systemic exposure, and potentially undesirable toxicity, is avoided.
• leukemic cells have reduced and/or downregulated expression of the costimulatory molecules CD80 and CD86 (Yaho, S. and Chen, L., Eur J. Immunol. 2013, 43(3): 576-579; and Hirano N, et ah, Leukemia. 1996, 10:1168-1176).
• a strong costimulatory signal is provided where it may be absent or downregulated, or if not absent, such as on dendritic cells, may augment or enhance existing costimulatory signals to provide a stronger synapse between T cells and leukemic and/or dendritic cells expressing the mRNA encoding OX40L and induce an anti-tumor immune response by T cells.
• mRNA encoding the costimulatory molecule e.g., mRNA encoding OX40L
• a strong anti-tumor effect resulted, compared to treatment without the co stimulatory molecule, thus demonstrating the theory as described herein.
• mRNAs encoding one or more cell-associated cytokines e.g., human IL-12, human IL-15/IL-15Ra
• an mRNA encoding a costimulatory molecule e.g., human OX40L
• a durable anti-cancer memory response in a disseminated cancer model that engrafts in hematopoietic tissues in a manner similar to that seen in AML patients.
• the anti-cancer immune response prevented relapse and recurrence of disease in this model. This effect is believed to be the first demonstration of an anti-cancer memory response by administration of mRNA therapeutics in a disseminated cancer model.
• fractionated dosing regimen provides greater or enhanced exposure to the mRNA encoded polypeptides in a subject, resulting in enhanced anti-tumor efficacy with reduced toxicity and better tolerability.
• biodistribution studies indicate that not only do the mRNA encapsulated lipid nanoparticles transfect leukemic cells, but a variety of immune cells, including dendritic cells. These studies suggest that the combination therapy of one or more mRNAs encoding cell-associated cytokines and an mRNA encoding a costimulatory molecule, such as human OX40L, would be useful for treating a variety of disseminated cancers, including cancers having significant myeloid populations such as AML, as well as multiple myeloma and B cell leukemias.
• the combination therapy described herein was also demonstrated to be efficacious in establishing anti-tumor immunity in solid tumors in animal models with various tumor microenvironments. Without being bound by theory, it is believed that the anti-tumor efficacy observed in immune checkpoint resistant tumor models and immunosuppressive tumor models demonstrates the effectiveness of T, NK, NKT and dendritic cell activation following administration of one or more mRNAs encoding cell-associated cytokines and an mRNA encoding a costimulatory molecule, such as human OX40L.
• mRNAs encoding one or more cell-associated cytokines e.g., human IL- 12, human IL-l5/IL-l5Ra
• an mRNA encoding a costimulatory molecule e.g., human OX40L
• a costimulatory molecule e.g., human OX40L
• a costimulatory molecule e.g., human OX40L
• NHP non-human primates
• immunostimulatory cytokines e.g., IFN-g and CXCL
• the anti-tumor efficacy mediated by the administration of one or more mRNAs encoding cell-associated cytokines and an mRNA encoding a costimulatory molecule, such as human OX40L requires CD8+ T cells, CD4+ T cells, as well as IFN-g, as experiments in animals depleted of CD8+ T cells, CD4+ T cells, or IFN-g resulted in increased disease burden and decreased survival.
• intratumoral administration of the combination therapy in solid tumors was found to induce an abscopal effect, indicating an anti-tumor immune response which is efficacious against distal, untreated tumors.
• compositions and methods for treating cancer by providing a combination of two or more mRNA(s) encoding cell-associated cytokines and an mRNA encoding a costimulatory molecule which activate particular immune cells, thereby enhancing an immune response against the cancer.
• cancers including myeloid malignancies, such as AML, are known to evade immune responses by a variety of mechanisms.
• compositions and methods of the disclosure are useful for activating innate immunity, activating adaptive immunity and/or activating memory responses against a cancer, e.g., a solid tumor and/or a disseminated cancer, e.g., a myeloid malignancy, such as AML.
• a cancer e.g., a solid tumor and/or a disseminated cancer, e.g., a myeloid malignancy, such as AML.
• the mRNA is a modified mRNA.
• the disclosure provides a method of treating cancer in a human patient, comprising administering to the patient:
• the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells.
• the disclosure provides a method of treating cancer in a human patient, comprising administering to the patient:
• the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells,
• first mRNA and at least one second mRNA are encapsulated in the same or different lipid nanoparticles.
• the at least one second mRNA is:
• an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain or (iii) an mRNA encoding a trans-presented human IL-15 and an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain.
• the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering at least two mRNAs selected from the group consisting of:
• the cancer is a disseminated cancer and the first mRNA and the at least one second mRNA are administered systemically.
• the disseminated cancer is a hematological cancer.
• the disseminated cancer is a myeloid malignancy.
• the myeloid malignancy is selected from the group consisting of myeloidysplastic syndrome (MDS), myeloproliferative disorder (MPD) and acute myeloid leukemia (AML).
• MDS myeloidysplastic syndrome
• MPD myeloproliferative disorder
• AML acute myeloid leukemia
• the cancer is a solid tumor and wherein the first mRNA and the at least one second mRNA are administered intratumorally.
• the disclosure provides a method of treating a solid tumor in a subject in need thereof, comprising administering (e.g., intratumorally) at least two mRNAs selected from the group consisting of:
• the disclosure provides a method of treating a disseminated cancer in a subject in need thereof, comprising administering (e.g., systemically, e.g., by intravenous injection) at least two mRNAs selected from the group consisting of:
• the disclosure provides a method of treating a myeloid malignancy in a subject in need thereof, comprising administering (e.g., systemically, e.g., by intravenous injection) at least two mRNAs selected from the group consisting of:
• the at least two mRNAs are selected from the group consisting of:
• an mRNA encoding a human OX40L polypeptide an mRNA encoding a human IL-15 polypeptide, an mRNA encoding a human IL-l5Ra polypeptide and an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain;
• the disclosure provides a method for treating a disseminated cancer in a human patient, comprising systemically administering to the patient:
• the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells,
• the method comprises administering a third mRNA encoding a second immune potentiator, wherein the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells.
• the second mRNA encodes a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain and the third mRNA encodes a trans-presented human IL-15.
• the disclosure provides a method of treating a disseminated cancer in a human patient, comprising systemically administering to the patient a pharmaceutical composition comprising a lipid nanoparticle (LNP) and a pharmaceutically acceptable carrier, wherein the LNP comprises:
• the method comprises administering a third mRNA encoding a second immune potentiator, wherein the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells.
• the second mRNA encodes a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain and the third mRNA encodes a trans-presented human IL-15.
• the disclosure provides a method of treating a disseminated cancer in a human patient, comprising administering to the patient a dosing regimen comprising: (i) a first fractionated dose of a pharmaceutical composition comprising a first mRNA encoding human OX40L, and at least one second mRNA encoding an immune potentiator, wherein the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells, and
• the first fractionated dose and second fractionated dose enhance anti-tumor efficacy of the treatment relative to a single dose of the same amount of mRNA. In some aspects, the first fractionated dose and second fractionated dose enhance anti-tumor efficacy with reduced toxicity and better tolerability.
• the method comprises administering a third mRNA encoding a second immune potentiator, wherein the immune potentiator is a cell- associated cytokine that activates T cells, NK cells, or both T cells and NK cells.
• the second mRNA encodes a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain and the third mRNA encodes a trans-presented human IL-15.
• the cell-associated cytokine is a human IL- 12 polypeptide operably linked to a membrane domain comprising a transmembrane domain.
• the cell-associated cytokine is a trans-presented human IL-15.
• the trans-presented human IL-15 is a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide.
• the trans-presented human IL-15 is encoded by a first mRNA encoding a human IL-15 polypeptide and a second mRNA encoding a human IL-l5Ra polypeptide.
• the mRNA is formulated in the same lipid nanoparticle (LNP). In any of the foregoing or related aspects, each mRNA is formulated in the same LNP. In other aspects, each mRNA is formulated in a separate LNP.
• LNP lipid nanoparticle
• the mRNAs of the disclosure are formulated in the same or different LNP(s), wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5- 25% phospholipid: 25-55% structural lipid; and 0.5-15% PEG-modified lipid.
• the LNP comprises a molar ratio of about 50% ionizable lipid: about 10% phospholipid: about 38.5% sterol; and about 1.5% PEG-modified lipid.
• the LNP comprises a molar ratio of 50:38.5:10:1.5 of ionizable lipid: cholesterol: DSPC: PEG-modified lipid.
• the mRNAs of the disclosure are formulated in the same or different LNP(s), wherein the LNP comprises about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % non-cationic helper lipid or phospholipid, about 18.5 mol % to about 48.5 mol % sterol or other structural lipid, and about 0 mol % to about 10 mol % PEG lipid.
• the LNP comprises about 50 mol % ionizable lipid, about 10 mol % non-cationic helper lipid or phospholipid, about 38.5 mol % sterol or other structural lipid, and about 1.5 mol % PEG lipid.
• the ionizable lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin- KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
• the ionizable lipid comprises Compound X.
• the LNP comprises a molar ratio of about 20-60% Compound X: 5-25% phospholipid: 25-55% cholesterol; and 0.5-15% PEG-modified lipid. In some aspects, the LNP comprises a molar ratio of about 50% Compound X: about 10% phospholipid: about 38.5% cholesterol; and about 1.5% PEG-modified lipid.
• the PEG-modified lipid is PEG-DMG or Compound P-428.
• the LNP comprises a molar ratio of 50:38.5:10:1.5 of Compound X: cholesterol: phospholipid: Compound P-428, or of Compound X: cholesterol: DSPC: Compound P-428.
• the LNP comprises a molar ratio of
• the LNP comprises a phytosterol or a combination of a phytosterol and cholesterol.
• the phytosterol is selected from the group consisting of b-sitosterol, stigmasterol, b-sitostanol, campesterol,
• the phytosterol comprises (i) a sitosterol or a salt or an ester thereof, or (ii) a stigmasterol or a salt or an ester thereof. In some aspects, the phytosterol is beta-sitosterol salt or an ester thereof.
• the phytosterol or a salt or ester thereof is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S- 160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175 and combinations thereof.
• the mol % sterol or other structural lipid is 18.5% phytosterol and the total mol % structural lipid is 38.5%. In other aspects, the mol% sterol or other structural lipid is 28.5% phytosterol and the total mol % structural lipid is 38.5%.
• the mRNAs of the disclosure are formulated in the same or different LNP(s), wherein the LNP comprises a molar ratio of 50:10:10:28.5:1.5 of Compound X:DSPC:cholesterol:beta-sitosterol:PEG-DMG. In some aspects, the mRNAs of the disclosure are formulated in the same or different LNP(s), wherein the LNP comprises a molar ratio of 50:10:20: 18.5:1.5 of Compound X:DSPC:cholesterol:beta-sitosterol:PEG- DMG.
• the cancer is a disseminated cancer. In some aspects, the disseminated cancer is a hematological cancer. In some aspects, the disseminated cancer is a myeloid malignancy.
• the myeloid malignancy is selected from the group consisting of myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD) and acute myeloid leukemia (AML). In some aspects, the myeloid malignancy is AML.
• the cancer is a solid tumor.
• the solid tumor is unresponsive to checkpoint inhibitor therapy.
• the solid tumor comprises an immunosuppressive tumor microenvironment.
• the at least two mRNAs are administered intratumorally. In some aspects, the at least two mRNAs are administered intravenously. In some aspects, the mRNAs are encapsulated in the same LNP and formulated in a solution suitable for intratumoral injection. In some aspects, the mRNAs are encapsulated in the same LNP and formulated in a solution suitable for intravenous injection. In other aspects, each mRNA is encapsulated in one or more separate LNPs and formulated in a solution suitable for intratumoral injection. In other aspects, each mRNA is encapsulated in one or more separate LNPs and formulated in a solution suitable for intravenous injection.
• the method for treating a cancer further comprises administering a checkpoint inhibitor polypeptide.
• a cancer e.g., solid tumor or disseminated cancer such as a myeloid malignancy
• the checkpoint inhibitor polypeptide inhibits PD-l, PD-L1, CTLA-4, or a combination thereof.
• the checkpoint inhibitor polypeptide is an antibody or an mRNA encoding the antibody.
• the antibody is an anti-CTLA-4 antibody or antigen-binding fragment thereof that specifically binds CTLA-4, an anti-PD-l antibody or antigen-binding fragment thereof that specifically binds PD-l, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
• the anti-PD-Ll antibody is atezolizumab, avelumab, or durvalumab.
• the anti-CTLA-4 antibody is tremelimumab or ipilimumab.
• the anti-PD-l antibody is nivolumab or pembrolizumab.
• an LNP comprising:
• the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells.
• an LNP comprising:
• the immune potentiator is a cell-associated cytokine that activates T cells, NK cells, or both T cells and NK cells;
• the disclosure provides an LNP comprising at least two encapsulated messenger RNAs (mRNAs), wherein the at least two mRNAs are selected from the group consisting of: (i) an mRNA encoding a human OX40L polypeptide;
• the disclosure provides a lipid nanoparticle, wherein the mRNAs are co-formulated in the same lipid nanoparticle, and wherein the mRNAs encoding human OX40L, tethered human IL-12 and cell-associated human IL-15 are co-formulated at a weight (mass) ratio of 1 : 1 : 1.
• the mRNAs of the disclosure are co-formulated in the same lipid nanoparticle, wherein the mRNAs encoding human OX40L, tethered human IL-12 and cell-associated human IL-15 are co-formulated at a weight (mass) ratio of 1:1:1, and wherein the mRNA encoding cell-associated human IL-15 is encoded by two mRNAs encoding human IL-15 and human IL-l5Ra, and wherein the two mRNAs are co-formulated at a molar ratio of 1:1.
• the at least two mRNAs are selected from the group consisting of:
• an mRNA encoding a human OX40L polypeptide an mRNA encoding a human IL-15 polypeptide, an mRNA encoding a human IL-l5Ra polypeptide and an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain;
• the disclosure provides a lipid nanoparticle comprising: an mRNA encoding a human OX40L polypeptide, an mRNA encoding a human IL-15 polypeptide, an mRNA encoding a human IL-l5Ra polypeptide and an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain.
• the disclosure provides a composition comprising: a first lipid nanoparticle encapsulating an mRNA encoding a human OX40L polypeptide, a second lipid nanoparticle encapsulating an mRNA encoding a human IL-15 polypeptide; a third lipid nanoparticle encapsulating an mRNA encoding a human IL-l5Ra polypeptide; and a fourth lipid nanoparticle encapsulating an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain.
• the disclosure provides a composition comprising: a first lipid nanoparticle encapsulating an mRNA encoding a human OX40L polypeptide, a second lipid nanoparticle encapsulating an mRNA encoding a human IL-15 polypeptide operably linked to an IL-l5Ra polypeptide; and a third lipid nanoparticle encapsulating an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a
• the mRNA encoding a human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1.
• the OX40L polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth SEQ ID NO: 11, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth SEQ ID NO: 11.
• the mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain comprises an IL-12 p40 subunit (IL-12B) polypeptide operably linked to an IL-12 p35 subunit (IL-12A) polypeptide.
• the IL-12B polypeptide is operably linked to the IL-12A polypeptide by a peptide linker.
• the IL-12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 39 or SEQ ID NO: 40.
• the IL-12 polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth SEQ ID NO: 46, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth SEQ ID NO: 46.
• the IL-12B polypeptide is located at the 5’ terminus of the IL-12A polypeptide, or the 5’ terminus of the peptide linker; or wherein the IL-12A polypeptide is located at the 5’ terminus of the IL-12B polypeptide, or the 5’ terminus of the peptide linker.
• the membrane domain is operably linked to the IL-12A polypeptide by a peptide linker. In other aspects, the membrane domain is operably linked to the IL-12B polypeptide by a peptide linker.
• the transmembrane domain comprises a transmembrane domain derived from a Type I integral membrane protein. In some aspects, the transmembrane domain is selected from the group consisting of: a Cluster of
• CD8 Differentiation 8
• PDGFR Platelet-Derived Growth Factor Receptor
• CD80 Cluster of Differentiation 80
• the PDGFR-beta transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 42.
• the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 41.
• the CD80 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 43.
• the membrane domain comprises an intracellular domain.
• the intracellular domain is derived from the same polypeptide as the transmembrane domain.
• the intracellular domain is derived from a different polypeptide than the transmembrane domain is derived from.
• the intracellular domain is selected from the group consisting of: a PDGFR intracellular domain, a truncated PDGFR intracellular domain, and a CD80 intracellular domain.
• the intracellular domain is a PDGFR intracellular domain comprising a PDGFR-beta intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 48.
• the intracellular domain is a truncated PDGFR intracellular domain comprising a PDGFR-beta intracellular domain truncated at E570 or G739.
• the truncated PDGFR-beta intracellular domain truncated at E570 comprises the amino acid sequence set forth in SEQ ID NO: 49.
• the truncated PDGFR-beta transmembrane truncated at G739 comprises the amino acid sequence set forth in SEQ ID NO: 50.
• the intracellular domain is a CD80 intracellular domain comprising the amino acid sequence set forth in SEQ ID NO: 47.
• the IL-12 polypeptide operably linked to a membrane domain comprises a membrane domain comprising:
• the mRNA encoding a human IL-l5Ra polypeptide comprises a sushi domain.
• the IL-l5Ra polypeptide comprises a sushi domain, an intracellular domain and a transmembrane domain.
• the intracellular domain and the transmembrane domain are derived from IL-l5Ra.
• the intracellular domain and the transmembrane domain are derived from a heterologous polypeptide.
• the mRNA encoding a human IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17.
• the IL-15 polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth SEQ ID NO: 122, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth SEQ ID NO: 122.
• the IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13.
• the IL-l5Ra polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth SEQ ID NO: 22, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth SEQ ID NO: 22.
• the IL-15 polypeptide operably linked to an IL-l5Ra polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27 and 123, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 23, 27 and 123.
• the IL-15 polypeptide operably linked to an IL-l5Ra polypeptide is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 24-26, 28-30 and 124-126, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the disclosure provides an LNP comprising:
• the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5- 25% phospholipid: 25-55% structural lipid; and 0.5-15% PEG-modified lipid.
• the mRNAs encoding human OX40L, tethered human IL-12 and cell- associated human IL- 15 are co-formulated in the LNP at a weight (mass) ratio of 1 : 1 : 1.
• the mRNAs encoding human OX40L, tethered human IL-12 and cell- associated human IL-15 are co-formulated at a weight (mass) ratio of 1:1:1, and wherein the mRNA encoding cell-associated human IL-15 is encoded by two mRNAs encoding human IL-15 and human IL-l5Ra, and wherein the two mRNAs are co-formulated at a molar ratio of 1: 1.
• the disclosure provides an LNP comprising:
• an mRNA encoding a human OX40L polypeptide comprising the nucleotide sequence set forth in SEQ ID NO: 11, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 11;
• an mRNA encoding a human IL-15 polypeptide comprising the nucleotide sequence set forth in SEQ ID NO: 122, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 122;
• an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 60, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID NO: 60, wherein the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5- 25% phospholipid: 25-55% structural lipid; and 0.5-15% PEG-modified lipid.
• the mRNAs encoding human OX40L, tethered human IL-12 and cell- associated human IL- 15 are co-formulated in the LNP at a weight (mass) ratio of 1 : 1 : 1. In some embodiments, the mRNAs encoding human OX40L, tethered human IL-12 and cell- associated human IL-15 are co-formulated at a weight (mass) ratio of 1:1:1, and wherein the mRNA encoding cell-associated human IL-15 is encoded by two mRNAs encoding human IL-15 and human IL-l5Ra, and wherein the two mRNAs are co-formulated at a molar ratio of 1: 1.
• the LNP comprises a range of 0.1-1: 0.1-1: 0.1-1 weight (mass) ratio of OX40L: IL-l5+IL-l5Ra:IL-l2. In one embodiment, the LNP comprises a 1:1:1 weight (mass) ratio of mRNAs encoding OX40L:IL-l5+IL-l5Ra:IL-l2. In some aspects, the LNP comprises a 1:1:1 weight (mass) ratio of mRNAs encoding OX40L:IL-l5/IL-l5Ra:IL- 12. In some aspects, the amount of mRNA encoding human OX40L polypeptide is 1/10* the amount of the remaining mRNAs in the LNP.
• the LNP is formulated for intratumoral delivery. In other aspects, the LNP is formulated for intravenous delivery.
• the LNP comprises a molar ratio of about 20-60% ionizable amino lipid: 5-25% phospholipid: 25-55% structural lipid; and 0.5-15% PEG-modified lipid.
• the lipid nanoparticle comprises a molar ratio of about 50% ionizable lipid: about 10% phospholipid: about 38.5% sterol; and about 1.5% PEG-modified lipid.
• the lipid nanoparticle comprises a molar ratio of 50:38.5:10:1.5 of ionizable lipid: cholesterol: DSPC: PEG-modified lipid.
• the ionizable lipid is selected from the group consisting of for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
• the ionizable lipid comprises Compound X.
• the LNP comprises a molar ratio of about 20-60% Compound X: 5- 25% phospholipid: 25-55% cholesterol; and 0.5-15% PEG-modified lipid.
• the lipid nanoparticle comprises a molar ratio of about 50% Compound X: about 10% phospholipid: about 38.5% cholesterol; and about 1.5% PEG-modified lipid.
• the PEG-modified lipid in the lipid nanoparticle is PEG-DMG or Compound P-428.
• the lipid nanoparticle comprises a molar ratio of 50:38.5:10:1.5 of Compound X: cholesterol: phospholipid: Compound P-428, or of Compound X: cholesterol: DSPC: Compound P-428.
• the lipid nanoparticle comprises a molar ratio of 40:38.5:20:1.5 of Compound X: cholesterol: phospholipid:
• the LNP comprises a phytosterol or a combination of a phytosterol and cholesterol.
• the phytosterol is selected from the group consisting of b- sitosterol, stigmasterol, b-sitostanol, campesterol, brassicasterol, and combinations thereof.
• the phytosterol comprises (i) a sitosterol or a salt or an ester thereof, or (ii) a stigmasterol or a salt or an ester thereof. In some aspects, the phytosterol is beta-sitosterol
• the phytosterol or a salt or ester thereof is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, Compound S-140, Compound S-151, Compound S-156, Compound S-157, Compound S-159, Compound S- 160, Compound S-164, Compound S-165, Compound S-170, Compound S-173, Compound S-175 and combinations thereof.
• the mol % sterol or other structural lipid is 18.5% phytosterol and the total mol % structural lipid is 38.5%. In other aspects, the mol% sterol or other structural lipid is 28.5% phytosterol and the total mol % structural lipid is 38.5%.
• the LNP comprises a molar ratio of 50:10:10:28.5:1.5 of Compound X:DSPC:cholesterol:beta-sitosterol:PEG-DMG.
• the mRNAs of the disclosure are formulated in the same or different LNP(s), wherein the LNP comprises a molar ratio of 50:10:20: 18.5:1.5 of Compound X:DSPC:cholesterol:beta-sitosterol:PEG- DMG.
• each mRNA comprises a 3’ untranslated region (UTR).
• the 3’UTR comprises at least one microRNA (miR) binding site.
• the at least one miR binding site is a miR-l22 binding site.
• the miR-l22 binding site is a miR-l22-3p or a miR-l22-5p binding site.
• the miR-l22-5p binding site comprises the nucleotide sequence set forth in SEQ ID NO: 83.
• the miR-l22-3p binding site comprises the nucleotide sequence set forth in SEQ ID NO: 74.
• each mRNA comprises a 3’UTR comprising the nucleotide sequence set forth in SEQ ID NO: 77 or SEQ ID NO: 121
• each mRNA comprises a 5’ untranslated region (UTR).
• the 5’UTR comprises the nucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 133.
• each mRNA includes at least one chemical modification.
• the chemical modification is selected from the group consisting of pseudouridine, Nl-methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5- methylcytosine, 2-thio- 1 -methyl- l-deaza-pseudouridine, 2-thio-l -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- l-methyl-pseudouridine, 4-thio-pseudouridine, 5 -aza- uridine, dihydropseudouridine, 5-methyluridine, 5- methyluridine, 5-methoxyuridine, and 2’
• At least 95% of uridines in each mRNA are Nl- methylpseudouridine. In some aspects, at least 99% of uridines in each mRNA are Nl- methylpseudouridine. In some aspects, 100% of uridines in each mRNA are Nl- methylpseudouridine.
• the disclosure provides methods for treating a cancer in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle as described herein. In some aspects, the disclosure provides methods for treating a
• the method comprising administering to the subject a lipid nanoparticle as described herein.
• the disclosure provides methods for treating a solid tumor in a subject in need thereof, the method comprising administering to the subject a lipid nanoparticle as described herein.
• the method further comprises administering a checkpoint inhibitor polypeptide or an mRNA encoding a checkpoint inhibitor polypeptide.
• the checkpoint inhibitor polypeptide inhibits PD-l, PD-L1, CTLA-4, or a combination thereof.
• the checkpoint inhibitor polypeptide is an antibody or an mRNA encoding the antibody.
• the antibody is an anti-CTLA-4 antibody or antigen-binding fragment thereof that specifically binds CTLA-4, an anti-PD-l antibody or antigen-binding fragment thereof that specifically binds PD-l, an anti-PD-Ll antibody or antigen-binding fragment thereof that specifically binds PD-L1, and a combination thereof.
• the anti-PD-Ll antibody is atezolizumab, avelumab, or durvalumab.
• the anti-CTLA-4 antibody is tremelimumab or ipilimumab.
• the anti-PD-l antibody is nivolumab or pembrolizumab.
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a cancer in an individual, wherein treatment comprises administration of the lipid
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual, wherein treatment comprises administration of the lipid nanoparticle.
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a solid tumor in an individual, wherein treatment comprises administration of the lipid nanoparticle.
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a cancer in an individual, wherein treatment comprises administration of the lipid
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual, wherein treatment comprises administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitory polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, for use in treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual, wherein treatment comprises administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitory polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier.
• composition for use in treating or delaying progression of a solid tumor in an individual, wherein treatment comprises administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitory polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a 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.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, 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.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a solid tumor 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.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a 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 an immune checkpoint inhibitor polypeptide or an mRNA encoding the immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, 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 an immune checkpoint inhibitor polypeptide or an mRNA encoding an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides use of a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, in the manufacture of a medicament for treating or delaying progression of a solid tumor 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 an immune checkpoint inhibitor polypeptide or an mRNA encoding an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the lipid nanoparticle for treating or delaying progression of a cancer in an individual.
• the package insert further comprises instructions for administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitor polypeptide, or an mRNA encoding an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a cancer in an individual.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the lipid nanoparticle for treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual.
• the package insert further comprises instructions for administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the lipid nanoparticle for treating or delaying progression of a solid tumor in an individual.
• the package insert further comprises instructions for administration of the lipid nanoparticle in combination with a composition comprising an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a solid tumor in an individual.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament alone, or in combination with a composition comprising an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a cancer in an individual.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament alone, or in combination with a composition comprising an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a disseminated cancer, such as a myeloid malignancy, in an individual.
• the disclosure provides a kit comprising a container comprising a lipid nanoparticle as described herein, and an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration of the medicament alone, or in combination with a composition comprising an immune checkpoint inhibitor polypeptide, and an optional pharmaceutically acceptable carrier, for treating or delaying progression of a solid tumor in an individual.
• the disclosure provides methods for enhancing an immune response in a subject, comprising administering to the subject a lipid nanoparticle or combination of mRNAs as described herein.
• the disclosure provides methods for enhancing immune cell activation in a subject, comprising administering to the subject a lipid nanoparticle or combination of mRNAs as described herein.
• the immune cell activation comprises T cell activation, NK cell activation, or both T cell and NK cell activation.
• the disclosure provides methods for enhancing NK cell activation in a subject, comprising administering to the subject a lipid nanoparticle or combination of mRNAs as described herein.
• the subject has a myeloid malignancy.
• the myeloid malignancy is AML.
• the subject has a solid tumor.
• the method further comprises administering a checkpoint inhibitor polypeptide or an mRNA encoding a checkpoint inhibitor polypeptide as described herein.
• the mRNA, pharmaceutical composition or LNP as described herein has one or more activities selected from the group consisting of (a) increasing NK, NKT, CD 8+ T, CD4+ T , and/or dendritic cell (DC) populations; (b) increasing proliferation of NK, NKT, CD8+ T cells, CD4+ T cells, and/or DCs; (c) increasing activation of NK, NKT, CD8+ T, CD4+ T, and/or dendritic cells; (d) increasing maturation of DCs; (e) decreasing disease burden in treated subject; (f) increasing survival in treated subject; (g) increasing expression of IFNy or IP10; and (h) any combinations of (a)-(g).
• DC dendritic cell
• the DC populations affected by the mRNA, pharmaceutical composition or LNP as described herein are CD8+ cDCl, CD103+ cDCl, cDC2, or iDC populations.
• FIG. 1 provides graphs showing transfection efficacy of an AML cell line (Kasumi-l) in vitro with lipid nanoparticles (LNPs) containing different PEG-modified lipids (PEG DMG or Compound 428) in the absence or presence of human serum.
• LNPs lipid nanoparticles
• FIG. 2 provides graphs showing transfection efficacy of primary AML samples in vitro with LNPs containing different PEG-modified lipids (PEG DMG or Compound 428) in presence of human serum.
• FIGs. 3A-3C are graphs showing tumor volume in mice implanted with P388D1 AML cells and treated with an mRNA encoding murine OX40L (mOX40L) formulated in an LNP (FIG. 3A), mRNAs encoding mOX40L and human IL-15 (hIL-l5) formulated in an LNP (FIG. 3B) and mRNAs encoding mOX40L, hIL-l5 and murine IL-12 (mIL-l2) formulated in an LNP (FIG. 3C). LNPs were administered intratumorally.
• mOX40L murine OX40L
• hIL-l5 human IL-15
• mIL-l2 murine IL-12
• FIGs. 4A-4D provide schematics of human IL-l5/IL-l5Ra constructs.
• FIG. 4A shows an IL-15 polypeptide (left) and an IL-l5Ra polypeptide comprising a sushi domain and a transmembrane domain (right), wherein IL-15 binds to IL-l5Ra sushi domain with high affinity, thereby restricting IL-15 to IL-l5Ra expressing cells.
• FIG. 4B shows a tethered IL- 15 construct, wherein an IL-15 polypeptide is linked to a full-length IL-l5Ra, thereby tethering IL-15 to the cell membrane.
• FIG. 4A shows an IL-15 polypeptide (left) and an IL-l5Ra polypeptide comprising a sushi domain and a transmembrane domain (right), wherein IL-15 binds to IL-l5Ra sushi domain with high affinity, thereby restricting IL-15 to IL-
• FIG. 4C shows a secreted IL-15 construct, wherein an IL-15 polypeptide is linked to the sushi domain of IL-l5Ra.
• FIG. 4D shows a tethered IL-15 constructs, wherein an IL-15 polypeptide is linked to the sushi domain of IL-l5Ra which is linked to a transmembrane domain and intracellular domain of a heterologous polypeptide (e.g. CD80).
• a heterologous polypeptide e.g. CD80
• FIGs. 5A-5D provide graphs comparing protein expression and T cell proliferation between human IL-l5/IL-l5Ra constructs described in FIGs. 4A-4C.
• FIG. 5A shows protein expression of IL-15 in the supernatant or the lysate when HeLa cells were transfected with mRNA encoding the indicated IL-l5/IL-l5Ra construct in Lipofectamine 2000.
• FIG. 5B shows proliferation of T cells when co-cultured with HeLa cells transfected with mRNA encoding the indicated IL-l5/IL-l5Ra constructs in Lipofectamine 2000.
• FIG. 5A shows protein expression of IL-15 in the supernatant or the lysate when HeLa cells were transfected with mRNA encoding the indicated IL-l5/IL-l5Ra construct in Lipofectamine 2000.
• FIG. 5B shows proliferation of T cells when co-cultured with HeLa cells transfected with mRNA encoding the indicated
• FIG. 5C shows protein expression of IL-15 in the supernatant or the lysate when HeLa cells were transfected with different mRNA versions encoding the indicated IL-l5/IL-l5Ra constructs.
• FIG. 5D shows the percent of protein shed in the supernatant (supernatant expression/lysate expression+supematant expression).
• FIGs. 6A-6F are graphs showing tumor volume in mice implanted with C1498 AML cells and treated intratumorally with LNPs encapsulating mRNAs encoding NST-mOX40L (NST) (FIG. 6A), mOX40L (FIG. 6B), hIL-l5/IL-l5Ra (FIG. 6C), membrane tethered mlL- 12 (mIL-l2TM) (FIG. 6D), mOX40L + L-l5/IL-l5Ra (FIG. 6E) or mOX40L + L-15/IL- l5Ra + mIL-l2TM (FIG. 6F).
• FIGs. 7A-7C are graphs showing percent survival of mice with AML tumors treated intratumorally with LNPs encapsulating mRNAs encoding various single agents (mOX40L or L-l5/IL-l5Ra or mIL-l2TM mRNAs) (FIG. 7A), various mOX40L + L-l5/IL-l5Ra doublet mRNAs (FIG. 7B) or various mOX40L + L-l5/IL-l5Ra + mIL-l2TM triplet mRNAs (FIG. 7C).
• various single agents mOX40L or L-l5/IL-l5Ra or mIL-l2TM mRNAs
• FIGs. 8A-8B show disease burden in mice bearing a disseminated model of AML, and treated intravenously with a combination of mRNAs encoding mouse OX40L (i.e., mOX40L), cell-associated human IL-15 (i.e., L-15 + hIL-l5Ra) and tethered mouse IL-12 (mIL-l2 linked to a PDGFR transmembrane domain, i.e., mIL-l2TM) (2 mg/kg total mRNA), formulated in separate LNPs comprising Compound X and Compound 428.
• FIG. 8A shows bioluminescence imaging (BLI)
• FIG. 8B shows the number of GFP+ cells in the blood as determined by flow cytometry.
• FIG. 9 provides graphs showing a decrease in leukemia burden in blood of mice treated intravenously with a combination of mRNAs encoding mOX40L, cell-associated L-15, and tethered mIL-l2, formulated in separate LNPs comprising Compound X and Compound 428, 21 days after implant of AML cells.
• the number of GFP+ cells in the blood was determined (left), along with the % of GFP+ of CD45+ cells (right) by flow cytometry.
• FIG. 10 provides a Kaplan-Meier survival graph showing mice from FIG. 9, and mice treated with a combination of mRNAs encoding mOX40L, cell-associated L-15 and tethered mIL-l2, formulated in separate LNPs comprising Compound X and Compound 428, at varying dosing regimens (i.e., 2 mg/kg once (QDxl); 2 mg/kg once a week for three weeks (Q7Dx3); 0.67 mg/kg once a week for three weeks (Q7Dx3); 0.22 mg/kg three times a week for three weeks (TIWx3)).
• dosing regimens i.e., 2 mg/kg once (QDxl); 2 mg/kg once a week for three weeks (Q7Dx3); 0.67 mg/kg once a week for three weeks (Q7Dx3); 0.22 mg/kg three times a week for three weeks (TIWx3)).
• FIG. 11 provides a graph showing protective immunity in mice from FIG. 9 that completely responded to combination therapy of mRNAs encoding mOX40L, cell-associated L-15 and tethered mIL-l2 at various dosing regimens, and were re-challenged with AML cells, as determined by bioluminescence imaging (BLI).
• FIG. 12 provides a Kaplan-Meier survival graph of mice re-challenged with AML cells, as described in FIG. 11.
• FIG. 13 provides graphs showing the number of GFP+ cells as determined by flow cytometry in the blood of mice re-challenged with AML cells, as described in FIG. 11.
• FIG. 14 provides graphs showing the percentage of mOX40L+ cells in the indicated cell types isolated from the peripheral blood, spleen or bone marrow of mice bearing AML cells 24 hours after intravenous administration of the third TIW dose of 0.22 mg/kg, of an mRNA encoding mOX40L formulated in an LNP comprising Compound X and Compound 428 (LNP1) or an LNP comprising Compound X/DSPC/cholesterol/beta-sitosterol/PEG-DMG (LNP2).
• LNP1 Compound X and Compound 428
• LNP2 Compound X/DSPC/cholesterol/beta-sitosterol/PEG-DMG
• FIG. 15 provides graphs showing serum cytokine levels of mouse IFNy (left), endogenous mouse IL-15/15R (middle) and mouse IP-10 (right) at 6, 24, 48 and 54 hours after the first intravenous dose from mice that received a combination of mRNAs encoding mOX40L, cell-associated ML-15 and tethered mIL-l2, formulated in separate LNPs, at a dose of either 0.22 mg/kg three times a week (TIW) or 2 mg/kg single dose.
• TIW time a week
• FIG. 16 provides graphs showing serum cytokine levels of mouse IFNy (left) and endogenous mouse IL-15/IL-15R (right) 6 and 24 hours after the first and second intravenous TIW dose from mice that received mRNA encoding either mOX40L; cell-associated L-15; tethered mIL-l2; mOX40L + cell-associated L-15; mOX40L + tethered mIL-l2; cell- associated L-15 + tethered mIL-l2; or mOX40L + cell-associated L-15 + tethered mIL-l2, formulated in LNP1 or LNP2.
• FIGs. 17A-17D provide graphs showing the percentage of body weight change in mice bearing a disseminated model of AML, and treated intravenously with mRNAs encoding: mOX40L + cell-associated L-15 (FIG. 17A); mOX40L + tethered mIL-l2 (FIG. 17B); cell- associated L-15 + tethered mIL-l2 (FIG. 17C); or mOX40L + cell-associated ML-15 + tethered mTL-12 (FIG. 17D).
• mRNAs were formulated in separate LNPs comprising Compound X and Compound 428, and administered at a dose of 0.22 mg/kg three times a week for three weeks.
• FIGs. 18A-18B provide graphs showing tumor volume of mice bearing MC38-R tumors administered a single intratumoral dose of mRNAs encoding mOX40L, tethered mlL- 12, and cell-associated L-15 (FIG. 18A) in combination with an immune checkpoint inhibitor, i.e., an anti-mCTLA-4 antibody (FIG. 18B).
• an immune checkpoint inhibitor i.e., an anti-mCTLA-4 antibody
• FIGs. 19A-19D provide flow cytometry plots showing NK cells as a percentage of live CD45+ cells at 24 hours post- I st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 lh dose (l3d) in mice administered the mRNAs encoding mOX40L, tethered mIL-l2, and cell- associated L-15 or control mRNA (NST).
• the NK cell percentage in peripheral blood (PB) FIG. 19A
• SP spleen
• BM bone marrow
• LN inguinal lymph nodes
• FIGs. 20A-20D provide flow cytometry plots showing percentage of NK cells expressing the activation marker CD69 at 24 hours post- I st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 th dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hIL-l5 or control mRNA (NST).
• the percentage of NK cells expressing CD69 in (PB) (FIG. 20A), spleen (SP) (FIG. 20B), bone marrow (BM) (FIG. 20C), and inguinal lymph nodes (LN) (FIG. 20D) are provided.
• FIGS. 21A-21D provide flow cytometry plots showing NKT cells as a percentage of live CD45+ cells at 24 hours post-l st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post- 6 th dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL-l2, and cell- associated hIL-15 or control mRNA (NST).
• the NK cell percentage in peripheral blood (PB) (FIG. 21A), spleen (SP) (FIG. 21B), bone marrow (BM) (FIG. 21C), and inguinal lymph nodes (LN) (FIG. 21D) are provided.
• FIGS. 22A-22D provide flow cytometry plots showing percentage of NKT cells expressing the activation marker CD69 at 24 hours post- I st dose (24 h), 24 hours post-3 rd dose (6 d) and 24 hours post-6 111 dose (l3d) in mice administered the mRNAs encoding mOX40L, tethered mIL- 12, and cell-associated hIL- 15 or control mRNA (NST).
• the percentage of NKT cells expressing CD69 in (PB) (FIG. 22A), spleen (SP) (FIG. 22B), bone marrow (BM) (FIG. 22C), and inguinal lymph nodes (LN) (FIG. 22D) are provided.
• FIGS. 23A-23D provide flow cytometry plots showing CD8+ T cells as a percentage of live CD45+ cells at 24 hours post- I st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 111 dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hIL-15 or control mRNA (NST).
• the CD8+ T cell percentage in peripheral blood (PB) (FIG. 23A), spleen (SP) (FIG. 23B), bone marrow (BM) (FIG. 23C), and inguinal lymph nodes (LN) (FIG. 23D) are provided.
• FIGS. 24A-24D provide flow cytometry plots showing percentage of CD8+ T cells expressing the activation marker CD69 at 24 hours post- I st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 th dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL- 12, and cell-associated hIL- 15 or control mRNA (NST).
• the percentage of CD8+ T cells expressing CD69 in (PB) (FIG. 24A), spleen (SP) (FIG. 24B), bone marrow (BM) (FIG. 24C), and inguinal lymph nodes (LN) are provided.
• FIGS. 25A-25D provide flow cytometry plots showing CD4+ T cells as a percentage of live CD45+ cells at 24 hours post- 1 st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 111 dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hIL-15 or control mRNA (NST).
• the CD4+ T cell percentage in peripheral blood (PB) (FIG. 25A), spleen (SP) (FIG. 25B), bone marrow (BM) (FIG. 25C), and inguinal lymph nodes (LN) (FIG. 25D) are provided.
• FIGS. 26A-26D provide flow cytometry plots showing percentage of CD4+ T cells expressing the activation marker CD69 at 24 hours post- I st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 th dose (l3d) in mice administered mRNAs encoding mOX40L, tethered mIL- 12, and cell-associated hIL- 15 or control mRNA (NST).
• the percentage of CD4+ T cells expressing CD69 in (PB) (FIG. 26A), spleen (SP) (FIG. 26B), bone marrow (BM) (FIG. 26C), and inguinal lymph nodes (LN) are provided.
• FIGS. 27A-27D provide flow cytometry plots showing splenic DC cell populations as a percentage of live CD45+ cells at 24 hours post-l st dose (24h), 24 hours post-3 rd dose (6d) and 24 hours post-6 lh dose (13d) in mice administered mRNAs encoding mOX40L, tethered mIL-12, and cell-associated hIL-15 or control mRNA (NST).
• the CD8+ cDCl cell numbers (FIG. 27A), CD103+ cDCl cell numbers (FIG. 27B), cDC2 cell numbers (FIG. 27C), and iDC cell numbers (FIG. 27D) are provided.
• FIGS. 28A-28D provide flow cytometry plots showing inguinal lymph node DC cell populations as a percentage of live CD45+ cells at 24 hours post- 1 st dose (24h), 24 hours post- 3 rd dose (6d) and 24 hours post-6 lh dose (13d) in mice administered mRNAs encoding mOX40L, tethered mIL-12, and cell-associated hIL-15 or control mRNA (NST).
• the CD8+ cDCl cell numbers (FIG. 28A), CD103+ cDCl cell numbers (FIG. 28B), cDC2 cell numbers (FIG. 28C), and iDC cell numbers (FIG. 28D) are provided.
• FIGS. 29A-29D provide flow cytometry plots showing expression of maturation marker, CD86, on splenic and inguinal lymph node CD8+ cDCl and CD 103+ cDCl cell populations at 24 hours post- 1 st dose (24 h), 24 hours post-3 rd dose (6d) and 24 hours post-6 lh dose (13d) in mice administered mRNAs encoding mOX40L, tethered mIL-12, and cell- associated hIL-15 or control mRNA (NST).
• the CD86 MF1 of splenic CD8+ cDCl cells (FIG. 29A), splenic CD103+ cDCl cells (FIG. 29B), inguinal lymph node CD8+ cDCl cells (FIG. 29C), and inguinal lymph node CD 103+ cDCl cells (FIG. 29D) are provided.
• FIGS. 30A-30D provide flow cytometry plots showing expression of maturation marker, CD86, on splenic and inguinal lymph node cDC2 and iDC cell populations at 24 hours post-l st dose (24 h), 24 hours post-3 rd dose (6d) and 24 hours post-6 lh dose (13d) in mice administered mRNAs encoding mOX40L, tethered mIL-12, and cell-associated hIL-15 or control mRNA (NST).
• the CD86 MFI of splenic cDC2 cells (FIG. 30A), splenic iDC cells (FIG. 30B), inguinal lymph node cDC2 cells (FIG. 30C), and inguinal lymph node iDC cells (FIG. 30D) are provided.
• FIG. 31 provides a graph showing percent survival of C57BL/6 or Batf3 KO mice bearing AML.
• the tumor-bearing C57BL/6 were untreated, administered control mRNA (NST) or mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated ML-15.
• the tumor-bearing Batf3 KO mice were administered 0.22 mg/kg three times a week for two weeks (TIWx3) of control mRNA (NST) or mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated ML-15.
• FIG. 32 provides a graph showing precent survival of mice administered control mRNA or mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated ML-15 and left untreated, treated with isotype mAh, or treated with anti-CD4+ mAh.
• FIG. 33 provides a graph showing precent survival of mice administered control mRNA or mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hIL-l5and left untreated, treated with isotype mAh, or treated with anti-IFNy mAh.
• FIGS. 34A-34B provide flow cytometry plots showing MHC II expression on monocytes, either as percent of monocytes (FIG. 34A) or MFI (FIG. 34B) after tumor bearing- mice were administered mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hlL- 15 or control mRNA, and further left untreated, treated with isotype mAh, or treated with anti- IFNy mAh.
• FIGS. 35A-35B provide flow cytometry plots showing PD-L1 expression on myeloid cells, either as percent of myeloid cells (FIG. 35A) or MFI (FIG. 35B) after tumor bearing- mice were administered mRNAs encoding mOX40L, tethered mIL-l2, and cell-associated hlL- 15 or control mRNA, and further left untreated, treated with isotype mAh, or treated with anti- IFNy mAh.
• FIGS. 36A-36B provide flow cytometry plots showing PD-L1 expression on granulocytes, either as percent of granulocytes (FIG. 36A) or MFI (FIG. 36B) after tumor bearing-mice were administered mRNAs encoding mOX40L, tethered mIL-l2, and cell- associated L-15 or control mRNA, and further left untreated, treated with isotype mAh, or treated with anti-IFNy mAh.
• FIGS. 37A-37B provide flow cytometry plots showing PD-L1 expression on monocytes, either as percent of monocytes (FIG. 37A) or MFI (FIG. 37B) after tumor bearing- mice were administered mRNAs encoding hOX40L, tethered ML-12, and cell-associated hlL- 15 or control mRNA, and further left untreated, treated with isotype mAh, or treated with anti- IFNy mAh.
• FIGS. 38A-38F provide graphs showing expression of IFNy (FIGS. 38A-38C) and IP- 10 (CXCL10) (FIGS. 38D-38F) in cynomolgus macaques after administration with mRNAs encoding hOX40L, tethered L-12, and cell-associated hIL-l5 formulated in LNP.
• FIGS. 39A-39C provides flow cytometry plots showing NK, NKT and CD8+ T cell numbers, respectively, (as percentage of live CD45+ cells) in spleen and bone marrow samples of macaques administered mRNAs encoding hOX40L, tethered ML-12, and cell-associated L-15 or given Tris/Sucrose control injections.
• FIGS. 40A-40D provides flow cytometry plots showing the percentage of CD8+ T cells, NK, CD4+ T cells and NKT cells, respectively, having the activation marker, CD69, in spleen and bone marrow samples of macaques administered hOX40L, tethered ML-12, and cell-associated L-15 or given Tris/Sucrose control injections.
• Immunotherapy also referred to in the art as immuno-oncology
• therapies that target not the tumor, but the host immune system.
• These therapies possess unique pharmacological response profiles, and thus represent therapies that might cure many distinct types of cancer.
• Cancers of the lungs, kidney, bladder and skin are among those that derive substantial efficacy from treatment with immuno- oncology in terms of survival or tumor response, with melanoma possibly showing the greatest benefits.
• Disseminated cancers are a significant health problem and are not effectively treated by conventional therapies.
• disseminated cancers including metastatic cancers and cancers of the blood which do not ordinarily form solid tumors, such as myeloid malignancies (e.g., AML)
• AML myeloid malignancies
• AML is known to evade NK cell lysis by upregulating NK inhibitor proteins, by suppressing NK activating ligands and/or by inducing NK cell anergy.
• compositions for use in treating cancer comprising one or more polynucleotides (e.g., mRNAs, e.g., modified mRNAs) to stimulate particular immune cell populations in a subject in need thereof.
• cancer e.g., solid tumors or disseminated cancers such as myeloid malignancies
• polynucleotides e.g., mRNAs, e.g., modified mRNAs
• compositions for use in treating cancer comprising at least two mRNAs (e.g., modified mRNAs), wherein the at least two mRNAs encode a human OX40L polypeptide, a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain, a human IL-15 polypeptide, a human IL-l5Ra polypeptide and combinations thereof.
• the composition comprises:
• an mRNA encoding an OX40L polypeptide an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain, an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-l5Ra polypeptide; or (g) an mRNA encoding a human OX40L polypeptide, an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain and an mRNA encoding a human IL-15 polypeptide operably linked to a human IL- l5Ra polypeptide.
• the mRNAs encoding human OX40L, tethered human IL-12 and cell-associated human IL-15 are co-formulated in the LNP at a weight (mass) ratio of 1:1:1. In some embodiments, the mRNAs encoding human OX40L, tethered human IL-12 and cell-associated human IL-15 are co-formulated at a weight (mass) ratio of 1:1:1, and wherein the mRNA encoding cell-associated human IL-15 is encoded by two mRNAs encoding human IL-15 and human IL-l5Ra, and wherein the two mRNAs are co-formulated at a molar ratio of 1 : 1.
• the mRNA of the disclosure encodes a human OX40L polypeptide, which is a human OX40L polypeptide comprising a cytoplasmic domain of OX40L. In some embodiments, the mRNA encodes a human OX40L polypeptide comprising a transmembrane domain of OX40L. In some embodiments, the mRNA encodes a human OX40L polypeptide comprising an extracellular domain of OX40L and a transmembrane of OX40L. In some embodiments, the mRNA encodes a human OX40L polypeptide comprising an extracellular domain of OX40L and a cytoplasmic domain of OX40L. In some embodiments, the mRNA encodes a human OX40L polypeptide comprising an extracellular domain of OX40L, a transmembrane of OX40L, and a cytoplasmic domain of OX40L.
• the mRNA encodes a human IL-12 polypeptide which is a membrane-tethered form of a human IL-12 polypeptide.
• an mRNA of the disclosure encodes a human IL-12 polypeptide operably linked to a membrane domain, wherein the membrane domain comprises a transmembrane domain.
• the membrane domain comprises a transmembrane domain and an intracellular domain.
• the transmembrane and intracellular domains are derived from the same polypeptide.
• the transmembrane and intracellular domains are derived from different polypeptides.
• the mRNA of the disclosure encodes a human IL-15 polypeptide which is a soluble human IL-15 polypeptide. In some embodiments, the mRNA of the disclosure encodes a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, thereby forming a membrane-tethered from (e.g., a complex) of IL-l5/IL-l5Ra upon expression of the mRNA in a cell.
• the disclosure provides a first mRNA encoding a human IL-15 polypeptide and a second mRNA encoding a human IL- l5Ra polypeptide, thereby providing a membrane-tethered form (e.g., a complex) of IL- l5/IL-l5Ra, encoded by separate mRNAs.
• the mRNA of the disclosure encodes a human IL-l5Ra polypeptide comprising a sushi domain, which has high affinity for IL-15.
• the mRNA of the disclosure encodes a human IL- l5Ra comprising a sushi domain, a transmembrane domain and an intracellular domain.
• the transmembrane and intracellular domains are the human IL-l5Ra transmembrane and intracellular domains. In some embodiments, the transmembrane and intracellular domains are heterologous to IL-l5Ra.
• Human OX40L was first identified on the surface of human lymphocytes infected with human T-cell leukemia virus type-I (HTLV-I) by Tanaka et al. (Tanaka et al. ,
• OX40L is the ligand for 0X40 (CD134). OX40L has also been designated CD252 (cluster of differentiation 252), tumor necrosis factor (ligand) superfamily, member 4, tax-transcriptionally activated glycoprotein 1, TXGP1, or gp34.
• Human OX40L is 183 amino acids in length and contains three domains: a cytoplasmic domain of amino acids 1 - 23; a transmembrane domain of amino acids 24 - 50, and an extracellular domain of amino acids 51 - 183.
• a composition or method of the disclosure comprises an mRNA encoding a mammalian OX40L polypeptide.
• the mammalian OX40L polypeptide is a murine OX40L polypeptide.
• the mammalian OX40L polypeptide is a human OX40L polypeptide.
• the OX40L polypeptide comprises an amino acid sequence set forth in SEQ ID NOs: 1-3.
• the mRNA encoding a human OX40L polypeptide encodes a human OX40L polypeptide comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 1-3 or an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs: 4-11, wherein the human OX40L polypeptide is capable of binding to an 0X40 receptor.
• a human OX40L polypeptide comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 1-3 or an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NOs: 4-11, wherein
• the mRNA encoding a human OX40L polypeptide encodes a human OX40L polypeptide comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 and is capable of binding to an 0X40 receptor.
• the mRNA encoding a human OX40L polypeptide encodes a human OX40L polypeptide that consists essentially of SEQ ID NO: 1 and is capable of binding to an 0X40 receptor.
• the mRNA encoding a human OX40L polypeptide encodes a human OX40L polypeptide comprising an amino acid sequence set forth in SEQ ID NOs: 1- 3, optionally with one or more conservative substitutions, wherein the conservative substitutions do not significantly affect the binding activity of the OX40L polypeptide to its receptor, i.e., the OX40L polypeptide binds to the 0X40 receptor after the substitutions.
• the mRNA encoding a human OX40L polypeptide encodes a human OX40L polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-3.
• an mRNA encoding a human OX40L polypeptide comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of the nucleic acid sequences set forth in SEQ ID NOs: 4-11.
• the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising a nucleotide sequence selected from any one of SEQ ID NOs: 9-11.
• the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to a nucleotide sequence selected from any one of SEQ ID NOs: 9-11. In some embodiments, the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 9.
• the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:
• the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO:
• the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the mRNA encoding a human OX40L polypeptide comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to the nucleotide sequence set forth in SEQ ID NO: 11.
• the mRNA useful for the methods and compositions described herein comprises an open reading frame encoding an extracellular domain of OX40L.
• the mRNA comprises an open reading frame encoding a cytoplasmic domain of OX40L.
• the mRNA comprises an open reading frame encoding a transmembrane domain of OX40L.
• the mRNA comprises an open reading frame encoding an extracellular domain of OX40L and a transmembrane domain of OX40L.
• the mRNA comprises an open reading frame encoding an extracellular domain of OX40L and a cytoplasmic domain of OX40L.
• the mRNA comprises an open reading frame encoding an extracellular domain of OX40L, a transmembrane of OX40L, and a cytoplasmic domain of OX40L.
• references to OX40L polypeptide or mRNA according to SEQ ID NOs: 1-11 encompass variants in which an alternative signal peptide (or encoding sequence) known in the art has been attached to said OX40L polypeptide (or mRNA).
• the methods and compositions described herein utilize mRNA encoding a cell-associated cytokine.
• Cytokines are small secreted proteins released by cells that have a specific effect on the interactions and communications between cells.
• the present disclosure utilizes at least one mRNA encoding a cell-associated cytokine.
• a cell-associated cytokine is one that either naturally or by design is associated with a cell surface.
• a soluble/secreted cytokine is modified to include a transmembrane domain such that the soluble/secreted cytokine will attach to a cell surface.
• by“anchoring” or“tethering” a cytokine to a cell surface systemic effects generally observed with administration of soluble cytokines are reduced.
• a cell-associated cytokine activates T cells, NK cells, or both T cells and NK cells.
• Methods for measuring T cell and NK cell activation are known to those of skill in the art.
• NK and T cell activation can be measured by analyzing surface expression of an activation marker (e.g., CD25 and CD69) on an NK cell or T cell by e.g., flow cytometry.
• a cytokine suitable as a cell-associated cytokine is an IL-12 family member.
• the IL-12 family member is a polypeptide selected from the group consisting of IL-12, IL-23, IL-l2p40 subunit, IL-23pl9 subunit, IL-27, IL-35, and combinations thereof.
• a cytokine suitable as a cell-associated cytokine is IL-15 as described herein.
• the methods and compositions described herein utilize mRNA encoding an IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain.
• Interleukin- 12 is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Gately, MK et al., Annu Rev Immunol. 16: 495-521 (1998). IL- 12 functions primarily as a 70 kDa heterodimeric protein consisting of two disulfide-linked p35 (IL-12A) and p40 (IL-12B) subunits. Due to its ability to activate both NK cells and cytotoxic T cells, IL-12 protein has been studied as a promising anti-cancer therapeutic since 1994. See Nastala, C. L. et al., J Immunol 153: 1697-1706 (1994).
• IL-12 membrane- anchored versions of IL-12 have been generated as IL- 12 is naturally soluble.
• PCT Application No. PCT/US2018/033436 describes mRNA encoding tethered IL-12 and is herein incorporated by reference in its entirety. Accordingly, in some embodiments the mRNA encoding a human IL-12 polypeptide encodes a tethered human IL-12 polypeptide, wherein human IL-12 is operably linked to a membrane domain.
• the IL-12 polypeptide is a murine IL-12 polypeptide. In some embodiments, the IL-12 polypeptide is a human IL-12 polypeptide. In some embodiments, the IL-12 polypeptide comprises an amino acid sequence set forth in SEQ ID NOs: 33, 35, 39 or 40.
• the IL-12 polypeptide comprises a single polypeptide chain comprising the IL-12B and IL-12A polypeptides fused directly or by a linker. In other embodiments, the IL-12 polypeptide comprises two polypeptides, the first polypeptide comprising IL-12B and the second polypeptide comprising IL-12A. In some embodiments, the disclosure provides an IL-12A polypeptide and an IL-12B polypeptide, wherein the IL- 12A and IL-12B polypeptides are on the same chain or different chains.
• IL-12 polypeptide refers to, e.g., an IL- 12r40 subunit of IL-12 (i.e., IL-12B), an IL-l2p35 subunit of IL-12 (i.e., IL-12A), or to a fusion protein comprising an IL-l2p40 subunit polypeptide and an IL-l2p35 subunit polypeptide, operably linked to a membrane domain comprising a transmembrane domain.
• the fusion protein comprises an IL-12B polypeptide selected from:
• the full-length IL-12B polypeptide e.g., having the same or essentially the same length as wild-type IL-12B
• a functional fragment of the full-length IL-12B polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-12B wild- type; but still retaining IL-12B functional activity;
• a variant thereof e.g., full-length or truncated IL-12B proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL- 12B activity of the polypeptide with respect to the wild type IL-12B polypeptide (such as, e.g., V33I, V298F, or any other natural or artificial variants known in the art); or
• a fusion protein comprising (i) a full-length IL-12B wild-type, a functional fragment or a variant thereof, and (ii) a heterologous protein;
• an IL-12A polypeptide selected from:
• the full-length IL-12A polypeptide e.g., having the same or essentially the same length as wild-type IL-12A
• a functional fragment of the full-length IL-12A polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-12A wild- type; but still retaining IL-12A functional activity
• a variant thereof e.g., full-length or truncated IL-12A proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL-12A activity of the polypeptide with respect to the wild type IL-12A polypeptide (such as natural or artificial variants known in the art); or
• a fusion protein comprising (i) a full-length IL-12A wild-type, a functional fragment or a variant thereof, and (ii) a heterologous protein.
• the mRNA encoding a human IL-12 polypeptide encodes a human IL-12 polypeptide comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in SEQ ID NOs: 33, 35, 39 or 40 or an amino acid sequence encoded by a nucleotide sequence listed in SEQ ID NOs: 34, 36 or 46, wherein the human IL-12 polypeptide is capable of binding to an IL-12 receptor.
• the IL-12 polypeptide encoded by an mRNA of the disclosure comprises an amino acid sequence listed in SEQ ID NOs: 33, 35, 39 or 40, with one or more conservative substitutions, wherein the conservative substitutions do not significantly affect the binding activity of the IL-12 polypeptide to its receptor, i.e., the IL-12 polypeptide binds to the IL-12 receptor after the substitutions.
• an mRNA encoding a human IL-12 polypeptide comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence listed in SEQ ID NOs: 34, 36 or 46.
• references to IL- 12 polypeptide or mRNA according to SEQ ID NOs: 33-40 and 46 encompass variants in which an alternative signal peptide (or encoding sequence) known in the art has been attached to said IL-12 polypeptide (or mRNA).
• the tethered IL-12 polypeptides encoded by the mRNAs of the disclosure comprise a membrane domain that tethers (i.e., anchors) the IL-12 polypeptide to a cell membrane (e.g. , a transmembrane domain).
• the tethered IL-12 polypeptides comprise a transmembrane domain.
• the tethered IL-12 polypeptides comprise a transmembrane domain, and optionally an intracellular domain. In some embodiments, the tethered IL-12 polypeptides comprise a transmembrane domain and an intracellular domain.
• the membrane domain is from an integral membrane protein.
• Integral membrane proteins can include, for example, integral polytopic proteins that contain a single-pass or multi-pass transmembrane domain that tethers the protein to a cell surface, including domains with hydrophobic a-helical or b-barrel (i.e. , b-sheet) structures.
• the amino-terminus (i.e ., N-terminus) of Type I integral membrane proteins is located in the extracellular space, while the carboxy-terminus (i.e. , C-terminus) of Type II integral membrane proteins is located in the extracellular space.
• a tethered IL-12 polypeptide of the disclosure comprises a transmembrane domain from an integral polytopic protein. In some embodiments, a tethered IL-12 polypeptide of the disclosure comprises a transmembrane domain from a Type I integral membrane protein. In some embodiments, a tethered IL-12 polypeptide comprises a transmembrane domain from a Type II integral membrane protein.
• the transmembrane domain comprises an intracellular domain (i.e., a domain that is localized to the intracellular space of a cell, e.g. , a domain that is localized to the cytoplasm of a cell). In some embodiments, an intracellular domain has been removed from the transmembrane domain. In some embodiments, the transmembrane domain comprises a membrane domain without an intracellular domain.
• Integral membrane proteins can also include, for example, integral monotopic proteins that contain a membrane domain that does not span the entire cell membrane but that tethers the protein to a cell surface.
• a tethered IL-12 polypeptide of the disclosure comprises a membrane domain from an integral monotopic protein.
• the membrane domain is derived from a Cluster of Differentiation (CD) protein, CD8, CD80, CD4, a receptor, Platelet-Derived Growth Factor Receptor (PDGF-R), Interleukin-6 Receptor (IL-6R), transferrin receptor, Tumor Necrosis Factor (TNF) receptor, erythropoietin (EPO) receptor, a T Cell Receptor (TCR), TCR b-chain, a Fc receptor, FcyRII, FcsRI, an interferon receptor, type I interferon receptor, a growth factor, Stem Cell Factor (SCF), TNF-a, B7-1, Asialoglycoprotein, c-erbB-2, ICAM-l, an immunoglobulin, an IgG, an IgM, a viral glycoprotein, rabies virus glycoprotein, respiratory syncytial vims glycoprotein G (RSVG), vesicular stomatis vims glycoprotein (VSVG),
• CD Cluster
• the membrane domain is selected from the group consisting of: a CD8 transmembrane domain, a PDGF-R transmembrane domain, a CD80 transmembrane domain, and any combination thereof.
• amino acid sequences of transmembrane domains are set forth in SEQ ID NOs: 41-43.
• a membrane domain comprises a transmembrane domain of T- cell surface glycoprotein CD8 alpha chain (also known as CD8A or T-lymphocyte differentiation antigen T8/Leu-2), e.g., a transmembrane of UniProtKB - P01732.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD8 transmembrane polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD8 transmembrane polypeptide as set forth in SEQ ID NO: 41.
• the mRNA encoding a tethered IL-12 comprising a CD8 transmembrane domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 69. In some embodiments, the mRNA encoding a tethered IL-12 comprising a CD8 transmembrane domain, comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 69.
• a membrane domain comprises a transmembrane domain of platelet-derived growth factor receptor beta (EC:2.7.l0.l) (also known as PDGF-R-beta, PDGFR-beta, beta platelet-derived growth factor receptor, beta-type platelet-derived growth factor receptor, CD140 antigen-like family member B, platelet-derived growth factor receptor 1, PDGFR-l, or CDl40b), e.g., a transmembrane domain of UniProtKB - P09619.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a PDGFR-beta transmembrane polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a PDGFR-beta transmembrane polypeptide as set forth in SEQ ID NO: 42.
• the mRNA encoding a tethered IL-12 comprising a PDGFR-beta transmembrane domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 62.
• the mRNA encoding a tethered IL-12 comprising a PDGFR-beta transmembrane domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 62.
• a membrane domain comprises a transmembrane domain of T- lymphocyte activation antigen CD80 (also known as activation B7-1 antigen, BB1, CTLA-4 counter-receptor B7.1, or B7), e.g., a transmembrane domain of UniProtKB - P33681.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD80 transmembrane polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD80 transmembrane polypeptide as set forth in SEQ ID NO: 43.
• the mRNA encoding a tethered IL-12 comprising a CD80 transmembrane domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 70.
• the mRNA encoding a tethered IL-12 comprising a CD80 transmembrane domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 70.
• the membrane domain in the tethered IL-12 polypeptide comprises an amino acid sequence at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% identical to SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, or any combination thereof.
• the membrane domain comprises a transmembrane domain and an intracellular domain.
• an intracellular domain is any oligopeptide or polypeptide known to act as a transmission signal in a cell.
• the membrane domain comprises an intracellular domain to stabilize the tethered IL-12 polypeptide.
• Intracellular domains useful in the methods and compositions of the present disclosure include at least those derived from any of the polypeptides in which transmembrane domains are derived, as described supra.
• suitable intracellular domains include, but are not limited to, an intracellular domain derived from CD80, PDGFR, or any combination thereof.
• a membrane domain comprises an intracellular domain of platelet-derived growth factor receptor beta (EC:2.7.l0.l) (also known as PDGF-R-beta, PDGFR-beta, beta platelet-derived growth factor receptor, beta-type platelet-derived growth factor receptor, CD140 antigen-like family member B, platelet-derived growth factor receptor 1, PDGFR-l, or CDl40b), e.g., an intracellular domain of UniProtKB - P09619.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a PDGFR-beta intracellular polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 48.
• a membrane domain comprises a truncated intracellular domain of PDGFR-beta.
• a truncated intracellular domain of PDGFR-beta stabilizes the tethered IL-12 polypeptide compared to the wild-type PDGFR-beta intracellular domain.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 49. In some embodiments, the mRNA encoding a tethered IL-12, comprises a nucleotide sequence encoding a truncated PDGFR-beta intracellular polypeptide as set forth in SEQ ID NO: 50.
• the mRNA encoding a tethered IL-12 comprising a truncated PDGFR-beta intracellular domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 63. In some embodiments, the mRNA encoding a tethered IL-12 comprising a truncated PDGFR-beta intracellular domain, comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 63.
• the mRNA encoding a tethered IL-12 comprising a truncated PDGFR-beta intracellular domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 64.
• the mRNA encoding a tethered IL-12 comprising a truncated PDGFR-beta intracellular domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 64.
• a membrane domain comprises an intracellular domain of T- lymphocyte activation antigen CD80 (also known as activation B7-1 antigen, BB1, CTLA-4 counter-receptor B7.1, or B7), e.g., an intracellular domain of UniProtKB - P33681.
• CD80 also known as activation B7-1 antigen, BB1, CTLA-4 counter-receptor B7.1, or B7
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD80 intracellular polypeptide.
• the mRNA encoding a tethered IL-12 comprises a nucleotide sequence encoding a CD80 intracellular polypeptide as set forth in SEQ ID NO: 47.
• the mRNA encoding a tethered IL-12 comprising a CD80 intracellular domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 71. In some embodiments, the mRNA encoding a tethered IL-12 comprising a CD80 intracellular domain, comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 71.
• the tethered IL-12 polypeptides described herein comprise a membrane domain comprising a transmembrane domain and an intracellular domain derived from the same polypeptide (i.e., homologous). In some embodiments, the tethered IL-12 polypeptides described herein comprise a membrane domain comprising a CD80 transmembrane domain and CD80 intracellular domain. In some embodiments, the tethered IL-12 polypeptide described herein comprise a membrane domain comprising a PDGFR-beta transmembrane domain and PDGFR-beta intracellular domain.
• the tethered IL-12 polypeptides described herein comprise a membrane domain comprising a transmembrane domain and an intracellular domain derived from different polypeptides (i.e., heterologous) (e.g., a CD80 transmembrane domain and a PDGFR-beta intracellular domain; a CD 8 transmembrane domain and a CD 80 intracellular domain; a CD 8 transmembrane domain and a PDGFR-beta transmembrane domain; or a PDGFR-beta transmembrane domain and a CD80 intracellular domain).
• heterologous e.g., a CD80 transmembrane domain and a PDGFR-beta intracellular domain; a CD 8 transmembrane domain and a CD 80 intracellular domain; a CD 8 transmembrane domain and a PDGFR-beta transmembrane domain and a CD80 intracellular domain.
• the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL-12 polypeptide is located C-terminal to any IL-12 amino acid sequence (i.e., any amino acid sequence of IL-12A, IL-12B, or both IL-12A and IL-12B when both are present in the tethered IL-12 polypeptide).
• IL-12 amino acid sequence i.e., any amino acid sequence of IL-12A, IL-12B, or both IL-12A and IL-12B when both are present in the tethered IL-12 polypeptide.
• located C- terminal to indicates location in a polypeptide with respect to other sequences in the polypeptide in relation to the C-terminus of the polypeptide.
• a membrane domain e.g., transmembrane domain, and optional intracellular domain
• a membrane domain that is "C-terminal to" any IL-12 amino acid sequences means that the membrane domain is located closer to the C-terminus of the tethered IL-12 polypeptide than any IL-12 amino acid sequences.
• the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL-12 polypeptide is located N-terminal to the IL- 12 polypeptide.
• a membrane domain that is "N-terminal to" any IL-12 amino acid sequences means that the membrane domain is located closer to the N-terminus of the tethered IL-12 polypeptide than any IL-12 amino acid sequences.
• the membrane domain (e.g., transmembrane domain, and optional intracellular domain) in the tethered IL-12 polypeptide is linked to the IL-12 polypeptide by a linker, which is referred to herein as a "membrane domain linker" or a "transmembrane domain linker” when the membrane domain is a transmembrane domain, and optionally an intracellular domain.
• linkers are disclosed elsewhere herein.
• the membrane domain in the tethered IL-12 polypeptide is fused directly to the IL-12 polypeptide.
• a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain encoded by an mRNA comprises the amino acid sequence set forth in SEQ ID NO: 53.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 52.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 52.
• a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain encoded by an mRNA comprises the amino acid sequence set forth in SEQ ID NO: 55.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 54.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO: 54.
• a tethered human IL-12 polypeptide comprising a human CD80 transmembrane domain and intracellular domain encoded by an mRNA comprises the amino acid sequence set forth in SEQ ID NO: 61.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD80 transmembrane domain and intracellular domain comprises an open reading frame comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 56-60.
• the mRNA encoding a tethered human IL-12 polypeptide comprising a human CD8 transmembrane domain comprises an open reading frame comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the nucleotide sequence selected from any one of SEQ ID NOs: 56-60.
• IL-15 is a member of the 4a-helix bundle family of cytokines and plays an important role in the development of an effective immune response. Waldmann, T.A., Cancer Immunol. Res. 3: 219-227 (2015). IL-15 is essential for the proper development of NK cells and long term maintenance of memory CD8+ T cells.
• the IL-15 gene encodes a 162 amino acid preprotein having a signal peptide of 48 amino acids, with the mature protein being 114 amino acids in length. Bamford, R.N., et al., Proc. Natl. Acad. Sci. USA 93: 2897-2902 (1996). See also, e.g., GenBank Accession Numbers NM_000585 for the Homo sapiens IL- 15 transcript variant 3 mRNA sequence and NP_000576 for the corresponding IL-15 isoform 1 preproprotein.
• IL-15 shares certain structural similarity to interleukin-2 (IL2). Like IL-2, IL-15 signals through the IL-2 receptor beta chain (CD 122) and the common gamma chain
• IL-15 cannot effectively bind CD122 and CD132 on its own.
• IL- 15 must first bind to the IL-15 alpha receptor subunit (IL-l5Ra).
• the IL-l5Ra gene encodes a 267 amino acid preprotein having a signal peptide of 30 amino acids, with the mature protein being 237 amino acids in length. See, e.g., GenBank Accession Numbers
• NM_002l89 for the Homo sapiens IL-l5Ra transcript variant 1 mRNA
• NP_002l80 for the Homo sapiens IL-l5Ra isoform 1 precursor amino acid sequence.
• Human IL-l5Ra is predominantly a transmembrane protein that binds to IL-15 on the surface of cells such as activated dendritic cells and monocytes. Waldmann, T.A., Cancer Immunol. Res. 3: 219-227 (2015). The membrane bound complex of IL-15/IL-15Ra then presents IL-15 in trans to CD122 and CD132 subunits. Accordingly, IL-15Ra is an essential component of IL-15 activity, such that IL-15 is a naturally cell-associated cytokine.
• the disclosure provides an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-15R0C polypeptide. In some embodiments, the disclosure provides an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-15Ra polypeptide.
• the IL-15 polypeptide and/or IL-15Ra polypeptide is a variant, a peptide or a polypeptide containing a substitution, and insertion and/or an addition, a deletion and/or a covalent modification with respect to a wild-type IL-15 and/or IL- 15Ra sequence.
• the term“IL-15 polypeptide” refers to the mature IL-15 polypeptide (i.e., without its signal peptide and propeptide).
• the IL-15 polypeptide includes a signal peptide and/or propeptide.
• IL-15Ra polypeptide as used herein includes at least a Sushi domain and a hinge region of a full-length human IL-15Ra polypeptide.
• the sushi domain of a full-length human IL-15Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 129.
• the IL-15R0C polypeptide comprises the extracellular domain of the full-length human IL-l5Ra polypeptide.
• the extracellular domain of the full-length human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 130.
• the IL-l5Ra polypeptide comprises the transmembrane region and/or intracellular domain of the full-length human IL- 15R0C polypeptide.
• the transmembrane region and/or intracellular domain of the full-length human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 131.
• the IL-l5Ra polypeptide comprises the transmembrane region and/or intracellular domain of a heterologous polypeptide.
• any of the transmembrane and/or intracellular domains described herein can be utilized as heterologous transmembrane and/or intracellular domains of the IL-l5Ra polypeptide.
• sequence tags or amino acids can be added to the sequences encoded by the polynucleotides of the invention (e.g., at the N-terminal or C-terminal ends), e.g., for localization.
• amino acid residues located at the carboxy, amino terminal, or internal regions of a polypeptide of the invention can optionally be deleted.
• the disclosure provides an mRNA encoding a human IL-15 polypeptide. In other aspects, the disclosure provides an mRNA encoding a human IL-l5Ra polypeptide. In some embodiments, the mRNA of the disclosure encodes a fusion protein comprising a human IL-15 polypeptide and a human IL-l5Ra polypeptide comprising at least a Sushi domain, which are operably linked. In other embodiments, the mRNA encodes two polypeptide chains, the first chain comprising a human IL-15 polypeptide and the second chain comprising a human IL-l5Ra polypeptide.
• the IL-15 polypeptide is selected from:
• the mature human IL-15 polypeptide e.g., having the same or essentially the same length as wild-type human IL-15 with or without a signal peptide;
• a functional fragment of the human IL-15 polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-15 wildtype; but still retaining IL-15 activity;
• a variant thereof e.g., full-length, mature, or truncated IL-15 proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL-15 activity of the polypeptide with respect to the wild- type IL-15 polypeptide; and
• a fusion protein comprising (a) a mature human IL-15 wild-type, a functional fragment or a variant thereof, with or without a signal peptide and (b) a heterologous protein; and/or
• the IL-l5Ra polypeptide is selected from:
• a functional fragment of the full-length human IL-l5Ra polypeptide e.g., a truncated (e.g., deletion of carboxy, amino terminal, or internal regions) sequence shorter than an IL-l5Ra wild-type; but still retaining IL-l5Ra activity;
• a variant thereof e.g., full-length or truncated IL-l5Ra proteins in which one or more amino acids have been replaced, e.g., variants that retain all or most of the IL-l5Ra activity of the polypeptide with respect to the wild-typeIL-l5Ra polypeptide (such as natural or artificial variants known in the art); and
• a fusion protein comprising (a) a full-length human IL-l5Ra wild-type, a functional fragment or a variant thereof, and (b) a heterologous protein.
• the mRNA encodes a mammalian IL-15 and/or IL-l5Ra polypeptide, such as a non-human (e.g., primate) IL-15 and/or IL-l5Ra polypeptide, a functional fragment or a variant thereof.
• a mammalian IL-15 and/or IL-l5Ra polypeptide such as a non-human (e.g., primate) IL-15 and/or IL-l5Ra polypeptide, a functional fragment or a variant thereof.
• the human IL-15 polypeptide comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in SEQ ID NOs: 15 and 17 or an amino acid sequence encoded by a nucleotide sequence listed in SEQ ID NOs: 16, 19, 20 and 122, wherein the human IL-15 polypeptide is capable of binding to a human IL-15 receptor.
• the human IL-15 polypeptide encoded by an mRNA of the disclosure comprises an amino acid sequence listed in SEQ ID NOs: 15 and 17 with one or more conservative substitutions, wherein the conservative substitutions do not significantly affect the binding activity of the IL-15 polypeptide to its receptor, /. ⁇ ? ., the IL-15 polypeptide binds to the IL-15 receptor after the substitutions.
• an mRNA encoding a human IL-15 polypeptide comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence listed in SEQ ID NOs: 16, 19, 20 and 122.
• the human IL-l5Ra polypeptide comprises an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in SEQ ID NO: 13 or an amino acid sequence encoded by a nucleotide sequence listed in SEQ ID NOs: 14, 21 and 22, wherein the human IL-l5Ra polypeptide is capable of binding to a human IL-15 polypeptide.
• the human IL-l5Ra polypeptide encoded by an mRNA of the disclosure comprises an amino acid sequence listed in SEQ ID NOs: 14, 21 and 22, with one or more conservative substitutions, wherein the conservative substitutions do not significantly affect the binding activity of the IL-l5Ra polypeptide to its ligand, i.e., the IL- l5Ra polypeptide binds to IL-15 after the substitutions.
• an mRNA encoding a human IL-l5Ra polypeptide comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence listed in SEQ ID NOs: 14, 21 and 22.
• an mRNA encodes a human IL-l5/IL-l5Ra fusion polypeptide comprising an amino acid sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to an amino acid sequence listed in SEQ ID NO: 13 or an amino acid sequence encoded by a nucleotide sequence listed in SEQ ID NOs: 14, 21 and 22..
• the IL-l5/IL-l5Ra fusion polypeptide encoded by an mRNA of the disclosure comprises an amino acid sequence listed in SEQ ID NOs: 23, 27 and 123, with one or more conservative substitutions, wherein the conservative substitutions do not significantly affect the binding activity of the IL-15 polypeptide to its receptor.
• an mRNA encoding an IL-l5/IL-l5Ra fusion polypeptide comprises a nucleotide sequence at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence listed in SEQ ID NOs: 24-26, 28-30 and 124-126 .
• the disclosure provides a composition (e.g., a lipid
• the disclosure provides a composition (e.g., a lipid nanoparticle) comprising two mRNAs described herein. In some embodiments, the disclosure provides a composition (e.g., a lipid nanoparticle) comprising three mRNAs described herein. In some embodiments, the disclosure provides a composition (e.g., a lipid nanoparticle) comprising four mRNAs described herein.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide; and (ii) an mRNA encoding a tethered human IL-12 polypeptide.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; and (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human tethered IL-12 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 53, 55, 61 and 66.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; and (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; and (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4,
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11; and (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 11; and (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 60.
• the disclosure provides a combination of an mRNA encoding a human OX40L polypeptide and an mRNA encoding a tethered human IL- 12 polypeptide, as described herein, wherein the two mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the two mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the two mRNAs are encapsulated in two different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide; (ii) an mRNA encoding a human IL-15 polypeptide; and (iii) an mRNA encoding a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the human IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 17; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQID NO: 13.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the human IL-15 polypeptide comprises the amino acid set forth in SEQ ID NO: 17; and (iii) an mRNA encoding a human IL- l5Rapolypeptide, wherein the human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19, 20 and 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 21 and 22.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19, 20 and 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 22.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 22.
• the disclosure provides a combination of an mRNA encoding a human OX40L polypeptide, an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-l5Ra polypeptide, as described herein, wherein the three mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the three mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the three mRNAs are encapsulated in three different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 27 and 123.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 11; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the disclosure provides a combination of an mRNA encoding a human OX40L polypeptide and an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, as described herein, wherein the two mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the two mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the two mRNAs are encapsulated in two different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide; (ii) an mRNA encoding a human IL-15 polypeptide; and (iii) an mRNA encoding a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 55, 61 and 66; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the human IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 17; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the human IL-15 polypeptide comprises the amino acid set forth in SEQ ID NO: 17; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19, 20 and 122; and (iii) an mRNA encoding a human IL- l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14, 21 and 22.
• the composition comprises (i) an mRNA encoding a tethered human IL- 12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19, 20 and 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%,
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 122; and (iii) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 22.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 60; (ii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 122; and (iii) an mRNA encoding a human IL-15R0C polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 22.
• the mRNA comprises an open reading frame compris
• the disclosure provides a combination of an mRNA encoding a tethered human IL-12 polypeptide, an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-l5Ra polypeptide, as described herein, wherein the three mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the three mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the three mRNAs are encapsulated in three different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 55, 61 and 66; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 27 and 123.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human tethered IL-12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-15R0C polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 27 and 123.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a tethered human IL- 12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 60; and (ii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the disclosure provides a combination of an mRNA encoding a tethered human IL-12 polypeptide and an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, as described herein, wherein the two mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the two mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the two mRNAs are encapsulated in two different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide; (ii) an mRNA encoding a tethered human IL-12 polypeptide; (iii) an mRNA encoding a human IL-15 polypeptide; and (iv) an mRNA encoding a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 55, 61 and 66; (iii) an mRNA encoding a human IL-15 polypeptide, wherein the human IL-15 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15 and 17; and (iv) an mRNA encoding a human IL-l5Ra polypeptide, wherein the human IL-l5Ra polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; (iii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19, 20 and 122; and (iv) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; (iii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to
• the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60; (iii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 122; and (iv) an mRNA encoding a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 22.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 60; (iii) an mRNA encoding a human IL-15 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 122; and (iv) an mRNA encoding
• the disclosure provides a combination of an mRNA encoding a human OX40L polypeptide, an mRNA encoding a tethered human IL-12 polypeptide, an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-l5Ra polypeptide, as described herein, wherein the four mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the four mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the four mRNAs are encapsulated in three different lipid nanoparticles.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide; (ii) an mRNA encoding a tethered human IL-12 polypeptide; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 55, 61 and 66; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 27 and 123.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the human OX40L polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the human IL-12 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 61; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 23, 27 and 123.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL- l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 4, 6 and 9-11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 54, 56-60 and 67; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence having at
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising the nucleotide sequence set forth in SEQ ID NO: 60; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the composition comprises (i) an mRNA encoding a human OX40L polypeptide, wherein the mRNA
• nucleotide sequence comprises an open reading frame comprising a nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 11; (ii) an mRNA encoding a tethered human IL-12 polypeptide, wherein the mRNA comprises an open reading frame comprising nucleotide sequence having least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 60; and (iii) an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, wherein the mRNA comprises an open reading frame a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 24-26, 28-30 and 124-126.
• the disclosure provides a combination of an mRNA encoding a human OX40L polypeptide, an mRNA encoding a tethered human IL-12 polypeptide and an mRNA encoding a human IL-15 polypeptide operably linked to a human IL-l5Ra polypeptide, as described herein, wherein the three mRNAs are encapsulated in the same or different lipid nanoparticles. In some embodiments, the three mRNAs are encapsulated in the same lipid nanoparticle. In some embodiments, the three mRNAs are encapsulated in two different lipid nanoparticles.
• 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“mrnRNA.”
• “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: 75.
• Another exemplary 5’ UTR for use in the constructs is shown in SEQ ID NO: 76.
• Another exemplary 5’ UTR for use in the constructs is shown in SEQ ID NO: 133.
• Another exemplary 5’ UTR for use in the constructs is shown in SEQ ID NO: 12.
• An exemplary 3’ UTR for use in the constructs is shown in SEQ ID NO: 77.
• An exemplary 3’ UTR comprising miR-l22 and miR-l42.3p binding sites for use in the constructs is shown in SEQ ID NO: 78.
• 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.
• nucleobases may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring.
• 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 poly A 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, m2 7 03 GpppG, m2 7 03 GppppG, m2 7 02 GppppG, m 7 Gpppm 7 G, m 7 3'dGpppG, m2 7 03 GpppG, m2 7 03’ GppppG, and m2 702’ 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
• 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 vims 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 vims (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, the Tkosea asigna vims 2A peptide, and the porcine teschovims- 1 2A peptide.
• the 2A peptides are used by several vimses 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 2 A peptide may have the protein sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 79), 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: 79) fragments or variants thereof.
• One example of a polynucleotide sequence encoding the 2 A peptide is:
• a 2A peptide is encoded by the following sequence: 5’-
• 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).
• the mRNA constructs described herein comprise a linker.
• the linker is a peptide linker, including from one amino acid to about 200 amino acids.
• the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 amino acids.
• the linker can be GS (Gly/Ser) linkers, for example, comprising (G n S) m , wherein n is an integer from 1 to 20 and m is an integer from 1 to 20.
• the GS linker can comprise (GGGGS) 0 (SEQ ID NO: 86), wherein o is an integer from 1 to 5.
• the GS linker can comprise GGSGGGGSGG (SEQ ID NO: 87), GGSGGGGG (SEQ ID NO: 88), or GSGSGSGS (SEQ ID NO: 89).
• the linker is GeS (GGGGGGS) (SEQ ID NO: 90).
• the linker can be a Gly-rich linker, for example, comprising (Gly) p , wherein p is an integer from 1 to 40.
• a Gly-rich linker can comprise GGGGG (SEQ ID NO: 91), GGGGGG (SEQ ID NO: 92), GGGGGGG (SEQ ID NO: 93) or GGGGGGGG (SEQ ID NO: 94).
• the linker can comprise (EAAAK) q (SEQ ID NO: 95), wherein q is an integer from 1 to 5.
• the linker can comprise (EAAAKp . i.e., EAAAKEAAAKEAAAK (SEQ ID NO: 96).
• linkers include, but not limited to, GGGGSLVPRGSGGGGS (SEQ ID NO: 97), GSGSGS (SEQ ID NO: 98), GGGGSLVPRGSGGGG (SEQ ID NO: 99), GGSGGHMGSGG (SEQ ID NO: 100), GGSGGSGGSGG (SEQ ID NO: 101), GGSGG (SEQ ID NO: 102), GSGSGSGS (SEQ ID NO: 103), GGGSEGGGSEGGGSEGGG (SEQ ID NO: 104), A AG A AT A A (SEQ ID NO: 105), GGSSG (SEQ ID NO: 106), GSGGGTGGGSG (SEQ ID NO: 107), GSGSGSGSGGSG (SEQ ID NO: 108), GSGGSGSGGSGGSG (SEQ ID NO: 109), and GSGGSGGSGGSGGS (SEQ ID NO: 110).
• Nucleotides encoding the linkers disclosed herein can be constructed to fuse the ORF or ORFs of a polynucleotide disclosed herein.
• 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 (y), 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
• 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 ( PC), 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), l-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 oc-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 oc-thio-guanosine, inosine (I), l-methyl-inosine (m 1 !), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-l4), 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 (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl- guanosine, 7-methyl-inosine, 6-methoxy-guanosine, l-methyl-guanosine (m'G), N2-methyl- gu
• 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 (y), Nl- methylpseudouridine (m ), 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 2-thio-l-methyl- l-deaza-pseudouridine, 2-thio-l -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-l -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2’-0-methyl uridine.
• an mRNA of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g.,
• 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 (ndA), 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), l-methyl-inosine (ndl), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m 7 G), 1- methyl-guanosine (m'G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
• I inosine
• ndl wyosine
• mim methylwyosine
• preQo 7-deaza-guanosine
• preQi 7-aminomethyl-7-deaza-guanosine
• m 7 G 1- methyl-gu
• 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 ), 5- methoxy-uridine (mo 5 U), 5-methyl-cytidine (m 5 C), pseudouridine (y), a-thio-guanosine, or a-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 (y). In some embodiments, the mRNA comprises pseudouridine (y). In some
• the mRNA comprises pseudouridine (y) and 5-methyl-cytidine (m 5 C). In some embodiments, the mRNA comprises 1 -methyl -pseudouridine (m ) ⁇ In some embodiments, the mRNA comprises 1 -methyl-pseudouridine (mV) 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’-0-methyl uridine. In some embodiments, the mRNA comprises 2’-0-methyl uridine and 5-methyl-cytidine (m 5 C). In some embodiments, the mRNA 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 of the disclosure is modified wherein at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of a specified nucleotide or nucleobase is modified.
• an mRNA can be uniformly modified with 5-methyl-cytidine (m 5 C), meaning that all cytosine residues in the mRNA sequence are replaced with 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 is uniformly modified with 1 -methyl pseudouridine (m ), meaning that all uridine residues in the mRNA sequence are replaced with l-methyl pseudouridine (m ) ⁇
• at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of uridines are l-methyl pseudouridine (mV) ⁇
• an mRNA of the disclosure may be modified in a coding region (e.g., an open reading frame encoding a polypeptide).
• a coding region e.g., an open reading frame encoding a polypeptide.
• an mRNA may be modified in regions besides a coding region.
• a 5'- UTR and/or a 3'-UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications.
• nucleoside modifications may also be present in the coding region.
• nucleoside modifications and combinations thereof that may be present in mmRNAs of the present disclosure include, but are not limited to, those described in PCT Patent Application Publications: W02012045075, W02014081507, 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.
• 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.
• 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 tools, algorithms and services are known in the art; non- limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park, CA) and/or proprietary methods.
• the 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.
• 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 intemucleoside 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.
• modified nucleic acids examples include Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).
• 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).
• Translation of a polynucleotide comprising an open reading frame encoding a polypeptide can be controlled and regulated by a variety of mechanisms that are provided by various cis-acting nucleic acid structures.
• cis-acting RNA elements that form hairpins or other higher-order (e.g., pseudoknot) intramolecular mRNA secondary structures can provide a translational regulatory activity to a polynucleotide, wherein the RNA element influences or modulates the initiation of polynucleotide translation, particularly when the RNA element is positioned in the 5' UTR close to the 5’-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):5l5-526; Kozak (1986) Proc Natl Acad Sci 83:2850-2854).
• Untranslated regions are nucleic acid sections of a polynucleotide before a start codon (5 UTR) and after a stop codon (3 UTR) that are not translated.
• a polynucleotide e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)
• RNA e.g., a messenger RNA (mRNA)
• ORF open reading frame
• UTR e.g., a 5' UTR or functional fragment thereof, a 3' UTR or functional fragment thereof, or a combination thereof.
• Cis-acting RNA elements can also affect translation elongation, being involved in numerous frameshifting events (Namy et ak, (2004) Mol Cell 13(2): 157-168).
• Internal ribosome entry sequences represent another type of cis-acting RNA element that are typically located in 5' UTRs, but have also been reported to be found within the coding region of naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet 16(10):469-473).
• IRES In cellular mRNAs, IRES often coexist with the 5 '-cap structure and provide mRNAs with the functional capacity to be translated under conditions in which cap-dependent translation is compromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol 4(7):a0l2245).
• Another type of naturally-occurring cis-acting RNA element comprises upstream open reading frames (uORFs).
• uORFs Naturally-occurring uORFs occur singularly or multiply within the 5' UTRs of numerous mRNAs and influence the translation of the downstream major ORF, usually negatively (with the notable exception of GCN4 mRNA in yeast and ATF4 mRNA in mammals, where uORFs serve to promote the translation of the downstream major ORF under conditions of increased eIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)). Additional exemplary translational regulatory activities provided by components, structures, elements, motifs, and/or specific sequences comprising
• polynucleotides include, but are not limited to, mRNA stabilization or destabilization (Baker & Parker (2004) Curr Opin Cell Biol l6(3):293-299), translational activation (Villalba et al., (2011) Curr Opin Genet Dev 2l(4):452-457), and translational repression (Blumer et al., (2002) Mech Dev 110(1-2):97-112). Studies have shown that naturally-occurring, cis-acting RNA elements can confer their respective functions when used to modify, by incorporation into, heterologous polynucleotides (Goldberg-Cohen et al.,
• the disclosure provides 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
• modifications are described in PCT Application No. PCT/US2018/033519, herein incorporated by reference in its entirety.
• 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 43 S pre-initiation complex (PIC) or ribosome at, or proximal to, the initiation codon.
• the desired translational regulatory activity is an increase in the initiation of polypeptide synthesis at or from the initiation codon. In some embodiments, the desired translational regulatory activity is an increase in the amount of polypeptide translated from the full open reading frame. In some embodiments, the desired translational regulatory activity is an increase in the fidelity of initiation codon decoding by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is inhibition or reduction of leaky scanning by the PIC or ribosome. In some embodiments, the desired translational regulatory activity is a decrease in the rate of decoding the initiation codon by the PIC or ribosome.
• 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 stmcture(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
• biological function and/or activity of the 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 an 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 an 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 a 5
• the sequence composition is >55% cytosine, >60% cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80% cytosine, >85% cytosine, or >90% cytosine.
• GC-motif [CCG]n 5 (SEQ ID NO: 112).
• the GC-rich RNA element is located about 30, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5’ UTR of the mRNA. In another embodiment, the GC- rich RNA element is located about 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensus sequence. In another embodiment, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5’ UTR of the mRNA.
• 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 provided herein.
• 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 some embodiments, the GC-rich RNA element is located immediately adjacent to a Kozak consensus sequence in the 5' UTR of the mRNA.
• the disclosure provides an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence set forth in SEQ ID NO: 113, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA.
• the GC-rich element comprises the sequence as set forth in SEQ ID NO: 113 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 as set forth in SEQ ID NO: 113 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 as set forth in SEQ ID NO: 113 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 an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence as set forth SEQ ID NO: 114, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA.
• the GC-rich element comprises the sequence as set forth SEQ ID NO: 114 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 as set forth SEQ ID NO: 114 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 as set forth SEQ ID NO: 114 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 an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence as set forth in SEQ ID NO: 115, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA.
• the GC-rich element comprises the sequence as set forth in SEQ ID NO: 115 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 as set forth in SEQ ID NO: 115 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 as set forth in SEQ ID NO: 115 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 an mRNA comprising at least one modification, wherein at least one modification is a GC-rich RNA element comprising the sequence set forth in SEQ ID NO: 113, or derivatives or analogs thereof, preceding a Kozak consensus sequence in the 5’ UTR of the mRNA, wherein the 5’ UTR comprises the sequence set forth in SEQ ID NO: 116.
• the GC-rich element comprises the sequence set forth in SEQ ID NO: 113 located immediately adjacent to and upstream of the Kozak consensus sequence in a 5’ UTR sequence described herein. In some embodiments, the GC-rich element comprises the sequence set forth in SEQ ID NO: 113 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 sequence shown in SEQ ID NO: 116.
• the GC-rich element comprises the sequence set forth in SEQ ID NO: 113 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 sequence set forth in SEQ ID NO: 116.
• the 5’ UTR comprises the sequence set forth in SEQ ID NO:
• the 5’ UTR comprises the sequence set forth in SEQ ID NO:
• the disclosure provides an 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. In another embodiment, the stable RNA secondary structure has a deltaG of about -30 kcal/mol, about -20 to -30 kcal/mol, about -20 kcal/mol, about -10 to -20 kcal/mol, about -10 kcal/mol, about -5 to -10 kcal/mol.
• 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 ak, (2009) Science
• the technique is based on protecting a region or segment of mRNA, by the PIC and/or ribosome, from nuclease digestion.
• 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 positions 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 ak, (2014) eLife 3:e03735). In some embodiments, residence time or the time of occupancy of the PIC or ribosome at a discrete position or location along a polynucleotide comprising any one or more of the RNA elements described herein is determined by ribosome profiling.
• a UTR can be homologous or heterologous to the coding region in a polynucleotide.
• the UTR is homologous to the ORF encoding the polypeptide.
• the UTR is heterologous to the ORF encoding the polypeptide.
• the polynucleotide comprises two or more 5' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
• the polynucleotide comprises two or more 3' UTRs or functional fragments thereof, each of which has the same or different nucleotide sequences.
• the 5' UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof is sequence optimized.
• the 5 'UTR or functional fragment thereof, 3' UTR or functional fragment thereof, or any combination thereof comprises at least one chemically modified nucleobase, e.g., Nl-methylpseudouracil or 5-methoxyuracil.
• UTRs can have features that provide a regulatory role, e.g., increased or decreased stability, localization and/or translation efficiency.
• a polynucleotide comprising a UTR can be administered to a cell, tissue, or organism, and one or more regulatory features can be measured using routine methods.
• a functional fragment of a 5’ UTR or 3’ UTR comprises one or more regulatory features of a full length 5’ or 3’ UTR, respectively.
• Natural 5 'UTRs bear features that play roles in translation initiation. They harbor signatures like Kozak sequences that are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ ID NO: 135), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another’G’. 5' UTRs also have been known to form secondary structures that are involved in elongation factor binding.
• liver-expressed mRNA such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, can enhance expression of polynucleotides in hepatic cell lines or liver.
• 5 'UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g., Tie-l, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF, CDllb, MSR, Fr-l, i- NOS), for leukocytes (e.g., CD45, CD18), for adipose tissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (e.g., SP-A/B/C/D).
• muscle e.g., MyoD, Myosin, Myoglobin, Myogenin, Herculin
• endothelial cells e.g., Tie-l, CD36
• myeloid cells e.g.
• UTRs are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
• an encoded polypeptide can belong to a family of proteins (i.e., that share at least one function, structure, feature, localization, origin, or expression pattern), which are expressed in a particular cell, tissue or at some time during development.
• the UTRs from any of the genes or mRNA can be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
• the 5' UTR and the 3' UTR can be heterologous. In some embodiments, the 5' UTR can be derived from a different species than the 3' UTR. In some embodiments, the 3' UTR can be derived from a different species than the 5' UTR.
• Exemplary UTRs of the application include, but are not limited to, one or more 5 'UTR and/or 3 'UTR derived from the nucleic acid sequence of: a globin, such as an a- or b- globin (e.g., a Xenopus, mouse, rabbit, or human globin); a strong Kozak translational initiation signal; a CYBA (e.g., human cytochrome b-245 a polypeptide); an albumin (e.g., human albumin7); a HSD17B4 (hydroxysteroid (17-b) dehydrogenase); a virus (e.g., a tobacco etch vims (TEV), a Venezuelan equine encephalitis vims (VEEV), a Dengue vims, a cytomegalovirus (CMV) (e.g., CMV immediate early 1 (IE1)), a hepatitis vims (e.g., hepati
• C0I6AI C0I6AI
• a ribophorin e.g., ribophorin I (RPNI)
• RPNI ribophorin I
• LRP1 low density lipoprotein receptor-related protein
• a cardiotrophin-like cytokine factor e.g., Nntl
• calreticulin Calr
• Plodl procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1
• nucleobindin e.g.,
• the 5' UTR is selected from the group consisting of a globin 5' UTR; a 5 'UTR containing a strong Kozak translational initiation signal; a cytochrome b- 245 a polypeptide (CYBA) 5’ UTR; a hydroxysteroid (17-b) dehydrogenase (HSD17B4) 5’ UTR; a Tobacco etch virus (TEV) 5’ UTR; a Venezuelan equine encephalitis virus (TEEV) 5’ UTR; a 5’ proximal open reading frame of rubella virus (RV) RNA encoding nonstructural proteins; a Dengue virus (DEN) 5’ UTR; a heat shock protein 70 (Hsp70) 5’ UTR; a eIF4G 5’ UTR; a GLUT1 5’ UTR; functional fragments thereof and any combination thereof.
• a globin 5' UTR a 5 'UTR containing a strong Kozak translational
• the 3’ UTR is selected from the group consisting of a globin 3’ UTR; a CYBA 3’ UTR; an albumin 3’ UTR; a growth hormone (GH) 3’ UTR; a VEEV 3’ UTR; a hepatitis B virus (HBV) 3’ UTR; a-globin 3'UTR; a DEN 3’ UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3’ UTR; an elongation factor 1 al (EEF1A1) 3’ UTR; a manganese superoxide dismutase (MnSOD) 3’ UTR; a b subunit of mitochondrial H(+)-ATP synthase (b-mRNA) 3’ UTR; a GLUT1 3’ UTR; a MEF2A 3’ UTR; a b-Fl-ATPase 3’ UTR; functional fragments thereof and combinations thereof.
• a globin 3’ UTR
• Wild-type UTRs derived from any gene or mRNA can be incorporated into the polynucleotides of the invention.
• a UTR can be altered relative to a wild type or native UTR to produce a variant UTR, e.g., by changing the orientation or location of the UTR relative to the ORF; or by inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.
• variants of 5' or 3' UTRs can be utilized, for example, mutants of wild type UTRs, or variants wherein one or more nucleotides are added to or removed from a terminus of the UTR.
• one or more synthetic UTRs can be used in combination with one or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat. Protoc. 2013 8(3):568-82, the contents of which are incorporated herein by reference in their entirety.
• the polynucleotide comprises multiple UTRs, e.g., a double, a triple or a quadruple 5' UTR or 3' UTR.
• a double UTR comprises two copies of the same UTR either in series or substantially in series.
• a double beta-globin 3'UTR can be used (see US2010/0129877, the contents of which are incorporated herein by reference in its entirety).
• the polynucleotides of the invention comprise a 5’ UTR and/or a 3’ UTR selected from any of the UTRs disclosed herein.
• the 5’ UTR comprises:
• the 5' UTR and/or 3' UTR sequence of the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5' UTR sequences comprising any of SEQ ID NOs: 75-76, 116-118, 132-134 or 136-147 and/or 3' UTR sequences comprises any of SEQ ID NOs: 4, 77-78 or 121, and any combination thereof.
• the 5' UTR and/or 3' UTR sequence of the invention comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a sequence selected from the group consisting of 5' UTR sequences comprising any of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 132 or SEQ ID NO: 134 and/or 3' UTR sequences comprises any of SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 121, and any combination thereof.
• the 5' UTR comprises a nucleotide sequence set forth SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 132 or SEQ ID NO: 134.
• the 3' UTR comprises a nucleotide sequence set forth in SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO: 121).
• the 5' UTR comprises a nucleotide sequence set forth in SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 132 or SEQ ID NO: 134 and the 3' UTR comprises nucleotide sequence set forth in SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO: 121.
• the polynucleotides of the invention can comprise combinations of features.
• the ORF can be flanked by a 5 'UTR that comprises a strong Kozak translational initiation signal and/or a 3 'UTR comprising an oligo(dT) sequence for templated addition of a poly- A tail.
• a 5 'UTR can comprise a first polynucleotide fragment and a second
• polynucleotide fragment from the same and/or different UTRs (see, e.g., US2010/0293625, herein incorporated by reference in its entirety).
• non-UTR sequences can be used as regions or subregions within the polynucleotides of the invention.
• introns or portions of intron sequences can be incorporated into the polynucleotides of the invention. Incorporation of intronic sequences can increase protein production as well as polynucleotide expression levels.
• the polynucleotide of the invention comprises an internal ribosome entry site (IRES) instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem. Biophys. Res. Commun. 2010 394(1): 189-193, the contents of which are incorporated herein by reference in their entirety).
• ITR internal ribosome entry site
• the polynucleotide comprises an IRES instead of a 5’ UTR sequence. In some embodiments, the polynucleotide comprises an ORF and a viral capsid sequence. In some embodiments, the polynucleotide comprises a synthetic 5’ UTR in combination with a non- synthetic 3’ UTR.
• the UTR can also include at least one translation enhancer polynucleotide, translation enhancer element, or translational enhancer elements
• TEE refers to nucleic acid sequences that increase the amount of polypeptide or protein produced from a polynucleotide.
• the TEE can be located between the transcription promoter and the start codon.
• the 5’ UTR comprises a TEE.
• a TEE is a conserved element in a UTR that can promote translational activity of a nucleic acid such as, but not limited to, cap-dependent or cap-independent translation.
• mRNAs 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.
• mRNAs including such regulatory elements are referred to as including“sensor sequences.”
• Non-limiting examples of sensor sequences are described in U.S. Publication 2014/0200261, the contents of which are incorporated herein by reference in their entirety.
• an mRNA of the disclosure comprises an open reading frame (ORF) encoding a polypeptide of interest and further comprises one or more miRNA binding site(s).
• ORF open reading frame
• Inclusion or incorporation of 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 KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell.
• an mRNA of the disclosure comprises one or more microRNA binding sites, microRNA target sequences, microRNA complementary sequences, or microRNA seed complementary sequences. Such 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 (miRNA or miR) binding site refers to a sequence within an mRNA 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.
• an mRNA 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 mRNA comprises the one or more miRNA binding site(s).
• a miRNA binding site having sufficient complementarity to a miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated regulation of an mRNA, e.g., miRNA-mediated translational repression or degradation of the mRNA.
• a miRNA binding site having sufficient complementarity to the miRNA refers to a degree of complementarity sufficient to facilitate miRNA-mediated degradation of the mRNA, e.g., miRNA-guided RN A- induced silencing complex (RISC)- mediated cleavage of mRNA.
• the miRNA binding site can have complementarity to, for example, a 19-25 nucleotide miRNA sequence, to a 19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence.
• a miRNA binding site can be complementary to only a portion of a miRNA, e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the full length of a naturally-occurring miRNA sequence.
• Full or complete complementarity e.g., full complementarity or complete complementarity over all or a significant portion of the length of a naturally-occurring miRNA is preferred when the desired regulation is mRNA degradation.
• a miRNA binding site includes a sequence that has complementarity (e.g., partial or complete complementarity) with a miRNA seed sequence.
• 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 a 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
• 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.
• 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.
• binding of the miRNA binding site to the corresponding miRNA in RISC degrades the mRNA
• 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.
• 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.
• 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 mRNA By engineering one or more miRNA binding sites into an mRNA of the disclosure, the mRNA 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 mRNA. For example, if an mRNA 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 mRNA.
• miRNA binding sites can be removed from mRNA 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 an mRNA to improve protein expression in tissues or cells containing the miRNA.
• an mRNA 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-l22), muscle (miR-l33, miR-206, miR- 208), endothelial cells (miR-l7-92, miR-l26), myeloid cells (miR-l42-3p, miR-l42-5p, miR- 16, miR-2l, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-ld, miR- 149), kidney (miR-l92, miR-l94, miR-204), and lung epithelial cells (let-7, miR-l33, miR- 126).
• liver miR-l22
• muscle miR-l33, miR-206, miR- 208
• endothelial cells miR-l7-92, miR-l26
• myeloid cells miR-l42-3p, 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-l42 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-l42 efficiently degrades exogenous polynucleotides in antigen presenting cells and suppresses cytotoxic elimination of transduced cells (e.g., Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med. 2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in its entirety).
• An antigen-mediated immune response can refer to an immune response triggered by foreign antigens, which, when entering an organism, are processed by the antigen presenting cells and displayed on the surface of the antigen presenting cells. T cells can recognize the presented antigen and induce a cytotoxic elimination of cells that express the antigen.
• Introducing a miR-l42 binding site into the 5’UTR and/or 3'UTR of an mRNA 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 mRNA.
• the mRNA 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 an mRNA 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.
• any miR-l22 binding site can be removed and a miR- 142 (and/or mirR-l46) binding site can be engineered into the 5’UTR and/or 3’UTR of an mRNA of the disclosure.
• an mRNA 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-l42 and/or miR-l46 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-l0a-3p, miR-l0a-5p, miR-H84, hsa-let-7f-l-3p, hsa-let-7f-2— 5p, hsa-let-7f-5p, miR-l25b-l-3p, miR-l25b-2-3p, miR-l25b-5p, miR-l279, miR-l30a-3p, miR-l30a-5p, miR-l32-3p, miR-l32-5p
• novel miRNAs can be identified in immune cell through micro-array hybridization and microtome analysis (e.g., Jima DD et al, Blood, 2010, H6:el l8-el27; Vaz C et al., BMC Genomics, 2010, 11,288, the content of each of which is incorporated herein by reference in its entirety.)
• an mRNA of the disclosure comprises a miRNA binding site, wherein the miRNA binding site comprises one or more nucleotide sequences selected from 72-74 and 82-83, including one or more copies of any one or more of the miRNA binding site sequences.
• an mRNA 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 SEQ ID NOs: 72-74 and 82-83, including any combination thereof.
• an mRNA of the disclosure comprises at least one miR-l22 binding site, at least two miR-l22 binding sites, at least three miR-l22 binding sites, at least four miR-l22 binding sites, or at least five miR-l22 binding sites.
• the miRNA binding site binds miR-l22 or is complementary to miR-l22.
• the miRNA binding site binds to miR-l22-3p or miR-l22-5p.
• 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: 74, wherein the miRNA binding site binds to miR-l22.
• 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: 83, wherein the miRNA binding site binds to miR-122.
• a miRNA binding site is inserted in the mRNA 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 mRNA can be anywhere in the mRNA as long as the insertion of the miRNA binding site in the mRNA 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 mRNA and the binding of the miRNA binding site to the corresponding miRNA are capable of degrading the mRNA or preventing the translation of the mRNA.
• a miRNA binding site is inserted in at least about 30 nucleotides downstream from the stop codon of an ORF in an mRNA 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 nucleotides downstream from
• 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 an mRNA 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 HA et al., Science, 2013, 340, 82-85, herein incorporated by reference in its entirety).
• the mRNAs 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 an mRNA 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 an mRNA 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 an mRNA of the disclosure.
• miRNA binding sites incorporated into an mRNA of the disclosure can be the same or can be different miRNA sites.
• a combination of different miRNA binding sites incorporated into an mRNA 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 an mRNA 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 an mRNA of the disclosure the degree of expression in specific cell types (e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.) can be reduced.
• 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 an mRNA 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.
• An mRNA 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, an mRNA of the disclosure can be designed for optimal protein expression in a tissue or cell, or in the context of a biological condition.
• an mRNA 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.
• an mRNA of the disclosure can include at least one miR-l22 binding site in order to dampen expression of an encoded polypeptide of interest in the liver.
• an mRNA of the disclosure can include at least one miR-142- 3p binding site, miR-l42-3p seed sequence, miR-l42-3p binding site without the seed, miR- l42-5p binding site, miR-l42-5p seed sequence, miR-l42-5p binding site without the seed, miR-l46 binding site, miR-146 seed sequence and/or miR-146 binding site without the seed sequence.
• an mRNA 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 an mRNA of the disclosure more unstable in antigen presenting cells.
• these miRNAs include mir- l42-5p, mir-l42-3p, mir-l46a-5p, and mir-l46-3p.
• an mRNA of the disclosure comprises at least one miRNA sequence in a region of the mRNA that can interact with an RNA binding protein.
• the mRNA of the disclosure e.g., a RNA, e.g., an mRNA
• a RNA e.g., an mRNA
• a sequence-optimized nucleotide sequence e.g., an ORF
• a miRNA binding site e.g., a miRNA binding site that binds to miR-l42.
• the mRNA 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-l42.
• 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.
• At least 95% of uracil in a uracil- modified sequence encoding a polypeptide is 5-methoxyuridine.
• the mRNA 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., Compound X.
• the present disclosure provides pharmaceutical compositions with advantageous properties.
• the lipid compositions described herein may be advantageously used in lipid nanoparticle compositions for the delivery of therapeutic and/or prophylactic agents, e.g., mRNAs, 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, e.g., mRNA, 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 present application provides pharmaceutical
• compositions comprising:
• an mRNA encoding a human IL-12 polypeptide operably linked to a membrane domain comprising a transmembrane domain, an mRNA encoding a human IL-15 polypeptide and an mRNA encoding a human IL-l5Ra polypeptide;
• compositions comprising:
• compositions comprising:
• LNPs comprise an (i) ionizable lipid; (ii) sterol or other structural lipid; (iii) a non-cationic helper lipid or phospholipid; a (iv) PEG lipid. These categories of lipids are set forth in more detail below.
• the lipid nanoparticles of the present disclosure include one or more ionizable lipids.
• the ionizable lipids of the disclosure comprise a central amine moiety and at least one biodegradable group.
• the ionizable lipids described herein may be advantageously used in lipid nanoparticles of the disclosure for the delivery of nucleic acid molecules to mammalian cells or organs.
• the structures of ionizable lipids set forth below include the prefix I to distinguish them from other lipids of the invention.
• R 1 is selected from the group consisting of C5-30 alkyl, C5-20 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 hydrogen, a C 3-6
• Ci -6 alkyl unsubstituted Ci -6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, - 0(CH 2 )nN(R) 2 , -C(0)0R, -0C(0)R, -CX3, -CX 2 H, -CXH 2 , -CN,
• each R 5 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H;
• each R 6 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H;
• M and M’ are independently selected
• 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 , Ci -6 alkyl, -OR, -S(0) 2 R, -S(0) 2 N(R) 2 , C2-6 alkenyl, C3-6 carbocycle and heterocycle;
• R 10 is selected from the group consisting of H, OH, C1-3 alkyl, and C2-3 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, (CH 2 ) q OR*, and H,
• each q is independently selected from 1, 2, and 3;
• each R’ is independently selected from the group consisting of Ci-i 8 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H;
• each R is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl
• each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;
• each Y is independently a C 3-6 carbocycle
• each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R 4
• Q 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.
• R 1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;
• R 2 and R 3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 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 hydrogen, a C3-6
• Ci -6 alkyl unsubstituted Ci -6 alkyl
• Q is selected from a carbocycle, heterocycle, -OR, -0(CH 2 ) n N(R) 2 , -C(0)0R, -0C(0)R, -CX 3 , -CX 2 H, -CXH 2 , -CN, -N(R) 2 , -C(0)N(R) 2 , -N(R)C(0)R, -N(R)S(0) 2 R, -N(R)C(0)N(R) 2 , -N(R)C(S)N(R) 2 ,
• each o is independently selected from 1, 2, 3, and 4, and each n is independently selected from 1, 2, 3, 4, and 5;
• R x is selected from the group consisting of Ci -6 alkyl, C 2-6 alkenyl, -(CH 2 ) v OH, and -(CH 2 ) V N(R) 2 ,
• v is selected from 1, 2, 3, 4, 5, and 6;
• each R 5 is independently selected from the group consisting of OH, Ci -3 alkyl, C 2-3 alkenyl, and H;
• each R 6 is independently selected from the group consisting of OH, Ci -3 alkyl, C 2-3 alkenyl, and H;
• M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R’)-, -N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0 ) 2 -, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, Ci-i 3 alkyl or C 2-i3 alkenyl;
• R 7 is selected from the group consisting of Ci -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, N0 2 , Ci-6 alkyl, -OR, -S(0) 2 R, -S(0) 2 N(R) 2 , C 2-6 alkenyl, C e carbocycle and heterocycle;
• R 10 is selected from the group consisting of H, OH, Ci -3 alkyl, and C 2-3 alkenyl; each R is independently selected from the group consisting of Ci -3 alkyl, C 2-3 alkenyl, (CH 2 ) q OR*, and H,
• each q is independently selected from 1, 2, and 3;
• each R’ is independently selected from the group consisting of Ci-i 8 alkyl, C 2-i8 alkenyl, -R*YR”, -YR”, and H;
• each R is independently selected from the group consisting of C 3-i5 alkyl and C 3-i5 alkenyl;
• each R* is independently selected from the group consisting of Ci-i 2 alkyl and C 2-i2 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.
• a subset of compounds of Formula (I) includes those of
• R 4 is hydrogen, unsubstituted Ci -3 alkyl, -(CH2) o C(R 10 )2(CH2)n- o Q, or -(CH2) n Q, in which Q is
• heteroaryl or heterocycloalkyl are independently selected
• R 2 and R 3 are independently selected from the group consisting of H, Ci-i 4 alkyl, and C2-14 alkenyl.
• m is 5, 7, or 9.
• Q is OH, -NHC(S)N(R) 2 , or -NHC(0)N(R) 2 .
• Q is -N(R)C(0)R, or -N(R)S(0) 2 R.
• a subset of compounds of Formula (I) includes those of Formula (IB):
• R 2 and R 3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl.
• m is 5, 7, or 9.
• a subset of compounds of Formula (I) includes those of Formula (II): (I II), or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R 4 is hydrogen, unsubstituted Ci-3 alkyl, -(CH2) o C(R 10 )2(CH2)n- o Q, or -(CH2) n Q, in which n is 2, 3, or 4, and Q is
• heteroaryl or heterocycloalkyl are independently selected
• R 2 and R 3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl.
• 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;
• each R 5 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H;
• each R 6 is independently selected from the group consisting of OH, C 1-3 alkyl, C 2-3 alkenyl, and H;
• M and M’ are independently selected from -C(0)0-, -OC(O)-, -0C(0)-M”-C(0)0-, -C(0)N(R’)-, -N(R’)C(0)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(0)(0R’)0-, -S(0 )2-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C 1-13 alkyl or C 2-13 alkenyl; R 7 is selected from the group consisting of Ci -3 alkyl, C2-3 alkenyl, and H;
• each R is independently selected from the group consisting of H, C1-3 alkyl, and C2-3 alkenyl;
• R N is H, or Ci-3 alkyl
• each R’ is independently selected from the group consisting of Ci-i 8 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H;
• each R is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl
• each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 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;
• X a and X b are each independently O or S;
• R 10 is selected from the group consisting of H, halo, -OH, R, -N(R)2, -CN, -N3, -C(0)OH, -C(0)OR, -OC(0)R, -OR, -SR, -S(0)R, -S(0)OR, -S(0) 2 OR, -NO2, -S(0) 2 N(R) 2 , -N(R)S(0) 2 R, -NH(CH 2 ),IN(R) 2 , -NH(CH2)piO(CH 2 ) qi N(R)2,
• n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
• r is 0 or 1 ;
• t 1 is selected from 1, 2, 3, 4, and 5;
• p 1 is selected from 1, 2, 3, 4, and 5;
• q 1 is selected from 1, 2, 3, 4, and 5;
• s 1 is selected from 1, 2, 3, 4, and 5.
• a subset of compounds of Formula (VI) includes those of Formula (VI- a):
• R la and R lb are independently selected from the group consisting of C 1-14 alkyl and C 2 14 alkenyl;
• 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.
• a subset of compounds of Formula (VI) includes those of Formula (VII):
• 1 is selected from 1, 2, 3, 4, and 5;
• Mi is a bond or M’ ;
• R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, and C2-14 alkenyl.
• a subset of compounds of Formula (I VI) includes those of Formula (I VIII):
• 1 is selected from 1, 2, 3, 4, and 5;
• Mi is a bond or M’ ;
• R a and R b are independently selected from the group consisting of Ci-i 4 alkyl and C 2- l4 alkenyl;
• R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, and C 2- l4 alkenyl.
• the compounds of any one of formula (I I), (I IA), (I VI), (I Vl-a), (I VII) or (I VIII) include one or more of the following features when applicable.
• Mi is M’.
• M and M’ are independently -C(0)0- or -OC(O)-.
• At least one of M and M’ is -C(0)0- or -OC(O)-.
• At least one of M and M’ is -OC(O)-.
• M is -OC(O)- and M’ is -C(0)0-. In some embodiments, M is -C(0)0- and M’ is -OC(O)-. In certain embodiments, M and M’ are each -OC(O)-. In some embodiments, M and M’ are each -C(0)0-.
• At least one of M and M’ is -0C(0)-M”-C(0)0-.
• M and M’ are independently -S-S-.
• At least one of M and M’ is -S-S.
• one of M and M’ is -C(0)0- or -OC(O)- and the other is -S-S-.
• M is -C(0)0- or -OC(O)- and M’ is -S-S- or M’ is -C(0)0-, or -OC(O)- and M is -S-S-.
• one of M and M’ is -0C(0)-M”-C(0)0-, in which M” is a bond, Ci-i3 alkyl or C2-13 alkenyl.
• M” is Ci-b alkyl or C2-6 alkenyl.
• M” is C M alkyl or C2-4 alkenyl.
• M” is Ci alkyl.
• M” is C2 alkyl.
• M” is C3 alkyl.
• M” is C 4 alkyl.
• M” is C2 alkenyl.
• M” is C3 alkenyl.
• M” is C 4 alkenyl.
• 1 is 1, 3, or 5.
• R 4 is hydrogen
• R 4 is not hydrogen
• R 4 is unsubstituted methyl or -(CFF Q, in which Q is
• Q is OH
• Q is -NHC(S)N(R)2.
• Q is -NHC(0)N(R) 2 .
• Q is -N(R)C(0)R.
• Q is -N(R)S(0) 2 R.
• Q is -0(CH 2 ) n N(R) 2 ⁇
• Q is -0(CH 2 ) n OR.
• Q is -N(R)R 8 .
• Q is -N(R)C(0)OR.
• n is 2.
• n 3.
• n 4.
• Mi is absent.
• At least one R 5 is hydroxyl.
• one R 5 is hydroxyl.
• At least one R 6 is hydroxyl.
• one R 6 is hydroxyl.
• one of R 5 and R 6 is hydroxyl.
• one R 5 is hydroxyl and each R 6 is hydrogen.
• one R 6 is hydroxyl and each R 5 is hydrogen.
• R x is Ci- 6 alkyl. In some embodiments, R x is Ci -3 alkyl. For example, R x is methyl. For example, R x is ethyl. For example, R x is propyl.
• R x is -(CFh) v OF[ and, v is 1, 2 or 3.
• R x is methanoyl.
• R x is ethanoyl.
• R x is propanoyl.
• R x is -(CH 2 ) V N(R) 2 , v is 1, 2 or 3 and each R is H or methyl.
• R x is methanamino, methylmethanamino, or dimethylmethanamino.
• R x is aminomethanyl, methylaminomethanyl, or dimethylaminomethanyl.
• R x is aminoethanyl, methylaminoethanyl, or dimethylaminoethanyl.
• R x is aminopropanyl, methylaminopropanyl, or dimethylaminopropanyl.
• R’ is Ci-i 8 alkyl, C 2-i8 alkenyl, -R*YR”, or -YR”.
• R 2 and R 3 are independently C 3-14 alkyl or C 3-14 alkenyl.
• R lb is C 1-14 alkyl. In some embodiments, R lb is C 2-14 alkyl. In some embodiments, R lb is C3-14 alkyl. In some embodiments, R lb is Ci-s alkyl. In some embodiments, R lb is C 1-5 alkyl. In some embodiments, R lb is C 1-3 alkyl. In some embodiments, R lb is selected from Ci alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl, and C 5 alkyl. For example, in some embodiments, R lb is Ci alkyl. For example, in some embodiments, R lb is C 2 alkyl. For example, in some embodiments, R lb is C 3 alkyl. For example, in some embodiments, R lb is C 4 alkyl. For example, in some embodiments, R lb is C 5 alkyl.
• R 1 is different from -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 .
• -CHR la R lb - is different from -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 .
• R 7 is H. In some embodiments, R 7 is selected from C 1-3 alkyl. For example, in some embodiments, R 7 is Ci alkyl. For example, in some embodiments, R 7 is C 2 alkyl. For example, in some embodiments, R 7 is C 3 alkyl. In some embodiments, R 7 is selected from C 4 alkyl, C 4 alkenyl, C 5 alkyl, C 5 alkenyl, Ce alkyl, Ce alkenyl, C 7 alkyl, C 7 alkenyl, C 9 alkyl, C 9 alkenyl, Cn alkyl, Cn alkenyl, C 17 alkyl, C 17 alkenyl, Ci 8 alkyl, and Ci 8 alkenyl.
• R b’ is C 1-14 alkyl. In some embodiments, R b’ is C 2-14 alkyl. In some embodiments, R b is C 3-14 alkyl. In some embodiments, R b is Ci -8 alkyl. In some embodiments, R b’ is C 1-5 alkyl. In some embodiments, R b is C 1-3 alkyl. In some
• R b’ is selected from Ci alkyl, C 2 alkyl, C 3 alkyl, C 4 alkyl and C 5 alkyl.
• R b’ is Ci alkyl.
• R b’ is C 2 alkyl.
• R b’ is C 3 alkyl.
• R b is C4 alkyl.
• the compounds of Formula (I) are of Formula (Ila):
• the compounds of Formula (I) are of Formula (lib):
• the compounds of Formula (I) are of Formula (He) or (He):
• M is -C(0)0- or -OC(O)-
• M is Ci-b alkyl or C2-6 alkenyl
• 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.
• the compounds of Formula (I I) are of Formula (lid):
• each of R 2 and R 3 may be independently selected from the group consisting of C 5-14 alkyl and C 5-14 alkenyl.
• the compounds of Formula (I) are of Formula (Ilg): (I Ilg), or their N-oxides, or salts or isomers thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; M and M’ are independently selected
• R 2 and R 3 are independently selected from the group consisting of H, Ci-i 4 alkyl, and C 2-i4 alkenyl.
• M is Ci -6 alkyl (e.g., Ci- 4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl).
• R 2 and R 3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
• a subset of compounds of Formula (I VI) includes those of Formula (I Vila):
• a subset of compounds of Formula (I VI) includes those of Formula (I Villa):
• a subset of compounds of Formula (I VI) includes those of Formula (I Vlllb): r its N- oxide, or a salt or isomer thereof.
• a subset of compounds of Formula (I VI) includes those of Formula (I Vllb-l):
• a subset of compounds of Formula (I VI) includes those of Formula (I VIIb-2):
• a subset of compounds of Formula (I VI) includes those of Formula (I VIIb-3):
• a subset of compounds of Formula (VI) includes those of Formula (Vile): Vile).
• a subset of compounds of Formula (I VI) includes those of
• Vlld Vlld
• N- oxide N- oxide
• salt or isomer thereof
• a subset of compounds of Formula (I VI) includes those of Formula (I VIIIc):
• a subset of compounds of Formula I VI) includes those of
• Vllld Vllld
• N- oxide N- oxide
• salt or isomer thereof
• R 4 is selected from the group consisting of a C 3-6
• each o is independently selected from 1, 2, 3, and 4
• each n is independently selected from 1, 2, 3, 4, and 5.
• R 4 is selected from the group consisting of a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -(CH 2 ) o C(R 10 ) 2 (CH 2 ) n-o Q, -CHQR, and -CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, a 5- to l4-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH 2 ) n N(R) 2 , -C(0)OR, - OC(0)R, -CX 3 ,
• R 4 is selected from the group consisting of a C3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -(CH 2 ) o C(R 10 ) 2 (CH 2 ) n-o Q, -CHQR, and -CQ(R) 2 , where Q is selected from a C3-6 carbocycle, a 5- to l4-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -0(CH 2 ) n N(R) 2 , -C(0)OR, -OC(0)R, - CX 3 , -CX 2 H, -CXH 2 , -CN, -C(0)N(R) 2 , -N(R)S(0) 2 R 8 , -N(R)C(0)R, -N(R)S(0) 2 R, -N(R)C(0)N(R) 2 , -N(R)C
• R 4 is selected from the group consisting of a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -(CH 2 ) o C(R 10 ) 2 (CH 2 ) n-o Q, -CHQR, and -CQ(R) 2 , where Q is selected from a C 3-6 carbocycle, a 5- to l4-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -0(CH 2 ) n N(R) 2 , -C(0)OR, - 0C(0)R, -CX 3 ,
• each o is independently selected from 1, 2, 3, and 4
• each n is independently selected from 1, 2, 3, 4, and 5.
• R 4 is -(CH 2 ) n Q, where Q is -N(R)S(0) 2 R 8 and n is selected from 1, 2, 3, 4, and 5.
• R 4 is -(CH 2 ) n Q, where Q is -N(R)S(0) 2 R 8 , in which R 8 is a C 3-6 carbocycle such as C 3-6 cycloalkyl, and n is selected from 1, 2, 3, 4, and 5.
• R 4 is -(CH 2 ) 3 NHS(0) 2 R 8 and R 8 is cyclopropyl.
• R 4 is -(CH 2 ) o C(R 10 ) 2 (CH 2 )n- o Q, where Q is -N(R)C(0)R, n is selected from 1, 2, 3, 4, and 5, and o is selected from 1, 2, 3, and 4.
• R 4 is -(CH 2 )oC(R 10 ) 2 (CH 2 )n- o Q, where Q is -N(R)C(0)R, wherein R is C1-C3 alkyl and n is selected from 1, 2, 3, 4, and 5, and o is selected from 1, 2, 3, and 4.
• R 4 is is -(CH 2 ) o C(R 10 ) 2 (CH 2 ) n-o Q, where Q is -N(R)C(0)R, wherein R is C 1 -C 3 alkyl, n is 3, and o is 1.
• R 10 is H, OH, C 1-3 alkyl, or C 2-3 alkenyl.
• R 4 is 3-acetamido-2,2-dimethylpropyl.
• one R 10 is H and one R 10 is C1-3 alkyl or C 2 -3 alkenyl. In another embodiment, each R 10 is is C1-3 alkyl or C 2 -3 alkenyl. In another embodiment, each R 10 is is Ci-3 alkyl (e.g. methyl, ethyl or propyl). For example, one R 10 is methyl and one R 10 is ethyl or propyl. For example, one R 10 is ethyl and one R 10 is methyl or propyl. For example, one R 10 is propyl and one R 10 is methyl or ethyl. For example, each R 10 is methyl. For example, each R 10 is ethyl. For example, each R 10 is propyl.
• one R 10 is H and one R 10 is OH. In another embodiment, each R 10 is is OH.
• R 4 is unsubstituted C 1-4 alkyl, e.g., unsubstituted methyl.
• R 4 is hydrogen
• 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-i4 alkyl, C 2-i4 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 -(CH2) n Q or -(CH2) 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 C2-14 alkyl, C2-14 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 2 and R 3 are independently selected from the group consisting of C2-14 alkyl, and C2 14 alkenyl.
• R 2 and R 3 are independently selected from the group consisting of -R*YR”, -YR”, and -R*OR”.
• R 1 is selected from the group consisting of C5-20 alkyl and C5-20 alkenyl. In some embodiments, R 1 is C5-20 alkyl substituted with hydroxyl.
• 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 Cx alkyl or Cx alkenyl.
• R” is C3-12 alkyl.
• R” may be C3 alkyl.
• R” may be C4-8 alkyl (e.g., C4, C5, Ce, C 7 , or Cx alkyl).
• R is (CFO q OR*, q is selected from 1, 2, and 3, and R* is C 1-12 alkyl substituted with one or more substituents selected from the group consisting of amino, C 1 -Ce alkylamino, and C 1 -Ce dialkylamino.
• R is (CFO q OR*, q is selected from 1, 2, and 3 and R* is C 1-12 alkyl substituted with C i -Ce dialkylamino.
• R is (CH 2 ) q OR*, q is selected from 1, 2, and 3 and R* is C 1-3 alkyl substituted with C 1 -Ce dialkylamino.
• R is (CFb) q OR*, q is selected from 1, 2, and 3 and R* is C 1-3 alkyl substituted with dimethylamino (e.g., dimethylaminoethanyl).
• R 1 is C5-20 alkyl. In some embodiments, R 1 is Ce alkyl. In some embodiments, R 1 is Cx alkyl. In other embodiments, R 1 is C9 alkyl. In certain embodiments, R 1 is C14 alkyl. In other embodiments, R 1 is Ci 8 alkyl.
• R 1 is C21-30 alkyl. In some embodiments, R 1 is C26 alkyl. In some embodiments, R 1 is C28 alkyl. In certain embodiments, R 1 is
• R 1 is C5-20 alkenyl. In certain embodiments, R 1 is Cis alkenyl. In some embodiments, R 1 is linoleyl. In certain embodiments, 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). In certain embodiments, R 1 is
• R 1 is unsubstituted C5-20 alkyl or C5-20 alkenyl.
• R’ is substituted C5-20 alkyl or C5-20 alkenyl (e.g. , substituted with a C3-6 carbocycle such as l-cyclopropylnonyl or substituted with OH or alkoxy).
• R 1 is
• R 1 is -R”M’R’.
• M
• x 1 is an integer between 1 and 13 (e.g., selected from 3, 4, 5, and 6)
• x 2 is an integer between 1 and 13 (e.g., selected from 1, 2, and 3)
• x 3 is an integer between 2 and 14 (e.g., selected from 4,
• x 1 is selected from 3, 4, 5, and 6
• x 2 is selected from 1, 2, and 3
• x 3 is selected from 4, 5, and 6.
• R 1 is different from -(CHR 5 R 6 ) m -M-CR 2 R 3 R 7 .
• R’ is selected from -R*YR” and -YR”.
• Y is C3-8 cycloalkyl.
• Y is C 6-i o aryl.
• Y is a cyclopropyl group.
• Y is a cyclohexyl group.
• R* is Ci alkyl.
• R is selected from the group consisting of C3-12 alkyl and C3-12 alkenyl. In some embodiments, R” is Cx alkyl. In some embodiments, R” adjacent to Y is Ci alkyl. In some embodiments, R” adjacent to Y is C4-9 alkyl (e.g., C 4 , C5, Ce, C7 or Cx or C 9 alkyl).
• R is substituted C3-12 (e.g., C3-12 alkyl substituted with, e.g., an hydroxyl).
• C3-12 alkyl substituted with, e.g., an hydroxyl e.g., C3-12 alkyl substituted with, e.g., an hydroxyl.
• R’ is selected from C 4 alkyl and C 4 alkenyl. In certain embodiments, R’ is selected from C5 alkyl and C5 alkenyl. In some embodiments, R’ is selected from Ce alkyl and Ce alkenyl. In some embodiments, R’ is selected from C7 alkyl and C7 alkenyl. In some embodiments, R’ is selected from Cy alkyl and Cy alkenyl.
• R’ is selected from C4 alkyl, C4 alkenyl, C 5 alkyl, C 5 alkenyl, Ce alkyl, Ce alkenyl, C7 alkyl, C7 alkenyl, C9 alkyl, C9 alkenyl, Cn alkyl, Cn alkenyl, C17 alkyl, C17 alkenyl, Cis alkyl, and Cis alkenyl, each of which is either linear or branched.
• R’ is linear. In some embodiments, R’ is branched.
• R’ is In some embodiments,
• R’ is selected from Cn alkyl and Cn alkenyl. In other embodiments, R’ is selected from C12 alkyl, C12 alkenyl, C13 alkyl, C13 alkenyl, Ci 4 alkyl, Ci 4 alkenyl, C15 alkyl, C15 alkenyl, Ci6 alkyl, Ci6 alkenyl, C17 alkyl, C17 alkenyl, Cis alkyl, and Ci 8 alkenyl. In certain embodiments, R’ is linear C4-18 alkyl or C4-18 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
• R’ is unsubstituted Ci-is alkyl.
• R’ is substituted Ci-is alkyl (e.g. , C1-15 alkyl substituted with, e.g., an alkoxy such as methoxy, or a C3-6 carbocycle such as l-cyclopropylnonyl, or C(0)0-alkyl or 0C(0)-alkyl such as
• R’ is branched Ci-is alkyl.
• R’ is
• R” is selected from the group consisting of C3-15 alkyl and C3- 15 alkenyl. In some embodiments, R” is C3 alkyl, C4 alkyl, C 5 alkyl, Ce alkyl, C7 alkyl, or Cs alkyl. In some embodiments, R” is C 9 alkyl, C 10 alkyl, Cn alkyl, C 12 alkyl, C 13 alkyl, C 14 alkyl, or C 15 alkyl.
• M’ is -C(0)0-. In some embodiments, M’ is -OC(O)-. In some embodiments, M’ is -0C(0)-M”-C(0)0-.
• M’ is -C(0)0-, -OC(O)-, or -0C(0)-M”-C(0)0-. In some embodiments wherein M’ is -0C(0)-M”-C(0)0-, M” is C M alkyl or C 2-4 alkenyl.
• M’ is an aryl group or heteroaryl group.
• M’ may be selected from the group consisting of phenyl, oxazole, and thiazole.
• M is -C(0)0-. In some embodiments, M is -OC(O)-. In some embodiments, M is -C(0)N(R’)-. In some embodiments, M is -P(0)(0R’)0-. In some embodiments, M is -0C(0)-M”-C(0)0-.
• M is -C(O). In some embodiments, M is -OC(O)- and M’ is -C(0)0-. In some embodiments, M is -C(0)0- and M’ is -OC(O)-. In some embodiments, M and M’ are each -OC(O)-. In some embodiments, M and M’ are each -C(0)0-.
• M is an aryl group or heteroaryl group.
• M may 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’.
• M is a bond. In some embodiments, M” is C 1-13 alkyl or C 2 13 alkenyl. In some embodiments, M” is Ci -6 alkyl or C 2-6 alkenyl. In certain
• M is linear alkyl or alkenyl. In certain embodiments, M” is branched, e.g., - CH(CH 3 )CH2-.
• each R 5 is H. In some embodiments, each R 6 is H. In certain such embodiments, each R 5 and each R 6 is 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 Ce alkyl. In some embodiments, R 2 and R 3 are C 7 alkyl.
• R 2 and R 3 are different.
• R 2 is Cx alkyl.
• R 3 is C 1-7 (e.g., Ci, C 2 , C 3 , C 4 , C 5 , Ce, or C 7 alkyl) or C 9 alkyl.
• R 3 is Ci alkyl.
• R 3 is C2 alkyl.
• R 3 is C3 alkyl.
• R 3 is C 4 alkyl.
• R 3 is C5 alkyl. In some embodiments, R 3 is Ce alkyl. In some embodiments, R 3 is C7 alkyl.
• R 3 is C 9 alkyl.
• R 7 and R 3 are H.
• R 2 is H.
• m is 5, 6, 7, 8, or 9. In some embodiments, m is 5, 7, or 9. For example, in some embodiments, m is 5. For example, in some embodiments, m is 7. For example, in some embodiments, m is 9.
• R 4 is selected from -(CH 2 ) n Q and -(CH 2 ) n CHQR.
• Q is selected from the group consisting of
• Q is -N(R)R 8 , -N(R)S(0) 2 R 8 , -0(CH 2 ) n 0R,
• Q is -N(0R)C(0)R, -N(0R)S(0) 2 R,
• Q is thiourea or an isostere thereof, e.g.,
• R 9 is -S(0) 2 N(R) 2 .
• Q is -OH.
• Q is a substituted or unsubstituted 5- to 10- membered heteroaryl, e.g., Q is a triazole, an imidazole, a pyrimidine, a purine, 2-amino- l,9-dihydro- 67/-purin-6-one-9-yl (or guanin-9-yl), adenin-9-yl, cytosin-l-yl, or uracil- l-yl, each of which is optionally substituted with one or more substituents selected from alkyl, OH, alkoxy, - alkyl-OH, -alkyl-O-alkyl, and the substituent can be further substituted.
• Q is a triazole, an imidazole, a pyrimidine, a purine, 2-amino- l,9-dihydro- 67/-purin-6-one-9-yl (or guanin-9-yl), adenin
• Q is 4-methylpiperazinyl, 4-(4-methoxybenzyl)piperazinyl, isoindolin-2-yl-l,3-dione, pyrrolidin-l-yl-2,5-dione, or imidazolidin-3-yl-2,4-dione.
• R 8 is cyclobutenyl, e.g., 3-(dimethylamino)- cyclobut-3-ene-4-yl-l,2-dione.
• R 8 is cyclobutenyl substituted with one or more of oxo, amino, and alkylamino, wherein the alkylamino is further substituted, e.g., with one or more of C1-3 alkoxy, amino, mono- or di- alkylamino, and halo.
• R 8 is 3-(((dimethylamino)ethyl)amino)cyclobut-3-enyl- l,2-dione.
• R 8 is cyclobutenyl substituted with one or more of oxo, and alkylamino.
• R 8 is 3-(ethylamino)cyclobut-3-ene-l,2-dione.
• R 8 is cyclobutenyl substituted with one or more of oxo, thio, and alkylamino.
• R 8 is 3- (ethylamino)-4-thioxocyclobut-2-en- 1 -one or 2-(ethylamino)-4-thioxocyclobut-2-en- 1 -one.
• R 8 is cyclobutenyl substituted with one or more of thio, and alkylamino.
• R 8 is 3-(ethylamino)cyclobut-3-ene-l,2-dithione.
• R 8 is cyclobutenyl substituted with one or more of oxo and dialkylamino.
• R 8 is 3- (diethylamino)cyclobut-3-ene-l,2-dione.
• R 8 is cyclobutenyl substituted with one or more of oxo, thio, and dialkylamino.
• R 8 is 2-(diethylamino)-4- thioxocyclobut-2-en-l-one or 3-(diethylamino)-4-thioxocyclobut-2-en-l-one.
• R 8 is cyclobutenyl substituted with one or more of thio, and dialkylamino.
• R 8 is 3-(diethylamino)cyclobut-3-ene-l,2-dithione.
• R 8 is cyclobutenyl substituted with one or more of oxo and alkylamino or dialkylamino, wherein alkylamino or
• dialkylamino is further substituted, e.g. with one or more alkoxy.
• R 8 is 3- (bis(2-methoxyethyl)amino)cyclobut-3-ene-l,2-dione.
• R 8 is cyclobutenyl substituted with one or more of oxo, and heterocycloalkyl.
• R 8 is cyclobutenyl substituted with one or more of oxo, and piperidinyl, piperazinyl, or morpholinyl.
• R 8 is cyclobutenyl substituted with one or more of oxo, and heterocycloalkyl, wherein heterocycloalkyl is further substituted, e.g., with one or more C1-3 alkyl.
• R 8 is cyclobutenyl substituted with one or more of oxo, and heterocycloalkyl, wherein heterocycloalkyl (e.g., piperidinyl, piperazinyl, or morpholinyl) is further substituted with methyl.
• Q is -NHR 8 , in which R 8 is a heteroaryl optionally substituted with one or more substituents selected from amino (NH 2 ), mono- or di- alkylamino, Ci -3 alkyl and halo.
• R 8 is thiazole or imidazole.
• R 9 is CN, Ci -6 alkyl, NO2, - S(0) 2 N(R) 2 , -OR, -S(0) 2 R, or H.
• R 9 is NO2, CN, Ci-6 alkyl, -S(0) 2 N(R) 2 , -OR, -S(0) 2 R, or H.
• Q is -0C(0)N(R) 2 , -N(R)C(0)0R, -N(0R)C(0)0R, such as -0C(0)NHCH 3 , -N(0H)C(0)0CH 3 , -N(0H)C(0)CH 3 , -N(0CH 3 )C(0)0CH 3 ,
• Q is -N(R)C(0)R, in which R is alkyl optionally substituted with Ci- 3 alkoxyl or S(0) z Ci- 3 alkyl, in which z is 0, 1, or 2.
• Q is an unsubstituted or substituted C 6-i o 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 may be -(CFF ⁇ OFl.
• R 4 may be -(CH2) 3 OH.
• R 4 may be -(CFF ⁇ OFl.
• R 4 may be benzyl.
• R 4 may be 4-methoxybenzyl.
• R 4 is a C e carbocycle. In some embodiments, R 4 is a C 3-6 cycloalkyl.
• R 4 may be cyclohexyl optionally substituted with e.g., OH, halo, Ci- 6 alkyl, etc.
• R 4 may be 2-hydroxycyclohexyl.
• R is H.
• R is Ci -3 alkyl substituted with mono- or di-alkylamino, e.g., R is ((dimethylamino)ethyl)amino.
• R is Ci -6 alkyl substituted with one or more substituents selected from the group consisting of Ci -3 alkoxyl, amino, and C 1 -C 3 dialkylamino.
• R is unsubstituted C 1-3 alkyl or unsubstituted C 2-3 alkenyl.
• R 4 may be -CH 2 CH(OH)CH 3 , -CH(CH 3 )CH 2 OH, or -CH 2 CH(OH)CH 2 CH 3 .
• R is substituted C 1-3 alkyl, e.g., CH 2 OH.
• R 4 may be -CH 2 CH(OH)CH 2 OH, -(CH 2 ) 3 NHC(0)CH 2 0H, - (CH 2 ) 3 NHC(0)CH 2 0Bn, -(CH 2 ) 2 0(CH 2 ) 2 0H, -(CH 2 ) 3 NHCH 2 OCH 3 , - (CH 2 )3NHCH 2 OCH 2 CH 3 , CH 2 SCH 3 , CH 2 S(0)CH 3 , CH 2 S(0) 2 CH 3 , or -CH(CH 2 OH) 2 .
• R 4 is selected from any of the following groups:
• R 4 is selected from any of the following groups:
• a compound of Formula (III) further comprises an anion.
• anion can be any anion capable of reacting with an amine to form an ammonium salt. Examples include, but are not limited to, chloride, bromide, iodide, fluoride, acetate, formate trifluoroacetate, difluoroacetate, trichloroacetate, and phosphate.
• the compound of any of the formulae described herein is suitable for making a nanoparticle composition for intramuscular administration.
• 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 Ce 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, may 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, may 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 -(CH2) n Q and -(CH2) n CHQR.
• Q is selected from the group consisting
• Q is selected from the group consisting of an imidazole, a pyrimidine, and a purine.
• 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 C3-6 carbocycle. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form a Ce carbocycle. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form a phenyl group. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form a cyclohexyl group. In some embodiments, R 2 and R 3 , together with the atom to which they are attached, form a heterocycle.
• the heterocycle or C3-6 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, may form a phenyl group bearing one or more C5 alkyl substitutions.
• At least one occurrence of R 5 and R 6 is C 1-3 alkyl, e.g., methyl.
• one of the R 5 and R 6 adjacent to M is C 1-3 alkyl, e.g., methyl, and the other is H.
• one of the R 5 and R 6 adjacent to M is C 1-3 alkyl, e.g., methyl and the other is H, and M is -OC(O)- or -C(0)0-.
• R 5 and R 6 is C 1-3 alkyl, e.g., methyl.
• one of the R 5 and R 6 adjacent to M is C 1-3 alkyl, e.g., methyl, and the other is H.
• one of the R 5 and R 6 adjacent to M is C 1-3 alkyl, e.g., methyl and the other is H, and M is -OC(O)- or -C(0)0-.
• At least one occurrence of R 5 and R 6 is methyl.
• the compounds of any one of formula (VI), (VI- a), (VII), (Vila), (Vllb), (Vile), (Vlld), (VIII), (Villa), (VUIb), (VIIIc) or (VUId) include one or more of the following features when applicable.
• r is 0. In some embodiments, r is 1.
• n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 4. In some embodiments, n is not 3. In some embodiments, R N is H. In some embodiments, R N is Ci -3 alkyl. For example, in some embodiments R N is Ci alkyl. For example, in some embodiments R N is C 2 alkyl. For example, in some embodiments R N is C 2 alkyl.
• X a is O. In some embodiments, X a is S. In some
• X b is O. In some embodiments, X b is S.
• R 10 is selected from the group consisting of N(R) 2 ,
• R 10 is selected from the group consisting of
• R 10 is-NH(CH2)oN(R)2, o is 2, 3, or 4.
• -NH(CH2) Pi O(CH2) qi N(R)2 p 1 is 2. In some embodiments wherein -NH(CH2) Pi O(CH2) qi N(R)2, q 1 is 2.
• R 10 is -N((CH 2 ) SI OR) 2
• s 1 is 2.
• R 10 is-NH(CH2) 0 N(R)2, -NH(CH2) P 0(CH2) q N(R)2, - NH(CH 2 ) S OR, or -N((CH 2 ) S OR) 2
• R is H or C 1 -C 3 alkyl.
• R is Ci alkyl.
• R is C 2 alkyl.
• R is H.
• R is H and one R is C 1 -C 3 alkyl.
• R is H and one R is Ci alkyl.
• R is H and one R is C 2 alkyl.
• each R is C2-C4 alkyl.
• one R is H and one R is C 2 -C 4 alkyl.
• R 10 is a heterocycle.
• R 10 is morpholinyl.
• R 10 is methyhlpiperazinyl.
• each occurrence of R 5 and R 6 is H.
• the compound of Formula (I) is selected from the group consisting of:
• the compound of Formula (I I) is selected from the group consisting of:
• the compound of Formula (I I) or Formula (I IV) is selected from the group consisting of:
• a lipid of the disclosure comprises Compound I-340A: (Compound I-340A).

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