US20200147176A1 - Therapeutic RNA - Google Patents

Therapeutic RNA Download PDF

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US20200147176A1
US20200147176A1 US16/552,248 US201916552248A US2020147176A1 US 20200147176 A1 US20200147176 A1 US 20200147176A1 US 201916552248 A US201916552248 A US 201916552248A US 2020147176 A1 US2020147176 A1 US 2020147176A1
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seq
protein
rna
nucleotides
tumor
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Friederike Gieseke
Ugur Sahin
Timothy R. Wagenaar
Dmitri G. Wiederschain
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Sanofi SA
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Sanofi SA
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Priority to US16/552,248 priority Critical patent/US20200147176A1/en
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Publication of US20200147176A1 publication Critical patent/US20200147176A1/en
Assigned to BIONTECH RNA PHARMACEUTICALS GMBH reassignment BIONTECH RNA PHARMACEUTICALS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GIESEKE, Friederike, SAHIN, UGUR
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WAGENAAR, Timothy R., WIEDERSCHAIN, DMITRI G.
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WAGENAAR, Timothy R., WIEDERSCHAIN, DMITRI G.
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BIONTECH RNA PHARMACEUTICALS GMBH
Assigned to BIONTECH RNA PHARMACEUTICALS GMBH reassignment BIONTECH RNA PHARMACEUTICALS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SAHIN, UGUR, GIESEKE, Friederike
Priority to US17/245,605 priority patent/US11865159B2/en
Assigned to SANOFI reassignment SANOFI ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BioNTech SE
Assigned to BioNTech SE reassignment BioNTech SE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HOTZ, CHRISTIAN
Priority to US18/516,006 priority patent/US20240173382A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • 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
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K38/19—Cytokines; Lymphokines; Interferons
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    • 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
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    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • 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
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    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/335—Modified T or U

Definitions

  • Solid tumors include benign and malignant (cancerous) sarcomas, carcinomas, and lymphomas, and can be physically located in any tissue or organ including the brain, ovary, breast, colon, and other tissues. Cancer is often divided into two main types: solid tumor cancer and hematological (blood) cancers. It is estimated that more than 1.5 million cases of cancer are diagnosed in the United States each year, and more than 500,000 people in the United States will die each year from cancer.
  • Solid tumor cancers are particularly difficult to treat.
  • Current treatments include surgery, radiotherapy, immunotherapy and chemotherapy.
  • Surgery alone may be an appropriate treatment for small localized tumors, but large invasive tumors and most metastatic malignancies are usually unresectable by surgery.
  • Other common treatments such as radiotherapy and chemotherapy are associated with undesirable side effects and damage to healthy cells.
  • compositions, uses, and methods that can overcome present shortcomings in treatment of solid tumors.
  • Administration of therapeutic RNAs disclosed herein can reduce tumor size, extend survival time, and/or protect against metastasis and/or recurrence of the tumor.
  • a composition comprising RNA encoding an IL-12sc protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 14 and RNA encoding a GM-CSF protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 27.
  • a composition comprising:
  • composition of any one of embodiments 8-9, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • composition of embodiment 10, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
  • composition of any one of embodiments 15-16, wherein the 5′ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.
  • composition of any one of embodiments 18-19, wherein the 3′ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
  • composition of any one of embodiments 21-22, wherein the poly-A tail comprises at least 100 nucleotides.
  • a method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering the composition of any one of embodiments 1-26 to the subject.
  • a method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering to the subject:
  • RNA encoding an IFN ⁇ 2b protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 19, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 22 or 23.
  • RNA encoding an IL-15 sushi protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 24, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NO: 26.
  • RNA encoding an IL-2 protein that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the amino acids of SEQ ID NO: 9, and/or comprising nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotides of SEQ ID NOs: 12 or 13.
  • the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
  • the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma, testicular tumor, carcinoma, adenocarcinoma, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
  • HL Hodgkin lymphoma
  • non-Hodgkin lymphoma prostate tumor
  • ovarian tumor renal cell tumor
  • renal cell tumor gastrointestinal tract tumor
  • hepatic tumor colorectal tumor
  • tumor with vasculature mesothelioma tumor
  • pancreatic tumor breast tumor
  • composition is administered intra-tumorally or peri-tumorally.
  • RNA comprises a modified nucleobase in place of at least one uridine.
  • each RNA comprises a modified nucleobase in place of each uridine.
  • modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
  • RNA further comprises a 5′ cap.
  • each RNA further comprises a 5′ cap.
  • RNA further comprises a 5′ UTR.
  • each RNA further comprises a 5′ UTR.
  • the 5′ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.
  • RNA further comprises a 3′ UTR.
  • each RNA further comprises a 3′ UTR.
  • the 3′ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
  • RNA further comprises a poly-A tail.
  • each RNA further comprises a poly-A tail.
  • RNA comprises a 5′ cap, 5′ UTR, 3′ UTR, and poly-A tail.
  • each RNA comprises a 5′ cap, 5′ UTR, 3′ UTR, and poly-A tail.
  • a codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 11.
  • the DNA of embodiment 63 comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 11.
  • a codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 83% identity to SEQ ID NO: 13.
  • RNA of embodiment 65 comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 13.
  • RNA produced from the DNA of any one of embodiments 63 or 64 is an RNA produced from the DNA of any one of embodiments 63 or 64.
  • a codon-optimized DNA comprising or consisting of:
  • part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 16; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 16.
  • a codon-optimized RNA comprising or consisting of:
  • RNA of embodiment 71 wherein the linker comprises nucleotides 985-1026 of SEQ ID NO: 18.
  • RNA of any one of embodiments 71 and 72 wherein part a) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity to nucleotides 1-984 of SEQ ID NO: 18; and part b) comprises contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to nucleotides 1027-1623 of SEQ ID NO: 18.
  • RNA produced from the DNA of any one of embodiments 68-70 is an RNA produced from the DNA of any one of embodiments 68-70.
  • a codon-optimized DNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 21.
  • the DNA of embodiment 75 comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 21.
  • a codon-optimized RNA comprising or consisting of contiguous nucleotides having at least 80% identity to SEQ ID NO: 23.
  • RNA of embodiment 77 comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 23.
  • a DNA comprising or consisting of:
  • a RNA comprising or consisting of:
  • a DNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 28.
  • RNA comprising or consisting of contiguous nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 29.
  • RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-85 wherein at least one uridine is replaced with a modified nucleobase.
  • RNA of embodiment 87 or 88, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
  • RNA of embodiment 89 wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
  • RNA of embodiment 91 wherein the 5′ cap is m 2 7,3′-O Gppp(m 1 2′-O ) ApG or 3′-O-Me-m 7 G(5′)ppp(5′)G.
  • RNA of embodiment 93 wherein the 5′ UTR comprises or consists of the nucleotides of SEQ ID NOs: 2, 4, or 6, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NOs: 2, 4, or 6.
  • RNA of embodiment 95 wherein the 3′ UTR comprises or consists of the nucleotides of SEQ ID NO: 8, or nucleotides having at least 99%, 98%, 97%, 96%, 95%, 90%, or 85% identity to SEQ ID NO: 8.
  • RNA of embodiment 97 wherein the poly-A tail comprises at least 100 nucleotides.
  • RNA of any one of embodiments 65-67, 71-74, 78-79, 81-82, and 84-97 further comprising a 5′ cap, 5′ UTR, 3′ UTR, and poly-A tail.
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • a DNA comprising or consisting of:
  • RNA of embodiment 116 wherein at least one uridine is replaced with a modified nucleobase.
  • each uridine is replaced with a modified nucleobase.
  • RNA of any one of embodiments 117-118, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
  • RNA of embodiment 119 wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
  • RNA of any one of embodiments 116-120 wherein the RNA further comprises a 5′ cap.
  • RNA of embodiment 121 wherein the 5′ cap is m 2 7,3′-O Gppp(m 1 2′-O )ApG or 3′-O-Me-m 7 G(5′)ppp(5′)G.
  • RNA of any of embodiments 116-122, wherein double stranded RNA has been removed from the RNA
  • a pharmaceutical formulation comprising any one of the DNA or RNAs of embodiments 63-125 and a pharmaceutically acceptable excipient.
  • a method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one or more of the RNAs or DNAs of embodiments 63-125, or the pharmaceutical formulation of embodiment 126.
  • a method of producing a polypeptide encoding IL-2, IL-12sc, IL-15 sushi, GM-CSF and IFN ⁇ 2b in vivo comprising administering to a subject one or more of the DNAs or RNAs of any one of embodiment 63-125, the composition of any one of embodiments 1-26, or the pharmaceutical formulation of embodiment 126.
  • a composition comprising at least two RNAs, wherein the RNAs encode different proteins, and wherein the RNAs are selected from the RNAs of any one of embodiments 65-67, 71-74, 78-79, 81-82, 84-100, and 116-125.
  • composition of embodiment 129 wherein the composition comprises two RNAs encoding GM-CSF and IL-12sc.
  • composition of embodiment 129 wherein the composition comprises three RNAs encoding GM-CSF, IL-12sc, and IFN ⁇ 2b.
  • composition of embodiment 129 wherein the composition comprises three RNAs encoding GM-CSF, IL-2, and IFN ⁇ 2b.
  • composition of embodiment 129 wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-2 and IFN ⁇ 2b.
  • composition of embodiment 129 wherein the composition comprises four RNAs encoding GM-CSF, IL-12sc, IL-15 sushi and IFN ⁇ 2b.
  • composition of embodiment 134, wherein the RNA encoding IL-15 sushi is selected from the RNA of any one of embodiments 81-82.
  • a composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acids of SEQ ID NO: 9, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a composition comprising an RNA encoding an IL-12sc protein having at least 95% identity to the amino acids of SEQ ID NO: 14, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a composition comprising an RNA encoding a GM-CSF protein having at least 95% identity to the amino acids of SEQ ID NO: 27, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a composition comprising an RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acids of SEQ ID NO: 19, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acids of SEQ ID NO: 24, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-12sc RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a GM-CSF RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IFN ⁇ 2b RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a uridine analog, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-15 sushi RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-2 RNA composition comprising or consisting of nucleotides having at least 95% identity to SEQ ID NOs: 12 or 13, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-12sc RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 17 or 18, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • a GM-CSF RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 29, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IFN ⁇ 2b RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 22 or 23, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-15 sushi RNA composition comprising or consisting of the nucleotides of SEQ ID NO: 26, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • An IL-2 RNA composition comprising or consisting of the nucleotides of SEQ ID NOs: 12 or 13, wherein each uridine is replaced with a modified nucleobase, and further comprising a 5′ UTR (SEQ ID NOs: 2, 4, or 6), a 3′ UTR (SEQ ID NO: 8), a 5′ cap, and a poly-A tail.
  • composition of any of embodiments 143-157, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
  • composition comprising an RNA encoding IFN ⁇ 2b, wherein the RNA is altered to have reduced immunogenicity as compared to un-altered RNA.
  • composition of embodiment 159, wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.
  • composition of embodiment 159, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ), or 5-methyl-uridine (m 5 U).
  • composition of embodiment 161, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
  • composition of embodiment 163, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.
  • composition of embodiment 166, wherein the 5′ cap is m 2 7,3′-O Gppp(m 1 2′-O )ApG or 3′-O-Me-m 7 G(5′)ppp(5′)G.
  • composition of embodiment 168 wherein the peptide or protein of interest is a peptide or protein selected or derived from cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, ⁇ -catenin inhibitors, activators of the STING pathway, activators of the retinoic inducible gene (RIG)-I pathway, agonists of toll-like receptor (TLR) pathways, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis.
  • cytokines cytokines
  • chemokines suicide gene products
  • immunogenic proteins or peptides apoptosis inducers
  • angiogenesis inhibitors heat shock proteins
  • tumor antigens ⁇ -catenin
  • composition of embodiment 170 wherein the alteration comprises substitution of at least one uridine with a modified nucleobase.
  • composition of embodiment 171, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • composition of embodiment 172, wherein the modified nucleobase is N1-methyl-pseudouridine (m 1 ⁇ ).
  • composition of any one of embodiments 168-173, wherein the alteration comprises a reduction in the amount of double-stranded RNA.
  • composition of embodiment 174, wherein the reduction in double-stranded RNA is the result of purification via HPLC or cellulose-based chromatography.
  • composition of any one of embodiments 168-173, wherein the alteration comprises addition of a 5′ cap to the RNA.
  • composition of embodiment 176, wherein the 5′ cap is m 2 7,3′-O Gppp(m 1 2′-O )ApG or 3′-O-Me-m 7 G(5′)ppp(5′)G.
  • composition of any one of embodiments 168-177, wherein the second RNA comprises:
  • a method for treating or preventing cancer, reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject comprising administering any one of the compositions of embodiments 159-178.
  • composition comprising an RNA encoding an IL-12sc protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 14.
  • composition comprising an RNA encoding a GM-CSF protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 27.
  • composition comprising an RNA encoding an IFN ⁇ 2b protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 19.
  • composition comprising an RNA encoding an IL-15 sushi protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 24.
  • composition comprising an RNA encoding an IL-2 protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 9.
  • composition comprising an RNA encoding an IL-12sc protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 17 or 18.
  • composition comprising an RNA encoding a GM-CSF protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 29.
  • composition comprising an RNA encoding an IFN ⁇ 2b protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 22 or 23.
  • composition comprising an RNA encoding an IL-15 sushi protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NO: 26.
  • composition comprising an RNA encoding an IL-2 protein, wherein the RNA comprises nucleotides having at least 95% identity to the nucleotides of SEQ ID NOs: 12 or 13.
  • composition comprising any two of the following RNAs:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • composition comprising any three of the following:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • composition comprising any four of the following:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a composition comprising:
  • a pharmaceutical formulation comprising any one of the compositions of embodiments 181-219.
  • a pharmaceutical formulation comprising any one of the compositions of embodiments 181-219 and a pharmaceutically acceptable excipient.
  • composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221, for use in a method of treating or preventing cancer for use in a method of treating or preventing cancer.
  • composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of reducing the size of a tumor are provided.
  • composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing the reoccurrence of cancer in remission is a composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing the reoccurrence of cancer in remission.
  • composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221 for use in a method of preventing cancer metastasis is provided.
  • a method for treating or preventing cancer comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
  • a method for reducing the size of a tumor comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
  • a method for preventing the reoccurrence of cancer in remission comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
  • a method for preventing cancer metastasis comprising administering the composition of any one of embodiments 181-219, or the pharmaceutical formulation of embodiment 220 or 221.
  • a medical preparation comprising RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.
  • embodiment A 1 The medical preparation of embodiment A 1, further comprising RNA encoding an IL-15 sushi protein.
  • embodiment A 1 or 2 The medical preparation of embodiment A 1 or 2, further comprising RNA encoding an IL-2 protein.
  • invention A 2 comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.
  • invention A 3 comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.
  • invention A 4 or 5 comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN ⁇ protein.
  • invention A 4 or 5 comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFN ⁇ protein.
  • invention A 4 or 5 comprising RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-2 protein, and RNA encoding an IFN ⁇ protein.
  • RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.
  • RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.
  • RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9.
  • RNA encoding an IFN ⁇ protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFN ⁇ protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • RNA comprises a modified nucleobase in place of at least one uridine.
  • each RNA comprises a modified nucleobase in place of at least one uridine.
  • each RNA comprises a modified nucleobase in place of each uridine.
  • each RNA comprises a 5′ cap.
  • each RNA comprises a 5′ UTR.
  • the 5′ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.
  • each RNA comprises a 3′ UTR.
  • embodiment A 27 or 28 wherein the 3′ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • each RNA comprises a poly-A tail.
  • RNA comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail.
  • each RNA comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail.
  • RNA is mRNA
  • checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.
  • any one of embodiments A 1 to 40 which is a kit comprising at least two containers, each container comprising at least one of said RNAs.
  • embodiment A 41 which comprises each RNA in a separate container.
  • any one of embodiments A 1 to 40 which is a pharmaceutical composition comprising the RNAs.
  • composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • RNA is present in a form selected from a liquid form, a solid form, or a combination thereof.
  • embodiment A 54 wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
  • embodiment A 54 or 55 wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
  • HL Hodgkin lymphoma
  • non-Hodgkin lymphoma prostate tumor
  • ovarian tumor renal cell tumor
  • renal cell tumor gastrointestinal tract tumor
  • hepatic tumor colorectal tumor
  • tumor with vasculature mesothelioma tumor, pancre
  • RNA is for intra-tumoral or peri-tumoral administration.
  • treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.
  • RNA for use in a method for treating or preventing cancer in a subject comprising administering RNA encoding an IL-12sc protein and RNA encoding a GM-CSF protein.
  • RNA of Embodiment B 1 wherein the method further comprises administering RNA encoding an IL-15 sushi protein.
  • RNA of Embodiment B 1 or 2 wherein the method further comprises administering RNA encoding an IL-2 protein.
  • RNA of Embodiment B 4 wherein the IFN ⁇ protein is an IFN ⁇ 2b protein.
  • RNA of Embodiment B 2 wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-15 sushi protein.
  • RNA of Embodiment B 3 wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IL-2 protein.
  • RNA of Embodiment B 4 or 5 wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, and RNA encoding an IFN ⁇ protein.
  • RNA of Embodiment B 4 or 5 wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-15 sushi protein, and RNA encoding an IFN ⁇ protein.
  • RNA of Embodiment B 4 or 5 wherein the method comprises administering RNA encoding an IL-12sc protein, RNA encoding a GM-CSF protein, RNA encoding an IL-2 protein, and RNA encoding an IFN ⁇ protein.
  • RNA of any one of embodiments B 1-10 wherein (i) the RNA encoding an IL-12sc protein comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17 or 18 and/or (ii) the IL-12sc protein comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 14.
  • RNA of any one of embodiments B 1-11 wherein (i) the RNA encoding a GM-CSF protein comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 29 and/or (ii) the GM-CSF protein comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 27.
  • RNA of any one of embodiments B 2-12 wherein (i) the RNA encoding an IL-15 sushi protein comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 26 and/or (ii) the IL-15 sushi protein comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 24.
  • RNA of any one of embodiments B 3-13 wherein (i) the RNA encoding an IL-2 protein comprises the nucleotide sequence of SEQ ID NO: 12 or 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12 or 13 and/or (ii) the IL-2 protein comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 9.
  • RNA of any one of embodiments B 4-14 wherein (i) the RNA encoding an IFN ⁇ protein comprises the nucleotide sequence of SEQ ID NO: 22 or 23, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 22 or 23 and/or (ii) the IFN ⁇ protein comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 19.
  • RNA of any one of embodiments B 1-15 wherein at least one RNA comprises a modified nucleobase in place of at least one uridine.
  • each RNA comprises a modified nucleobase in place of at least one uridine.
  • each RNA comprises a modified nucleobase in place of each uridine.
  • RNA of any one of embodiments B 16-18, wherein the modified nucleobase is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • RNA of Embodiment B 19 wherein the modified nucleobase is N1-methyl-pseudouridine (m1 ⁇ ).
  • RNA of any one of embodiments B 1-20 wherein at least one RNA comprises a 5′ cap.
  • each RNA comprises a 5′ cap.
  • each RNA comprises a 5′ UTR.
  • RNA of Embodiment B 24 or 25, wherein the 5′ UTR comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.
  • each RNA comprises a 3′ UTR.
  • RNA of embodiment B 27 or 28, wherein the 3′ UTR comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 8.
  • RNA of any one of embodiments B 1-29 wherein at least one RNA comprises a poly-A tail.
  • each RNA comprises a poly-A tail.
  • RNA of embodiment B 30 or 31, wherein the poly-A tail comprises at least 100 nucleotides.
  • each RNA comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail.
  • RNA of any one of embodiments B 1 to 36 wherein the method further comprises administering a further therapy.
  • RNA of embodiment B 37 wherein the further therapy comprises one or more selected from the group consisting of: (i) surgery to excise, resect, or debulk a tumor, (ii) immunotherapy, (iii) radiotherapy, and (iv) chemotherapy.
  • RNA of embodiment B 37 or 38 wherein the further therapy comprises administering a further therapeutic agent.
  • RNA of embodiment B 39 wherein the further therapeutic agent is an anti-cancer therapeutic agent.
  • RNA of embodiment B 39 or 40 wherein the further therapeutic agent is a checkpoint modulator.
  • RNA of embodiment B 41 wherein the checkpoint modulator is an anti-PD1 antibody, an anti-CTLA-4 antibody, or a combination of an anti-PD1 antibody and an anti-CTLA-4 antibody.
  • RNA of any one of embodiments B 1-42, wherein the cancer is a sarcoma, carcinoma, or lymphoma.
  • RNA of any one of embodiments B 1-43, wherein the cancer is a solid tumor is a solid tumor.
  • RNA of embodiment B 44 wherein the solid tumor is in the lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
  • RNA of embodiment B 44 or 45 wherein the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
  • HL Hodgkin lymphoma
  • non-Hodgkin lymphoma prostate tumor
  • ovarian tumor renal cell tumor
  • renal cell tumor gastrointestinal tract tumor
  • hepatic tumor colorectal tumor
  • tumor with vasculature mesothelioma tumor
  • RNA of any one of embodiments B 1-50 wherein the RNAs are administered by administering a composition comprising a combination of the RNAs.
  • RNA of any one of embodiments B 1-49 wherein at least two of the RNAs are administered at different times.
  • RNA of any one of embodiments B 1-49 and 52 wherein the RNAs are administered by administering at least two compositions, each composition comprising at least one of said RNAs.
  • RNA of any one of embodiments B 1 to 53, wherein treating or preventing cancer comprises reducing the size of a tumor, preventing the reoccurrence of cancer in remission, or preventing cancer metastasis in a subject.
  • RNA of any one of embodiments B 1 to 54 which is or comprises one or more of the RNAs administered in said method.
  • the RNA of embodiment B 55 which is or comprises one or more selected from the group consisting of the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN ⁇ protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding a GM-CSF protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-15 sushi protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-2 protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IFN ⁇ protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein and the RNA encoding a GM-CSF protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-15 sushi protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IL-2 protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, and the RNA encoding an IFN ⁇ protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-15 sushi protein, and the RNA encoding an IFN ⁇ protein.
  • RNA of embodiment B 55 or 56 which is or comprises the RNA encoding an IL-12sc protein, the RNA encoding a GM-CSF protein, the RNA encoding an IL-2 protein, and the RNA encoding an IFN ⁇ protein.
  • FIGS. 1A-1G shows results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 8, 10, 12, 14 and individual tumor growth was monitored to day 41.
  • FIG. 1A and FIG. 1D show results when using IL-2, IL-12sc, and GM-CSF (ModA) mRNA.
  • FIG. 1B and FIG. 1E show IL-2, IL-12sc, and GM-CSF (ModB) mRNA.
  • FIG. 1C and FIG. 1F show results when using luciferase mRNA (ModA).
  • FIG. 1G shows results when using luciferase mRNA (ModB).
  • FIGS. 2A-2D show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 19, 21, 24, 26, 28 and 31 and individual tumor growth was monitored to day 48.
  • FIG. 2A shows GM-CSF, IL-2, IL-12sc (ModA).
  • FIG. 2B shows GM-CSF, IL-2, IL-12sc (ModB).
  • FIG. 2C shows luciferase mRNA (ModA) as a control.
  • FIG. 2D shows Ringer's solution as a control.
  • FIGS. 3A-3C show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on day 13, 15, 18, 20 and 22 and tumor growth was monitored to day 42.
  • FIG. 3A shows IL-2, GM-CSF, IL-12sc (ModB).
  • FIG. 3B shows IL-15 sushi, GM-CSF, IL-12sc (ModB).
  • FIG. 3C shows luciferase mRNA (ModB) as a control.
  • FIGS. 4A-4F show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45.
  • FIG. 4A shows IL-2, IL-12sc, and GM-CSF (ModA).
  • FIG. 4B is a duplicate in the same experiment as described in FIG. 5A , showing IL-2, IL-12sc, and GM-CSF (ModB).
  • FIG. 4C shows IL-15 sushi, IL-12sc, and GM-CSF (ModA).
  • FIG. 4D is a duplicate in the same experiment as described in FIG. 5B , showing IL-15 sushi, IL-12sc, and GM-CSF (ModB).
  • FIG. 4E shows control luciferase mRNA (ModA).
  • FIG. 4F is a duplicate in the same experiment as described in FIGS. 5D and 6D showing control luciferase mRNA (
  • FIGS. 5A-5D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 13, 15, 17 and individual tumor growth was monitored to day 45.
  • FIG. 5A is a duplicate in the same experiment as described in FIG. 4B , showing IL-2, IL-12sc, and GM-CSF (ModB).
  • FIG. 5B is a duplicate in the same experiment as described in FIG. 4D , showing IL-15 sushi, IL-12sc, and GM-CSF (ModB).
  • FIG. 5C is a duplicate in the same experiment as described in FIG. 6C , showing IL-2, IL-12sc, GM-CSF, and IFN ⁇ (ModB).
  • FIGS. 6A and 6B show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted.
  • FIG. 6A is a duplicate in the same experiment as described in FIG. 7A , showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIG. 6B is a duplicate in the same experiment as described in FIG. 7C , showing luciferase mRNA (ModB).
  • N 8 mice/group.
  • FIGS. 6C and 6D show results of experiments where B16F10 tumor bearing mice were injected intratumorally with mRNA on days 11, 13, 15, 17 and individual tumor growth was plotted.
  • FIG. 6C is a duplicate in the same experiment as described in FIG. 5C , showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIG. 6D is a duplicate in the same experiment as described in FIGS. 4F and 5D , showing luciferase mRNA (ModB).
  • N 8 mice/group.
  • FIGS. 6E and 6F show results of experiments where MC38 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 11, 15, 19, 23 and individual tumor growth was plotted.
  • FIG. 6E shows GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIGS. 7A-7F show results of experiments where CT26 tumor bearing mice were injected intratumorally with cytokine mRNA mixtures on days 13, 15, 17, 19, 21, 23 and individual tumor growth was plotted.
  • FIG. 7A is a duplicate in the same experiment as described in FIG. 6A , showing IL-2, IL-12sc, GM-CSF, IFN ⁇ (ModB).
  • FIG. 7B shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
  • FIG. 7C is a duplicate in the same experiment as described in FIG. 6B , showing a luciferase mRNA (ModB) control.
  • FIG. 7D is a duplicate of the same experiment as described in FIG. 9A , showing IL-2, IL-12sc, GM-CSF, IFN ⁇ (ModB).
  • FIG. 7E shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
  • FIG. 7F is a duplicate of the same experiment as described in FIG. 9F , showing a luciferase mRNA (ModB) control.
  • FIGS. 8A-8H show results of experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 12, 15, 19 and 22 and individual tumor growth was monitored and plotted to day 35.
  • FIG. 8A shows IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB).
  • FIG. 8B shows IL-15 sushi, IL-12sc, IFN ⁇ (ModB).
  • FIG. 8C shows IL-15 sushi, GM-CSF, IFN ⁇ (ModB).
  • FIG. 8D shows GM-CSF, IL-12sc, IFN ⁇ (ModB).
  • FIG. 8E shows IL-15 sushi, GM-CSF, IL-12sc (ModB).
  • FIGS. 8G and 8H show tumor growth kinetics of the study shown in FIGS. 8A-8F .
  • FIG. 8G shows mean tumor volumes up to day 33 for all treatment groups.
  • FIG. 8H shows tumor growth repression.
  • T/C Tumor/Control based on mean tumor volume
  • FIGS. 9A-9F show experiments where CT26 tumor bearing mice were injected intratumorally with mRNA on days 19, 21, 23, 26, 28 and 30 and tumor growth was monitored to day 50.
  • FIG. 9A is a duplicate in the same experiment as described in FIG. 7D , showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIG. 9B shows IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIG. 9C shows GM-CSF, IL-2, IFN ⁇ (ModB).
  • FIG. 9D shows GM-CSF, IL-12sc, IFN ⁇ (ModB).
  • FIG. 9E shows GM-CSF, IL-2, IL-12sc (ModB).
  • FIG. 9A is a duplicate in the same experiment as described in FIG. 7D , showing GM-CSF, IL-2, IL-12sc, IFN ⁇ (ModB).
  • FIG. 9B shows IL-2, IL-12sc
  • FIGS. 10A-10B shows tumor growth kinetics of the study shown in FIG. 9 .
  • FIG. 10A shows mean tumor volumes up to day 36 for all treatment groups.
  • FIG. 10B shows tumor growth repression.
  • T/C Tumor/Control based on mean tumor volume
  • FIG. 11 shows a bar graph of data from the experiments shown in FIG. 9 showing mRNA mixtures with significant reduction in tumor volume, where the number of mice in each of the treatment groups with significant tumor reduction was compared to the luciferase control group based on Z score of tumor volume and the ratio between tumor volume change and the mean of the control group.
  • FIGS. 12A-12D show the results of experiments where mice that were 1) tumor na ⁇ ve, or 2) had been previously injected subcutaneously with 5 ⁇ 10 5 B16F10 cells and rejected the original tumor following intratumoral cytokine mRNA treatment. Both groups were re-challenged with B16F10 tumors.
  • FIG. 12A shows tumor na ⁇ ve host mice.
  • FIG. 12B shows mice that had previously rejected B16F10 tumors following intratumoral cytokine mRNA treatment with GM-CSF, IL-15sushi, IL-12sc, IFN ⁇ (ModB). Mice were monitored for 55 days following B16F10 injection and tumor growth for each mouse was plotted.
  • FIG. 12A All nine na ⁇ ve mice engrafted with B16F10 cells developed tumors ( FIG. 12A ), whereas all eight tumor-free mice rejected the B16F10 cells and did not exhibit growth of B16F10 tumors ( FIG. 12B ).
  • the graph in FIG. 12B has no visible data trace because all observations were zero, i.e., overlapping the horizontal axis.
  • FIG. 12C shows an example of localized vitiligo at the tumor site.
  • FIG. 12D shows the results of experiments where mice that were tumor na ⁇ ve (triangle symbol), or had been previously injected subcutaneously with CT26 tumor cells and rejected the original tumor following intratumoral cytokine mRNA treatment (circle symbol).
  • CT26-WT CT26 tumor cells
  • CT26- ⁇ gp70 tumor cells CT26- ⁇ gp70 tumor cells
  • Mice were monitored for 21 days following tumor cell injection. All nine but one na ⁇ ve mice engrafted with CT26-WT cells and all na ⁇ ve mice engrafted with CT26- ⁇ gp70 cells developed tumors, whereas all three tumor-free mice rejected the CT26 tumor cells and did not exhibit growth of CT26 and CT26- ⁇ gp70 tumors, respectively.
  • FIGS. 13A-13D show the results of experiments where mice were implanted with B16F10 tumor cells on day 0 on the right (injected) and left flanks (uninjected) ( FIG. 13A ).
  • Mice received a series of 4 intratumoral injections with ModB cytokine mRNA (IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ ) or ModB control mRNA (luciferase) in the right tumor on days 11, 15, 19, and 23.
  • FIG. 13C Median survival is shown in FIG. 13D .
  • FIGS. 14A-14F show results of experiments where human HEK293 ( FIG. 14B ) and melanoma cell lines (A101D ( FIG. 14C ), A2058 ( FIG. 14D ), A375 ( FIG. 14E ), and Hs294T ( FIG. 14F )) were transfected with human cytokine mRNA mixture (IL-12sc, GM-CSF, IL-15 sushi and IFN ⁇ 2b) in a range of mRNA doses. Supernatants were collected 24 hrs after transfection and protein concentrations were determined with cytokine specific ELISAs.
  • FIG. 14A shows a schematic of the experiment.
  • FIGS. 16A-16E show the results of experiments where immune compromised mice bearing human A375 tumor xenografts received a single injection with the ModB mRNA mixture encoding the human cytokines (IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b; “the IL15 sushi mixture”) or (IL-2, IL-12sc, GM-CSF and IFN ⁇ 2b; “the IL2 mixture”).
  • FIG. 16A shows IFN ⁇ 2b
  • FIG. 16B shows IL-2
  • FIG. 16C shows IL-12sc
  • FIG. 16D shows IL-15 sushi
  • FIG. 16E shows GM-CSF.
  • FIGS. 17A-17C show the results of experiments where mRNA was isolated from A375 tumors at 2 hrs, 4 hrs, 8 hrs, 24 hrs, 48 hrs, and 72 hrs after injection of ModB cytokine mRNA mixture (IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ 2b) or (IL-2, IL-12sc, GM-CSF, IFN ⁇ 2b). Expression of interferon alpha response genes were monitored by qPCR.
  • FIG. 17A shows human ISG15
  • FIG. 17B shows human ISG54
  • FIG. 17C shows human MX1.
  • FIGS. 18A-18E show the results of experiments where mice were implanted with B16F10 tumor cells and treated with mRNA mixtures (FLT3L, IL-2, 41BBL, and CD27L-CD40L) with or without IFN ⁇ .
  • mRNA mixtures without IFN ⁇ in standard (ModA, FIG. 18B ) and modified forms (ModB, FIG. 18C ) were compared to those including IFN ⁇ in standard (ModA, FIG. 18D ) and modified forms (ModB, FIG. 18E ).
  • FIG. 18A is a negative control where Ringer's media without mRNA was provided.
  • FIGS. 19A-19E show the results of experiments where mice were implanted with tumors on one flank and received an IV injection of luciferase-expressing tumor cells that homed to the lung ( FIG. 19A ).
  • Mice in the treatment group received intratumoral injections of mRNA mixtures IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ into the flank tumor only while tumors in the lung were untreated.
  • FIG. 19B shows exemplarily bioluminescence measurements in lungs and pictures of the according lungs taken out on the same day (day 20); tumor nodes are visual as black marks;
  • FIG. 19C shows mean tumor volume of flank tumors as determined by caliper measurements;
  • FIG. 19D shows total flux analysis of bioluminescence measurements on day 20;
  • FIG. 19E shows lung weights.
  • FIGS. 20A-20G show the results of experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic administration of antibodies in dual flank tumor models.
  • Mice implanted with either the B16F10 tumor on the left and right flank or MC38 tumors on the left and right flank received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (Mod B) into only one flank tumor, while the other flank tumor was untreated. Mice also received intraperitoneal (systemic) injection of an anti-PD1 antibody.
  • FIG. 20 shows overall survival in the B16F10 ( FIG. 20A ) and MC38 ( FIG. 20B ) tumor models.
  • FIG. 20C-G show the results of an experiment evaluating the anti-PD-1 antibody where mice were implanted with B16F10 tumors on one flank and received an IV injection of luciferase-expressing B16F10 tumor cells that homed to the lung. Mice received three intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ and also received three intraperitoneal (systemic) injection of an anti-PD-1 antibody. Tumor growth of the SC tumors is depicted in FIG. 20C-F .
  • FIG. 20C shows control mRNA and control antibody
  • FIG. 20D shows control mRNA plus anti-PD1 antibody
  • FIG. 20E shows cytokine mRNA mixture plus isotype control antibody.
  • FIG. 20F shows cytokine mRNA plus anti-PD-1 antibody.
  • FIG. 20G shows percent survival of all four treatment groups until day 70 after IV tumor inoculation; the treatment group that received mRNA plus anti-PD-1 antibody showed strongest anti-tumoral activity with 6 out of 15 mice being tumor-free on day 40 after tumor inoculation.
  • FIGS. 21A-21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with systemic administration of antibodies.
  • Mice bearing CT26 tumors received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ . Mice also received intraperitoneal (systemic) injection of an anti-CTLA-4 antibody.
  • FIG. 21A shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 12 out of 16 mice being tumor-free on day 55 after tumor inoculation.
  • FIG. 21B shows cytokine mRNA mixture plus isotype control antibody; FIG.
  • FIG. 21C shows control mRNA plus anti-CTLA-4 antibody
  • FIG. 21D shows control mRNA and control antibody.
  • FIGS. 21E-21I show the results of additional experiments designed to assess the effect of intratumoral injection of mRNA mixtures in combination with anti-CTLA-4 antibody in B16F10 tumor model. Mice bearing B16F10 tumors received intratumoral injections with an mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ . Mice also received intraperitoneal (systemic) injection of an anti-CTLA-4 antibody.
  • FIG. 21E shows that the combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 6 out of 9 mice being tumor-free on day 70 after tumor inoculation.
  • FIG. 21F shows cytokine mRNA mixture plus isotype control antibody;
  • FIG. 21G shows control mRNA plus anti-CTLA-4 antibody;
  • FIG. 21H shows control mRNA and control antibody.
  • FIG. 21I shows percent survival of all four treatment groups until day 70 after tumor inoculation.
  • FIGS. 22A-22D shows the results of experiments designed to evaluate the effect of intratumoral injection of cytokine mRNA (Mod B) in human tumor xenografts of different human cancers. Intratumoral expression of each of the 4 mRNA encoded cytokines is shown: IL-12sc ( FIG. 22A ), IFN ⁇ 2b ( FIG. 22B ), GM-CSF ( FIG. 22C ), and IL-15 sushi ( FIG. 22D ).
  • FIGS. 23A-23D show the results of experiments designed to evaluate the effect of different intratumoral mRNA doses on the expression of the encoded cytokines: IL-15 sushi ( FIG. 23A ), IL-12sc ( FIG. 23B ), GM-CSF ( FIG. 23C ) and IFN ⁇ 2b ( FIG. 23D ).
  • FIGS. 24A-24G show the results of experiments where mice were implanted with B16F10 tumor, and treated with four intratumoral injections of cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ (ModB) or mRNA encoding a single cytokine. Tumor volume out to approximately day 70 was measured.
  • FIG. 24A shows luciferase control;
  • FIG. 24B shows the four-cytokine mixture;
  • FIG. 24C shows IL-12sc mRNA only;
  • FIG. 24D shows GM-CSF mRNA only;
  • FIG. 24E shows IFN ⁇ mRNA only; and
  • FIG. 24F shows IL-15 sushi only.
  • FIG. 24G shows overall survival of B16F10 tumors treated with cytokine mRNA mixture or individual mRNA encoded cytokines. Survival data is from experiment presented in FIG. 24A-F .
  • FIG. 25 shows CD8+ immune cell infiltrate in subcutaneous tumors after control mRNA (“placebo”) and cytokine mRNA treatment.
  • FIGS. 26A-26C show results of measurements of CD8+ T cells specific for the gp70 tumor antigen of gp70 in blood of CT26 tumor bearing mice that had received intratumoral administration of cytokine mRNA treatment and control mRNA, respectively.
  • FIG. 26C shows exemplarily a FACS histogram of CD8+ T cells stained with anti-mouse CD8 antibody and with the gp70-specific tetramer derived from an animal that had received control mRNA and
  • FIG. 26B shows the example from one animal treated with cytokine mRNA.
  • FIG. 26C shows the analysis of percentage of gp70-specific CD8+ T-cells in blood 13 days after treatment start from 9 mice that had received four injections of control mRNA and 10 mice that had received 4 injections of cytokine mRNA.
  • FIGS. 27A-27C show experiments where mice bearing B16F10 tumors on the left and right flanks received a single intratumoral mRNA injection with cytokine mRNA or control mRNA in only one tumor. On day 7 following the mRNA injection the left and right tumors were collected and subjected to RNA sequencing.
  • FIG. 27B shows the results of ingenuity pathway analysis comparing the gene expression changes between the cytokine mRNA treatment vs control mRNA treated tumors.
  • Causal network analysis for treated tumor side (Column 1) and untreated tumor side (Column 2) was performed and Activation Z score (Top half) and Inhibition Z score (Lower half) was analyzed to define pathways up and down regulated, respectively.
  • FIGS. 28A-28D show fluorescence micrographs of cells from a B16F10 dualtumor model.
  • Panel A shows the injected tumor treated with cytokine mRNA and panel B shows the corresponding uninjected tumor.
  • Panel C shows the injected tumor treated with control mRNA and panel D shows the corresponding uninjected tumor.
  • the slides were stained for CD4+, CD8+, and FoxP3+ cells.
  • FIGS. 28E-G show frequency of CD4+, CD8+ and FOXP3+ cells quantified in the immunofluorescent images.
  • the frequency of CD4+ and CD8+ cells/mm 2 is presented in FIGS. 28E and 28F .
  • the ratio of the CD8+ frequency divided by FOXP3+ frequency is presented in FIG. 28G .
  • FIGS. 29A-29G show mice with a single B16F10 tumor received a single injection with either mRNA encoding the Thy1.1 cell surface protein or vehicle alone (Ringer's solution). At approximately 16-18 hours following intratumoral injection the tumor was excised, digested, stained with a panel of antibodies and analyzed by flow cytometry. The cell type and frequency of cells expressing Thy1.1 were characterized.
  • FIGS. 30A-30F show expression of the indicated proteins following various doses of cytokine mRNA or luciferase control mRNA detected in tumor lysates as described in Example 15. “IFNy” in FIG. 30E indicates IFN ⁇ .
  • FIGS. 31A-31B show flow cytometry results for CD8+ and FOXP3+(Treg) cells following control or cytokine mRNA treatments as described in Example 15. The observed ratio of CD8+ to Treg cells is shown in each panel.
  • FIGS. 31C-31D show flow cytometry results for polyfunctional CD8+ T cells following control or cytokine mRNA treatments as described in Example 15. The proportion of polyfunctional CD8+ T cells is shown in each panel.
  • FIG. 31E shows the level of PD-L1 on infiltrating myeloid cells following control or cytokine mRNA treatments as described in Example 15.
  • FIG. 31F shows the level of PD-1 on infiltrating CD8+ cells following control or cytokine mRNA treatments as described in Example 15.
  • FIGS. 31G-H show the frequency of intratumoral Granzyme B CD8+ T cells following control or cytokine mRNA treatments as described in Example 15.
  • FIGS. 32A-32B show luciferase expression in various tissues following intratumoral injection of 50 ⁇ g mRNA encoding firefly luciferase as described in Example 16.
  • FIG. 33 shows data relating to an experiment essentially as shown in FIG. 12 .
  • FIG. 34 shows the effect of depleting CD8+ T cells, CD4+ T cells or NK cells before treatment with cytokine mRNAs on survival in mice bearing B16F10 tumors as described in Example 17.
  • FIG. 35 shows survival of WT and IFN ⁇ KO mice implanted with B16F10 tumor cells as described in Example 1 and treated with control or cytokine mRNAs as described in Example 18.
  • FIG. 36 shows a “peri-tumorally,” or “peri-tumoral,” area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery.
  • the peri-tumoral area comprises host tissue.
  • Tables 1 and 2 provide a listing of certain sequences referenced herein.
  • ModB describes RNA comprising a modified nucleobase in place of at least one (e.g., every) uridine and further comprising a Cap1 structure at the 5′ end of the RNA.
  • the 5′ UTR of a ModB RNA comprises SEQ ID NOs: 4 or 6.
  • ModB RNA has been processed to reduce double-stranded RNA (dsRNA).
  • the “Cap1” structure may be generated after in-vitro translation by enzymatic capping or during in-vitro translation (co-transcriptional capping).
  • the building block cap for ModB modified RNA is as follows, which is used when co-transcriptionally capping:
  • m 2 7,3′-O Gppp(m 1 2′-O )ApG also sometimes referred to as m 2 7,3′O G(5′)ppp(5′)m 2′-O ApG, which has the following structure:
  • Cap1 RNA after co-transcriptional capping which comprises RNA and m 2 7,3′O G(5′)ppp(5′)m 2′-O ApG:
  • ModA describes RNA without dsRNA reduction that does not comprise a modified nucleobase in place of at least one uridine.
  • ModA RNA comprises a Cap0 structure at the 5′ end of the RNA.
  • the 5′ UTR of a ModA RNA may comprise SEQ ID NO: 2.
  • “Cap0” structures are generated during in-vitro translation (co-transcriptional capping) using, in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m 2 7,3′O G(5′)ppp(5′)G)) with the structure:
  • Cap0 RNA comprising RNA and m 2 7,3′O G(5′)ppp(5′)G:
  • the “Cap0” structures are generated during in-vitro translation (co-transcriptional capping) using the cap analog Beta-S-ARCA (m 2 7,2′O G(5′)ppSp(5′)G) with the structure:
  • Cap0 RNA comprising Beta-S-ARCA (m 2 7,2′O G(5′)ppSp(5′)G) and RNA.
  • uracil describes one of the nucleobases that can occur in the nucleic acid of RNA.
  • the structure of uracil is:
  • uridine describes one of the nucleosides that can occur in RNA.
  • the structure of uridine is:
  • UTP uridine 5′-triphosphate
  • Pseudo-UTP (pseudouridine 5′-triphosphate) has the following structure:
  • Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Pseudouridine is described, for example, in Charette and Gray, Life; 49:341-351 (2000).
  • N1-methylpseudouridine (m1 ⁇ ), which has the structure:
  • N1-Methylpseudo-UTP has the following structure:
  • poly-A tail refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3′ end of an RNA molecule.
  • Poly-A tails or poly-A sequences are known to those of skill in the art, and may follow the 3′ UTR in the RNAs described herein.
  • An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical.
  • RNAs disclosed herein can have a poly-A tail attached to the free 3′ end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5′) of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).
  • the poly-A tail may be of any length.
  • a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly-A tail typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in the present invention.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency. Consequently, in one embodiment of the present invention, the poly-A tail contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly-A tail at its 3′ end, i.e., the poly-A tail is not masked or followed at its 3′ end by a nucleotide other than A.
  • a poly-A tail comprises the sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • RNA and “mRNA” are used interchangeably herein.
  • IFN ⁇ is used generically herein to describe any interferon alpha Type I cytokine, including IFN ⁇ 2b and IFN ⁇ 4.
  • human IFN ⁇ 2b and mouse IFN ⁇ 4 were utilized. Any IFN ⁇ may be incorporated into the compositions and used in the methods described herein.
  • treatment covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of the disease.
  • treatment of a solid tumor may comprise alleviating symptoms of the solid tumor, decreasing the size of the solid tumor, eliminating the solid tumor, reducing further growth of the tumor, or reducing or eliminating recurrence of a solid tumor after treatment.
  • Treatment may also be measured as a change in a biomarker of effectiveness or in an imaging or radiographic measure.
  • prevention means inhibiting or arresting development of cancer, including solid tumors, in a subject deemed to be cancer free.
  • Methodastasis means the process by which cancer spreads from the place at which it first arose as a primary tumor to other locations in the body.
  • intra-tumorally means into the tumor.
  • intra-tumoral injection means injecting the therapeutic at any location that touches the tumor.
  • peripheral is an area that is about 2-mm wide and is adjacent to the invasive front of the tumor periphery.
  • the peri-tumoral area comprises host tissue. See, for example, FIG. 36 .
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • the disclosure describes nucleic acid sequences and amino acid sequences having a certain degree of identity to a given nucleic acid sequence or amino acid sequence, respectively (a reference sequence).
  • Sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • % identical refers, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing said sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • the degree of identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.
  • the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments in continuous nucleotides.
  • the degree of identity is given for the entire length of the reference sequence.
  • Nucleic acid sequences or amino acid sequences having a particular degree of identity to a given nucleic acid sequence or amino acid sequence, respectively, may have at least one functional property of said given sequence, e.g., and in some instances, are functionally equivalent to said given sequence.
  • One important property includes the ability to act as a cytokine, in particular when administered to a subject.
  • a nucleic acid sequence or amino acid sequence having a particular degree of identity to a given nucleic acid sequence or amino acid sequence is functionally equivalent to said given sequence.
  • IL-2 Interleukin-2
  • the composition comprises a DNA sequence encoding interleukin-2 (IL-2) (SEQ ID NO: 9).
  • IL-2 interleukin-2
  • the DNA sequence encoding IL-2 is provided in SEQ ID NO: 10.
  • the composition comprises a codon-optimized DNA sequence encoding IL-2.
  • the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NOs: 11.
  • the DNA sequence comprises a codon-optimized DNA sequence with 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11.
  • the composition comprises an RNA sequence transcribed from a DNA sequence encoding IL-2.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 10 or 11.
  • the RNA sequence comprises or consists of SEQ ID NOs: 12 or 13.
  • the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 12 or 13.
  • one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the IL-2 RNA comprises an altered nucleotide at the 5′ end. In some embodiments, the IL-2 RNA comprises a 5′ cap. Any 5′ cap known in the art may be used. In some embodiments, the 5′ cap comprises a 5′ to 5′ triphosphate linkage. In some embodiments, the 5′ cap comprises a 5′ to 5′ triphosphate linkage including thiophosphate modification. In some embodiments, the 5′ cap comprises a 2′-O or 3′-O-ribose-methylated nucleotide. In some embodiments, the 5′ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5′ cap comprises 7-methylguanylate. In some embodiments, the 5′ cap is Cap0 or Cap1.
  • Exemplary cap structures include m7G(5′)ppp(5′)G, m7,2′ O-mG(5′)ppsp(5′)G, m7G(5′)ppp(5′)2′ O-mG, and m7,3′ O-mG(5′)ppp(5′)2′ O-mA.
  • the IL-2 RNA comprises a 5′ untranslated region (UTR).
  • the 5′ UTR is upstream of the initiation codon.
  • the 5′ UTR regulates translation of the RNA.
  • the 5′ UTR is a stabilizing sequence.
  • the 5′ UTR increases the half-life of RNA. Any 5′ UTR known in the art may be used.
  • the 5′ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 1, 3, or 5.
  • the 5′ UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6.
  • the 5′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.
  • the IL-2 RNA comprises a 3′ UTR.
  • the 3′ UTR follows the translation termination codon.
  • the 3′ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3′ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 7.
  • the 3′ UTR RNA sequence comprises or consists of SEQ ID NO: 8.
  • the 3′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IL-2 composition comprises both a 5′ UTR and a 3′ UTR. In some embodiments, the composition comprises only a 5′ UTR. In some embodiments, the composition comprises only a 3′ UTR.
  • the IL-2 RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50, at least about 70, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 78.
  • the RNA comprises a 5′ cap, a 5′ UTR, a nucleic acid encoding IL-2, a 3′ UTR, and a poly-A tail, in that order.
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the composition comprises an RNA sequence, that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 10 or 11; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 12 or 13; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IL-2 RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence encoding interleukin-12 single-chain (IL-12sc) (e.g., SEQ ID NO: 14), which comprises IL-12 p40 (sometimes referred to as IL-12B), a linker, such as a GS linker, and IL-12 p35 (sometimes referred to as IL-12A).
  • IL-12sc interleukin-12 single-chain
  • SEQ ID NO: 14 IL-12 single-chain
  • IL-12p40 sometimes referred to as IL-12B
  • linker such as a GS linker
  • IL-12 p35 sometimes referred to as IL-12A
  • the IL-12p40, linker, and IL-12p35 are consecutive with no intervening nucleotides.
  • An exemplary DNA sequence encoding IL-12sc is provided in SEQ ID NO: 15.
  • codon optimized IL-12 p40 The alignment of codon optimized IL-12 p40 to native IL-12 p40 is shown below, where the “S” is native IL-12 p40 (NM_002187.2; nucleotides 1-984 of SEQ ID NO: 15) and the “Q” is codon optimized IL-12 p40 (nucleotides 1-984 of SEQ ID NO: 16). The percent identity is 77%.
  • codon optimized IL-12 p35 to native IL-12 p35 is shown below, where the “S” is native IL-12 p35 (NM_00882.3; nucleotides 1027-1623 of SEQ ID NO: 15) and the “Q” is codon optimized IL-12 p35 (nucleotides 1027-1623 of SEQ ID NO: 16).
  • the percent identity is 80%.
  • the composition comprises a codon-optimized DNA sequence encoding IL-12sc. In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-12 p40. In some embodiments, the composition comprises a codon-optimized DNA sequence encoding IL-12 p35. In some embodiments, the codon-optimized DNA sequence comprises or consists of SEQ ID NO: 16. In some embodiments, the DNA sequence comprises a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16.
  • the codon-optimized DNA sequence encoding IL-12 p40 comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16). In some embodiments, the codon-optimized DNA sequence encoding IL-12 p35 comprises the nucleotides encoding the IL-12sc-p35 (nucleotides 1027-1623 of SEQ ID NO: 16).
  • the codon-optimized DNA sequence encoding IL-12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 16) and -p35 (nucleotides 1027-1623 of SEQ ID NO: 16) portions of SEQ ID NO: 16 and further comprises nucleotides between the p40 and p35 portions (e.g., nucleotides 985-1026 of SEQ ID NO: 16) encoding a linker polypeptide connecting the p40 and p35 portions. Any linker known to those of skill in the art may be used.
  • the p40 portion may be 5′ or 3′ to the p35 portion.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-12sc.
  • the RNA may also be recombinantly produced.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 15 or 16.
  • the RNA sequence comprises or consists of SEQ ID NOs: 17 or 18.
  • the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 17 or 18.
  • the RNA sequence comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NOs: 17 or 18) and -p35 (nucleotides 1027-1623 of SEQ ID NOs: 17 or 18) portions of SEQ ID NOs: 17 or 18.
  • the codon-optimized RNA sequence encoding IL-12sc comprises the nucleotides encoding the IL-12sc-p40 (nucleotides 1-984 of SEQ ID NO: 18) and -p35 (nucleotides 1027-1623 of SEQ ID NO: 18) portions of SEQ ID NO: 18 and further comprises nucleotides between the p40 and p35 portions encoding a linker polypeptide connecting the p40 and p35 portions. Any linker known to those of skill in the art may be used.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IL-12sc RNA comprises an altered nucleotide at the 5′ end.
  • the RNA comprises a 5′ cap. Any 5′ cap known in the art may be used.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage including thiophosphate modification.
  • the 5′ cap comprises a 2′-O or 3′-O-ribose-methylated nucleotide.
  • the 5′ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5′ cap comprises 7-methylguanylate. In some embodiments, the 5′ cap is Cap0 or Cap1.
  • Exemplary cap structures include m7G(5′)ppp(5′)G, m7,2′ O-mG(5′)ppsp(5′)G, m7G(5′)ppp(5′)2′ O-mG, and m7,3′ O-mG(5′)ppp(5′)2′ O-mA.
  • the IL-12sc RNA comprises a 5′ untranslated region (UTR).
  • the 5′ UTR is upstream of the initiation codon.
  • the 5′ UTR regulates translation of the RNA.
  • the 5′ UTR is a stabilizing sequence.
  • the 5′ UTR increases the half-life of RNA. Any 5′ UTR known in the art may be used.
  • the 5′ UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5.
  • the 5′ UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6.
  • the 5′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.
  • the IL-12sc RNA comprises a 3′ UTR.
  • the 3′ UTR follows the translation termination codon.
  • the 3′ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3′ UTR RNA sequence is transcribed from SEQ ID NO: 7.
  • the 3′ UTR RNA sequence comprises or consists of SEQ ID NO: 8.
  • the 3′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IL-12sc composition comprises both a 5′ UTR and a 3′ UTR. In some embodiments, the IL-12sc composition comprises only a 5′ UTR. In some embodiments, the IL-12sc composition comprises only a 3′ UTR.
  • the IL-12sc RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 78.
  • the RNA comprises a 5′ cap, a 5′ UTR, a nucleic acid encoding IL-12sc, a 3′ UTR, and a poly-A tail, in that order.
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 15 or 16; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 17 or 18; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IL-12sc RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the composition comprises a DNA sequence encoding interferon alpha (IFN ⁇ ) (e.g., SEQ ID NO: 19).
  • IFN ⁇ interferon alpha
  • SEQ ID NO: 19 An exemplary DNA sequence encoding this IFN ⁇ is provided in SEQ ID NO: 20.
  • the composition comprises a codon-optimized DNA sequence encoding IFN ⁇ .
  • the codon-optimized DNA sequence comprises or consists of the nucleotides of SEQ ID NO: 21.
  • the DNA sequence comprises or consists of a codon-optimized DNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IFN ⁇ .
  • the RNA may also be recombinantly produced.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 20 or 21.
  • the RNA sequence comprises or consists of SEQ ID NOs: 22 or 23.
  • the RNA sequence comprises or consists of an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 22 or 23.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • each uridine in the RNA is modified.
  • each uridine in the RNA is modified with N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IFN ⁇ RNA comprises an altered nucleotide at the 5′ end.
  • the IFN ⁇ RNA comprises a 5′ cap. Any 5′ cap known in the art may be used.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage including thiophosphate modification.
  • the 5′ cap comprises a 2′-O or 3′′-O-ribose-methylated nucleotide.
  • the 5′ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5′ cap comprises 7-methylguanylate. In some embodiments, the 5′ cap is Cap0 or Cap1.
  • Exemplary cap structures include m7G(5′)ppp(5′)G, m7,2′ O-mG(5′)ppsp(5′)G, m7G(5′)ppp(5′)2′ O-mG and m7,3′ O-mG(5′)ppp(5′)2′ O-mA.
  • the IFN ⁇ RNA comprises a 5′ untranslated region (UTR).
  • the 5′ UTR is upstream of the initiation codon.
  • the 5′ UTR regulates translation of the RNA.
  • the 5′ UTR is a stabilizing sequence.
  • the 5′ UTR increases the half-life of RNA. Any 5′ UTR known in the art may be used.
  • the 5′ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NOs: 1, 3, or 5.
  • the 5′ UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6.
  • the 5′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.
  • the IFN ⁇ RNA comprises a 3′ UTR.
  • the 3′ UTR follows the translation termination codon.
  • the 3′ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3′ UTR RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 7.
  • the 3′ UTR RNA sequence comprises or consists of SEQ ID NO: 8.
  • the 3′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IFN ⁇ composition comprises both a 5′ UTR and a 3′ UTR. In some embodiments, the composition comprises only a 5′ UTR. In some embodiments, the composition comprises only a 3′ UTR.
  • the IFN ⁇ RNA comprises a poly-A tail. In some embodiments, the IFN ⁇ RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides. In some embodiments, the poly-A tail comprises 200 or more nucleotides. In some embodiments, the poly-A tail comprises or consists of SEQ ID NO: 78.
  • the RNA comprises a 5′ cap, a 5′ UTR, a nucleic acid encoding IFN ⁇ , a 3′ UTR, and a poly-A tail, in that order.
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 20 or 21; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 22 or 23; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the term “IL-15 sushi” describes a construct comprising the soluble interleukin 15 (IL-15) receptor alpha sushi domain and mature interleukin alpha (IL-15) as a fusion protein.
  • the composition comprises a DNA sequence encoding IL-15 sushi (SEQ ID NO: 24), which comprises the soluble IL-15 receptor alpha chain (sushi) followed by a glycine-serine (GS) linker followed by the mature sequence of IL-15.
  • SEQ ID NO: 24 DNA sequence encoding IL-15 sushi
  • GS glycine-serine
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding IL-15 sushi.
  • the RNA may also be recombinantly produced.
  • the RNA sequence is transcribed from a nucleotide sequence comprising SEQ ID NO: 25.
  • the nucleotides encoding the linker may be completely absent or replaced in part or in whole with any nucleotides encoding a suitable linker.
  • the RNA sequence comprises or consists of SEQ ID NO: 26.
  • the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 26.
  • the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g., nucleotide 382-729 of SEQ ID NO: 25 or 26).
  • the DNA or RNA sequence encoding IL-15 sushi comprises the nucleotides encoding the sushi domain of IL-15 receptor alpha (e.g., nucleotide 1-321 of SEQ ID NOs: 25 or 26) and mature IL-15 (e.g., nucleotide 382-729 of SEQ ID NOs: 25 or 26) and further comprises nucleotides between these portions encoding a linker polypeptide connecting the portions.
  • the linker comprises nucleotides 322-381 of SEQ ID Nos: 25 or 26. Any linker known to those of skill in the art may be used.
  • one or more uridine in the IL-15 sushi RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IL-15 sushi RNA comprises an altered nucleotide at the 5′ end.
  • the IL-15 sushi RNA comprises a 5′ cap. Any 5′ cap known in the art may be used.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage including thiophosphate modification.
  • the 5′ cap comprises a 2′-O or 3′-O-ribose-methylated nucleotide.
  • the 5′ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide.
  • the 5′ cap comprises 7-methylguanylate. In some embodiments, the 5′ cap is Cap0 or Cap1.
  • Exemplary cap structures include m7G(5′)ppp(5′)G, m7,2′ O-mG(5′)ppsp(5′)G, m7G(5′)ppp(5′)2′ O-mG and m7,3′ O-mG(5′)ppp(5′)2′ O-mA.
  • the IL-15 sushi RNA comprises a 5′ untranslated region (UTR).
  • the 5′ UTR is upstream of the initiation codon.
  • the 5′ UTR regulates translation of the RNA.
  • the 5′ UTR is a stabilizing sequence.
  • the 5′ UTR increases the half-life of RNA. Any 5′ UTR known in the art may be used.
  • the 5′ UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5.
  • the 5′ UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6.
  • the 5′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.
  • the IL-15 sushi RNA comprises a 3′ UTR.
  • the 3′ UTR follows the translation termination codon.
  • the 3′ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3′ UTR RNA sequence is transcribed from SEQ ID NO: 7.
  • the 3′ UTR RNA sequence comprises or consists of SEQ ID NO: 8.
  • the 3′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the IL-15 sushi composition comprises both a 5′ UTR and a 3′ UTR. In some embodiments, the IL-15 sushi composition comprises only a 5′ UTR. In some embodiments, the IL-15 sushi composition comprises only a 3′ UTR.
  • the IL-15 sushi RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 78.
  • the RNA comprises a 5′ cap, a 5′ UTR, a nucleic acid encoding IL-15 sushi, a 3′ UTR, and a poly-A tail, in that order.
  • the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IL-15 sushi composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the IL-15 sushi composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the IL-15 sushi composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the IFN ⁇ RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
  • the composition comprises a DNA sequence encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) (e.g., SEQ ID NO: 27).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the DNA sequence encoding GM-CSF is provided in SEQ ID NO: 28.
  • the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence encoding GM-CSF.
  • the RNA sequence is transcribed from SEQ ID NO: 28.
  • the RNA may also be recombinantly produced.
  • the RNA sequence comprises or consists of SEQ ID NO: 29.
  • the RNA sequence comprises an RNA sequence with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NOs: 29.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the GM-CSF RNA comprises an altered nucleotide at the 5′ end.
  • the RNA comprises a 5′ cap.
  • the 5′ cap comprises a 5′ to 5′ triphosphate linkage. In some embodiments, the 5′ cap comprises a 5′ to 5′ triphosphate linkage including thiophosphate modification. In some embodiments, the 5′ cap comprises a 2′-O or 3′′-O-ribose-methylated nucleotide. In some embodiments, the 5′ cap comprises a modified guanosine nucleotide or modified adenosine nucleotide. In some embodiments, the 5′ cap comprises 7-methylguanylate. In some embodiments, the 5′ cap is Cap0 or Cap1.
  • Exemplary cap structures include m7G(5′)ppp(5′)G, m7,2′ O-mG(5′)ppsp(5′)G, m7G(5′)ppp(5′)2′ O-mG and m7,3′ O-mG(5′)ppp(5′)2′ O-mA.
  • the GM-CSF RNA comprises a 5′ untranslated region (UTR).
  • the 5′ UTR is upstream of the initiation codon.
  • the 5′ UTR regulates translation of the RNA.
  • the 5′ UTR is a stabilizing sequence.
  • the 5′ UTR increases the half-life of RNA. Any 5′ UTR known in the art may be used.
  • the 5′ UTR RNA sequence is transcribed from SEQ ID NOs: 1, 3, or 5.
  • the 5′ UTR RNA sequence comprises or consists of SEQ ID NOs: 2, 4, or 6.
  • the 5′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 2, 4, or 6.
  • the GM-CSF RNA comprises a 3′ UTR.
  • the 3′ UTR follows the translation termination codon.
  • the 3′ UTR regulates polyadenylation, translation efficiency, localization, or stability of the RNA.
  • the 3′ UTR RNA sequence is transcribed from SEQ ID NO: 7.
  • the 3′ UTR RNA sequence comprises or consists of SEQ ID NO: 8.
  • the 3′ UTR RNA sequence is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8.
  • the GM-CSF composition comprises both a 5′ UTR and a 3′ UTR. In some embodiments, the composition comprises only a 5′ UTR. In some embodiments, the composition comprises only a 3′ UTR.
  • the GM-CSF RNA comprises a poly-A tail.
  • the RNA comprises a poly-A tail of at least about 25, at least about 30, at least about 50 nucleotides, at least about 70 nucleotides, or at least about 100 nucleotides.
  • the poly-A tail comprises 200 or more nucleotides.
  • the poly-A tail comprises or consists of SEQ ID NO: 78.
  • the GM-CSF RNA comprises a 5′ cap, a 5′ UTR, nucleotides encoding GM-CSF, a 3′ UTR, and a poly-A tail, in that order.
  • the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the GM-CSF composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the GM-CSF composition comprises a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the composition comprises an RNA sequence that is, for example, transcribed from a DNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 1, 3, or 5; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the RNA may also be recombinantly produced.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • the RNA comprises a modified nucleoside in place of each uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (m 1 ⁇ ).
  • the GM-CSF composition comprises an RNA sequence comprising or consisting of a nucleic acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 4, or 6; and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8.
  • one or more uridine in the GM-CSF RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • RNAs and compositions described herein may be modified in any way known to those of skill in the art. In some embodiments, the modifications are “ModA” or “ModB” modified as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • the modified uridine replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m1 ⁇ ), or 5-methyl-uridine (m5U).
  • one or more cytosine, adenine or guanine in the RNA is replaced by modified nucleobase(s).
  • the modified nucleobase replacing cytosine is 5-methylcytosine (m 5 C).
  • the modified nucleobase replacing adenine is N 6 -methyladenine (m 6 A).
  • any other modified nucleobase known in the art for reducing the immunogenicity of the molecule can be used.
  • the modified nucleoside replacing one or more uridine in the RNA may be any one or more of 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 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), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl
  • the invention comprises a composition comprising more than one RNA as described herein.
  • the composition comprises two RNAs.
  • the composition comprises three RNAs.
  • the composition comprises four RNAs.
  • the composition comprises five RNAs.
  • any or all of the RNAs encoding IL-2, IL12sc, IL-15 sushi, GM-CSF, or IFN ⁇ may be replaced by IL-2, IL12sc, IL-15 sushi, GM-CSF, and/or IFN ⁇ polypeptides, e.g., in any of the compositions and formulations comprising these RNAs described herein.
  • the modified or unmodified RNAs encoding IL-2, IL12sc, IL-15 sushi, GM-CSF, and/or IFN ⁇ may be replaced by modified or unmodified polycistronic RNAs encoding two or more polypeptides selected from IL-2, IL12sc, IL15 sushi, GM-CSF and IFN ⁇ polypeptides, e.g., in any of the compositions and formulations comprising these RNAs described herein.
  • any of the combination compositions may further comprise an excipient or diluent.
  • the excipient or diluent may be pharmaceutically acceptable for administration to a subject.
  • a combination composition comprises RNAs with the same modifications. In some embodiments, a combination composition comprises RNAs with different modifications. In some embodiments, a combination composition comprises RNAs with ModA modification. In some embodiments, a combination composition comprises RNAs with ModB modification. In some embodiments, a combination composition comprises RNAs with ModA and ModB modifications.
  • a composition comprising DNA or RNA encoding IL-2 and one or more of a DNA or RNA encoding IL-12sc, IFN ⁇ , IL-15 sushi, and GM-CSF is encompassed.
  • the composition comprises a DNA or RNA encoding IL-2 or codon-optimized IL-2 (SEQ ID NOs: 10-13) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID Nos: 15-18), IFN ⁇ or optimized IFN ⁇ (SEQ ID Nos: 20-23), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • one or more of the RNAs in the composition further comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail as described herein in the composition section.
  • a composition comprising DNA or RNA encoding IL-12sc and one or more of a DNA or RNA encoding IL-2, IFN ⁇ , IL-15 sushi, and GM-CSF is encompassed.
  • the composition comprises a DNA or RNA encoding IL-12sc or codon-optimized IL-12sc (SEQ ID NOs: 15-18) and one or more of a DNA or RNA encoding IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), IFN ⁇ or optimized IFN ⁇ (SEQ ID NOs: 20-23), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • one or more of the RNAs in the composition further comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail, as described herein in the composition section.
  • a composition comprising DNA or RNA encoding IFN ⁇ and one or more of a DNA or RNA encoding IL-2, IL-12sc, IL-15 sushi, and GM-CSF is encompassed.
  • the composition comprises a DNA or RNA encoding IFN ⁇ or codon-optimized IFN ⁇ (SEQ ID NOs: 20-23) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), IL-15 sushi (SEQ ID NOs: 25-26), and GM-CSF (SEQ ID NOs: 28-29), as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • one or more of the RNAs in the composition further comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail as described herein in the composition section.
  • a composition comprising DNA or RNA encoding IL-15 sushi and one or more of a DNA or RNA encoding IL-2, IL-12sc, IFN ⁇ , and GM-CSF is encompassed.
  • the composition comprises a DNA or RNA encoding IL-15 sushi (SEQ ID NOs: 25-26) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IFN ⁇ or optimized IFN ⁇ (SEQ ID NOs: 20-23), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), and GM-CSF (SEQ ID NOs: 28-29), as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • one or more of the RNAs in the composition further comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail as described herein in the composition section.
  • a composition comprising DNA or RNA encoding GM-CSF and one or more of a DNA or RNA encoding IL-2, IL-12sc, IFN ⁇ , and IL-15 sushi is encompassed.
  • the composition comprises a DNA or RNA encoding GM-CSF (SEQ ID NOs: 28-29) and one or more of a DNA or RNA encoding IL-12sc or optimized IL-12sc (SEQ ID NOs: 15-18), IFN ⁇ or optimized IFN ⁇ (SEQ ID NOs: 20-23), IL-2 or optimized IL-2 (SEQ ID NOs: 10-13), and IL-15 sushi (SEQ ID NOs: 25-26), as described herein.
  • one or more uridine in the RNA is replaced by a modified nucleoside as described herein.
  • the modified nucleoside replacing uridine is pseudouridine ( ⁇ ), N1-methyl-pseudouridine (m 1 ⁇ ) or 5-methyl-uridine (m 5 U).
  • one or more of the RNAs in the composition further comprises a 5′ cap, a 5′ UTR, a 3′ UTR, and a poly-A tail as described herein in the composition section.
  • the composition comprises GM-CSF, IL-2, and IL-12sc RNA.
  • the composition is modified, for example, as ModA or ModB.
  • the IL-12sc RNA is optimized as shown in SEQ ID NO: 18.
  • the composition comprises GM-CSF, IL-15 sushi, and IL-12sc RNA.
  • the composition is modified, for example, as ModA or ModB.
  • the IL-12sc RNA is optimized as shown in SEQ ID NO: 18.
  • the composition comprises GM-CSF, IL-2, IL-12sc, and IFN ⁇ RNA.
  • the composition is modified, for example, as ModA or ModB.
  • the IL-12sc RNA and IFN ⁇ RNA is optimized as shown in SEQ ID NOs: 18 and 23, respectively.
  • the composition comprises GM-CSF, IL-15 sushi, IL-12sc, and IFN ⁇ RNA.
  • the composition is modified, for example, as ModA or ModB.
  • the IL-12sc RNA and IFN ⁇ RNA is optimized as shown in SEQ ID NOs: 18 and 23, respectively.
  • the composition comprises GM-CSF, IL-15 sushi, IL-12sc, and IFN ⁇ RNA, wherein the RNAs comprise or consist of the nucleotides shown in SEQ ID Nos: 18 (IL-12sc), 23 (IFN ⁇ ), 26 (IL-15 sushi), or 29 (GM-CSF).
  • the composition is modified, for example, as ModA or ModB.
  • combinations of RNA are administered as a 1:1, 1:1:1, or 1:1:1:1 ratio based on equal RNA mass.
  • the ratio is adjusted so that different ratios by mass are administered, for example, 1:10:1:10 ratio (20 ⁇ g, 200 ⁇ g, 20 ⁇ g, 200 ⁇ g).
  • a ratio of 1:2:3:4 (20 ⁇ g, 40 ⁇ g, 60 ⁇ g, 80 ⁇ g) is used.
  • the ratio may be based on the molarity of the RNA.
  • a mixture of RNAs is administered with an equal ratio of each RNA of the mixture.
  • a mixture of RNAs is administered with an unequal ratio of each RNA of the mixture. In some embodiments, one or more RNAs are administered at a ratio that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than another RNA in the mixture. In some embodiments, one or more RNAs are administered at a ratio that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times less than another RNA in the mixture.
  • the compositions described herein may be a medical preparation.
  • the medical preparation comprises a kit, wherein the included RNAs may be in the same or separate vials.
  • the medical preparation further comprising instructions for use of the composition for treating or preventing a solid tumor.
  • kits comprising the compositions described herein is provided, wherein the included RNAs may be in the same or separate vials. In some embodiments, the kit further comprising instructions for use of the composition for treating or preventing a solid tumor.
  • RNA can activate the immune system through stimulating various pattern recognition receptors (PRR) leading to production of Type I interferons (like IFN ⁇ ).
  • PRR pattern recognition receptors
  • the incorporation of various modified nucleotides, or other alterations like reducing the amount of dsRNA administered, can reduce the immune stimulatory effects of RNA.
  • inclusion of nucleotide-modified and dsRNA-reduced mRNA encoding interferon alpha improved anti-tumor activity relative to that of mRNA that was not nucleotide modified and dsRNA-reduced.
  • the addition of mRNA encoding interferon alpha restored a portion of the immune stimulatory effects removed by the inclusion of modified nucleotides and the dsRNA purification.
  • RNA encoding IFN (in any form or subtype) is provided, wherein the IFN RNA is altered to have reduced immunogenicity compared to un-altered RNA.
  • the administration of this IFN improves the anti-tumor response of non-IFN encoding RNA.
  • RNA encoding IFN ⁇ improves the anti-tumor response of other RNAs, so long as the other RNAs have been altered to reduce immunogenicity.
  • the alteration to reduce immunogenicity is a reduction in the amount of dsRNA.
  • the alteration to reduce immunogenicity is the replacement of one or more uridines with a modified nucleoside.
  • the alteration to reduce immunogenicity is both a reduction in the amount of dsRNA and the replacement of one or more uridines with modified nucleoside.
  • the IFN is IFN ⁇ .
  • IFN RNA improves the anti-tumor response of modified RNAs. In some embodiments, IFN RNA improves the anti-tumor response of RNAs comprising modified nucleotides. In some embodiments, IFN RNA improves the anti-tumor response of mRNAs comprising pseudouridine. In some embodiments, IFN RNA improves the anti-tumor response of RNAs with ModB modifications.
  • IFN RNA improves the anti-tumor response of RNAs of IL-2 (SEQ ID NO: 12 or 13), IL-12sc (SEQ ID NO: 17 or 18), IL-15 sushi (SEQ ID NO: 26) or GM-CSF (SEQ ID NO: 29).
  • the RNAs comprise ModB modifications.
  • the IFN is IFN ⁇ .
  • the IFN RNA construct is SEQ ID NO: 22 or 23.
  • RNAs, compositions, medical preparations and combination compositions described herein may be administered to a subject to treat cancer or a solid tumor.
  • the cancer is a solid tumor.
  • the solid tumor is an abnormal mass of tissue that does not contain cysts or liquid areas.
  • the solid tumor may be benign or malignant.
  • the solid tumor is a pre-cancerous lesion.
  • the solid tumor occurs in lung, colon, ovary, cervix, uterus, peritoneum, testicles, penis, tongue, lymph node, pancreas, bone, breast, prostate, soft tissue, connective tissue, kidney, liver, brain, thyroid, or skin.
  • the solid tumor is a sarcoma, carcinoma, or lymphoma.
  • the solid tumor is an epithelial tumor, Hodgkin lymphoma (HL), non-Hodgkin lymphoma, prostate tumor, ovarian tumor, renal cell tumor, gastrointestinal tract tumor, hepatic tumor, colorectal tumor, tumor with vasculature, mesothelioma tumor, pancreatic tumor, breast tumor, sarcoma tumor, lung tumor, colon tumor, brain tumor, melanoma tumor, basal cell carcinoma, squamous cell carcinoma, small cell lung tumor, neuroblastoma tumor, testicular tumor, carcinoma tumor, adenocarcinoma tumor, glioma tumor, seminoma tumor, retinoblastoma, or osteosarcoma tumor.
  • the solid tumor is a precancerous lesion such as actinic keratosis.
  • the RNA compositions may be delivered via injection into (e.g., intra-tumorally) or near (peri-tumorally) the tumor. In some embodiments, the RNA compositions may be delivered at or near the site of a tumor removal.
  • the RNA compositions may be delivered via a topical solution, ointment, or cream.
  • the RNA compositions may be delivered via a virus. In some embodiments, the RNA compositions may be delivered by infection with a virus encoding the RNA compositions, such as an oncolytic virus. In some embodiments, the RNA compositions may be delivered by an oncolytic virus.
  • a catheter is placed into or near the site of the tumor for multiple administrations. In some embodiments, a catheter is placed at the site of removal of a tumor for multiple administrations.
  • the subject is human. In some embodiments, the subject is a non-human mammal such as a dog, cat, mouse, rat, rabbit, sheep, cattle, horse and pig.
  • RNA compositions are combined with another therapy. In some embodiments, RNA compositions are combined with more than one other therapy. In some embodiments, RNA compositions are combined in a multi-modal therapy.
  • the other therapy is surgery to excise, resect, or debulk the tumor.
  • therapeutic RNA compositions are administered during a surgery to excise, resect, or debulk the tumor.
  • the other therapy is radiotherapy.
  • the radiotherapy is external beam radiation therapy or particle beam radiation.
  • the radiotherapy is brachytherapy involving temporary or permanent implantation of radioactive isotopes directly into the tumor via catheter or large bore needle.
  • the radioactive isotope is 137Cesium, 192Iridium, or radioactive iodine.
  • the radiotherapy is radioisotope preparations administered intravenously.
  • the radioisotope preparations are radioactive iodine (131I) Strontium (89Sr), or Samarium (153Sm).
  • the other therapy is chemotherapy.
  • the chemotherapy is an alkylating agent, an antimetabolite, an anti-microtubule agent, a topoisomerase inhibitor, or a cytotoxic antibody.
  • the chemotherapy comprises anti-invasion agents (e.g., metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function).
  • the chemotherapy comprises inhibitors of growth factor function (e.g., platelet derived growth factor and hepatocyte growth factor), growth factor antibodies, or growth factor receptor antibodies, (e.g., anti-erbb2 antibody trastuzumab [HerceptinTM] and the anti-erbb1 antibody CetuximabTM).
  • the chemotherapy is a farnesyl transferase inhibitor.
  • the chemotherapy is a tyrosine kinase inhibitor such as inhibitors of the epidermal growth factor family (e.g., EGFR family tyrosine kinase inhibitors such as gefitinib (IressaTM), erlotinib (TarcevaTM), and Canertinib (CI 1033), or a serine/threonine kinase inhibitor).
  • EGFR family tyrosine kinase inhibitors such as gefitinib (IressaTM), erlotinib (TarcevaTM), and Canertinib (CI 1033)
  • serine/threonine kinase inhibitor e.g., a serine/threonine kinase inhibitor.
  • the chemotherapy comprises antiproliferative/antineoplastic drugs such as antimetabolites (e.g., antifolates like methotrexate, fluoropyrimidines like 5-fluorouracil, tegafur, purine and adenosine analogues, cytosine arabinoside); antitumour antibiotics (e.g., anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin); platinum derivatives (e.g., cisplatin, carboplatin); alkylating agents (e.g., nitrogen mustard, melphalan, chlorambucil, busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa); antimitotic agents (e.g., vinca alkal
  • the chemotherapy is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the ADC is an antibody linked to a cytotoxic (anticancer) drug.
  • the ADC allows targeted delivery of cytotoxic drugs to tumor cells.
  • the ADC allows preferential deliver of cytotoxic drugs to tumor cells versus normal tissue.
  • the chemotherapy is combination chemotherapy with a combination of different agents.
  • the combination comprises different agents that have different mechanisms of action and/or different, non-overlapping toxicities.
  • the other therapy is an immune stimulator or immunotherapy, such as, for example, a checkpoint modulator/inhibitor.
  • Checkpoint modulators/inhibitors are well known in the art to prevent the host immune system from attacking itself, and include, for example, CTLA-4, PD1, PDL1, GITR, OX40, LAG-3, and TIM-3.
  • the immune stimulator, immunotherapy, or checkpoint modulator/inhibitor is a monoclonal antibody.
  • the monoclonal antibody is an antibody against PD1, PDL1, CTLA-4, LAG3, OX40, CD40, CD40L, 41BB, 41BBL, GITR, CD3, CD28, CD38, or TGFbeta.
  • the monoclonal antibody is a bispecific antibody.
  • the immune stimulator is a cell-based immunotherapy.
  • the immune stimulator is a cytokine or chemokine.
  • the immune stimulator is a cancer vaccine.
  • the invention is not limited to a specific combination with a particular immune stimulator.
  • any of the RNAs, RNA compositions, medical preparations, and RNA combination compositions described herein may be administered in combination with an immune stimulator, immunotherapy, or checkpoint modulator.
  • the RNAs, RNA compositions, and RNA combination compositions described herein are administered in combination with an antibody to a subject to treat cancer, including solid tumors.
  • the antibody is an anti-PD1 antibody, an anti-CTLA4 antibody, or a combination of an anti-PD1 antibody and anti-CTLA4 antibody.
  • the antibody is a multi-specific antibody such as, for example, a tri-specific or bi-specific antibody.
  • the anti-PD1 antibody is a chimeric, humanized or human antibody.
  • the anti-PD-1 antibody is isolated and/or recombinant.
  • anti-PD-1 antibodies are nivolumab, pembrolizumab, cemiplimab, MEDI0608 (formerly AMP-514; see, e.g., WO 2012/145493 and U.S. Pat. No. 9,205,148), PDR001 (see, e.g., WO 2015/112900), PF-06801591 (see, e.g., WO 2016/092419), BGB-A317 (see, e.g., WO 2015/035606).
  • the anti-PD-1 antibody is one of those disclosed in WO 2015/112800 (such as those referred to as H1M7789N, H1M7799N, H1M7800N, H2M7780N, H2M7788N, H2M7790N, H2M7791N, H2M7794N, H2M7795N, H2M7796N, H2M7798N, H4H9019P, H4xH9034P2, H4xH9035P2, H4xH9037P2, H4xH9045P2, H4xH9048P2, H4H9057P2, H4H9068P2, H4xH9119P2, H4xH9120P2, H4xH9128P2, H4xH9135P2, H4xH9145P2, H4xH8992P, H4xH8999P and H4xH9008P in Table 1 of the PCT publication, and those referred to as H4H7798N, H
  • WO 2015/112800 The disclosure of WO 2015/112800 is incorporated by reference herein in its entirety.
  • the antibodies disclosed in WO 2015/112800 and related antibodies including antibodies and antigen-binding fragments having the CDRs, VH and VL sequences, or heavy and light chain sequences disclosed in that PCT publication, as well as antibodies and antigen-binding fragments binding to the same PD-1 epitope as the antibodies disclosed in that PCT publication, can be used in conjunction with the RNA compositions of the present invention to treat and/or prevent cancer.
  • the anti-PD-1 antibody may comprise the heavy and light chain amino acid sequences shown below as SEQ ID NOs: 79 and 80, respectively; the VH and VL sequences in SEQ ID NOs: 87 and 88 (shown in italics), or one or more (e.g., all six) CDRs in SEQ ID NOs: 79 and 80 (shown in bold boxes).
  • an antibody comprising the following CDRs is encompassed:
  • HCDR1 GFTFSNFG (SEQ ID NO: 82)
  • HCDR2 ISGGGRDT (SEQ ID NO: 83)
  • HCDR3 VKWGNIYFDY (SEQ ID NO: 84)
  • LCDR1 LSINTF (SEQ ID NO: 85)
  • LCDR2 AAS (SEQ ID NO: 86)
  • LCDR3 QQSSNTPFT.
  • An exemplary antibody comprising a heavy chain comprising the VH and VL sequences in SEQ ID NOs: 87 and 88 (shown in italics) is the fully human anti-PD-1 antibody known as REGN2810 (cemiplimab).
  • Anti-PD-1 Mab heavy chain (SEQ ID NO: 79) KGPSVFPLAP CSRSTSESTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH NHYTQKSLSL SLGK (SEQ ID NO: 81)
  • the RNAs, RNA compositions, and RNA combination compositions may be delivered via injection into the tumor (e.g., intratumorally), near the tumor (peri-tumorally), or near the site of a tumor removal, and the antibody may be delivered in the same manner or systemically, such as, for example, enteral or parenteral, including, via injection, infusion, and implantation.
  • “Administered in combination” includes simultaneous or sequential administration. If sequential, administration can be in any order and at any appropriate time interval known to those of skill in the art.
  • the other therapy is hormonal therapy.
  • the hormonal therapy is antiestrogen drugs for treatment of breast cancer or anti-androgen drugs for treating prostate cancer.
  • Example agents include antiestrogens (e.g., tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene), estrogen receptor down regulators (e.g., fulvestrant), progestogens (e.g., megestrol acetate), aromatase inhibitors (e.g., anastrozole, letrazole, vorazole, exemestane), antiprogestogens, antiandrogens (e.g., flutamide, nilutamide, bicalutamide, cyproterone acetate), LHRH agonists and antagonists (e.g., goserelin acetate, luprolide, buserelin), and inhibitors of 5-alpha-reducta
  • the other therapy is a targeted therapy.
  • the targeted therapy is a kinase inhibitor.
  • the targeted therapy is one that inhibits activity of a gene product of a proto-oncogene.
  • the targeted therapy is an anti-angiogenic agent.
  • the targeted therapy is one directed to modulate activity of VEGF, BCR-ABL, BRAF, EGFR, c-Met, MEK, ERK, mTOR, or ALK.
  • the other therapy is stem cell transplantation.
  • therapeutic RNA compositions are delivered at the same time as another therapy.
  • therapeutic RNA compositions are delivered before another therapy.
  • therapeutic RNA compositions are delivered after another therapy.
  • therapeutic RNA compositions are delivered directly into the tumor, or near the tumor or the site of tumor removal together with another therapy. In some embodiments, therapeutic RNA compositions are delivered directly into the tumor, or near the tumor or site of tumor removal while another agent is delivered systemically.
  • any of the DNAs, RNAs, and compositions described herein are pharmaceutical formulations.
  • the pharmaceutical formulations comprise a diluent, excipient, or other pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising the DNA, RNA, compositions, or combinations thereof provided herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable excipient is an aqueous solution.
  • the aqueous solution is a saline solution.
  • pharmaceutically acceptable excipients are understood to be sterile.
  • a pharmaceutical composition is administered in the form of a dosage unit.
  • a dosage unit is in the form of a tablet, capsule, implantable device, or a bolus injection.
  • the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents.
  • the compositions may also contain additional, compatible, pharmaceutically-inactive materials such as excipients, diluents, and carriers.
  • compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
  • Lipid moieties may be used to deliver the RNAs provided herein.
  • the RNA is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • RNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a pharmaceutical composition provided herein comprises a polyamine compound or a lipid moiety complexed with the DNA or RNA provided herein.
  • a pharmaceutical composition provided herein comprises a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • a pharmaceutical composition provided herein comprises a RNA or combination of RNAs in a therapeutically effective amount.
  • the therapeutically effective amount is sufficient to treat or prevent cancer in the subject being treated.
  • mice Female C57BL/6J mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and housed on 12 hours light/dark cycle at 22° C. ⁇ 2° C. with a relative humidity of 55% ⁇ 15%.
  • B16F10 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, Va. USA) (Cat No. CRL-6475) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Life technologies, Cat No.
  • ATCC American Type Culture Collection
  • DMEM Dulbecco's Modified Eagle's Medium
  • mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and housed on 12 hours light/dark cycle at 22° C. ⁇ 2° C. with a relative humidity of 55% ⁇ 10%.
  • B16F10 cells were obtained from the American Type Culture Collection (ATCC® CRL-6475TM) and cultured in DMEM, high glucose, GlutaMAXTM (Life technologies, Cat No. 31966047) supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom, Cat No. S 0115) in 7.5% CO2 at 37° C. The cells were harvested using StemPro® Accutase® Cell Dissociation Reagent (Life technologies, Cat No.
  • B16F10_luc-gfp cells were used for seeding of distant lung tumors. These cells were derived from B16F10 by stable transfection with a plasmid coding for Luciferase and GFP.
  • B16F10_luc-gfp cells were cultured with the same conditions as the B16F10; only 0.5 ⁇ g/mL Puromycin was added to the culture medium.
  • One day after SC implantation 0.3 ⁇ 10 ⁇ circumflex over ( ) ⁇ 6 cells/200 ⁇ l of B16F10_luc-gfp cells were injected per mouse intravenously (IV) into the tail vein.
  • Intratumoral mRNA injections were initiated 10-14 days after SC inoculation of the tumors. Tumor growth was assessed by caliper measurements every 2-3 days and is expressed as the product of the perpendicular diameters using the following formula: a2*b/2, with a ⁇ b.
  • Engraftment of luciferase-positive tumor cells in the lung was analyzed by in vivo bioluminescence imaging using the Xenogen IVIS Spectrum imaging system (Caliper Life Sciences).
  • An aqueous solution of L-luciferin (2500, 1.6 mg; BD Biosciences) was injected intraperitoneally. Emitted photons from live animals quantified 10 min later with an exposure time of 1 min.
  • Regions of interest (ROI) were quantified as average radiance (photons s ⁇ 1 cm ⁇ 2 'sr ⁇ 1 , represented by as color-scaled images superimposed on grayscale photos of mice using the Living Image software from Caliper Life Sciences).
  • mice Female Balb/c Rj mice (Janvier, Genest-St.-Isle, France), 6-8 weeks of age, with a weight between 17 and 24 g, were acclimated for at least six days prior to study enrollment. Mice had free access to food (ssniff M-Z autoclavable Soest, Germany) and sterile water and housed on 12 hours light/dark cycle at 22° C. ⁇ 2° C. with a relative humidity of 55% ⁇ 10%.
  • CT26 cells were obtained from the (ATCC® CRL-2638TM) and cultured in Roswell Park Memorial Institute medium (RPMI) 1640 Medium, GlutaMAXTM (Life technologies, Cat No. 61870-044) supplemented with 10% Fetal Bovine Serum (FBS) (Biochrom, Cat No. S 0115) in 5% CO2 at 37° C.
  • the cells were harvested using StemPro® Accutase® Cell Dissociation Reagent (Life technologies, Cat No. A1110501), resuspended in DPBS (Life technologies, Cat No. 14190-169), and 0.5 ⁇ 10 ⁇ circumflex over ( ) ⁇ 6 cells/100 ⁇ l per mouse SC implanted into the right shaven flank of female Balb/c Rj mice.
  • Intratumoral RNA injections were initiated 13-19 days after inoculation of the tumors. Tumor growth was assessed by caliper measurements every 2-3 days and is expressed as the product of the perpendicular diameters using the following formula: a2*b/2 where b is the longer of the two diameters (a ⁇ b).
  • CT26 cells were obtained from the ATCC (Manassas, Va. USA) (Cat No. CRL-2638) and cultured in RPMI-1640 (Life technologies, Cat No. 11875-093) supplemented with 10% HI FBS (Life technologies, Cat No.
  • mice Female C57BL/6J mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA) and sterile water and housed on 12 hours light/dark cycle at 22° C. ⁇ 2° C. with a relative humidity of 55% ⁇ 15%.
  • MC38 cells were generous gifts from Dr S. A. Rosenberg (National Institute of Health, Bethesda, Md., USA). The cell line was cultured in RPMI-1640 with L-glutamine (Gibco, Cat No. 11875) supplemented with 10% HI FBS (Gibco, Cat No.
  • mice Female severe combined immune deficiency mice (SCID) mice (Jackson Laboratory; Bar Harbor, Me.), 6-8 weeks-old and weighing between 17.0 and 20.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and housed on 12 hours light/dark cycle at 22° C. ⁇ 2° C. with a relative humidity of 55% ⁇ 15%. A375 cells were obtained from the ATCC (Manassas, Va. USA) (Cat No. CRL-1619). The cell line was cultured in DMEM (Life technologies, Cat No. 11995) supplemented with 10% HI FBS (Life technologies, Cat No.
  • KM12 (CRC) Xenograft Model Female NOD.CB17-Prkdcscid/SCID mice (Jackson Laboratory, Bar Harbor, Me.), 10-weeks-old and weighing between 17.3 g and 21.9 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22 ⁇ 2° C.) with a relative humidity (55 ⁇ 15%). KM-12 cells were obtained from the American National Cancer Institute (NCI) (Cat No. 507345). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No.
  • RPMI8226 Myeloma Xenograft Model: Female NSG mice (Jackson Laboratory, Bar Harbor, Me.), 12-weeks-old and weighing between 19.8 g and 26.6 g were acclimated for at least three days prior to study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22 ⁇ 2° C.) with a relative humidity (55 ⁇ 15%). RPMI8226 cells were obtained from the ATCC (Cat No. CCL-155). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No.
  • NCI-N87 (Gastric) Xenograft Model Female NOD.CB17-Prkdcscid/SCID (Jackson Laboratory, Bar Harbor, Me.), 11-weeks-old and weighing between 18.3 and 22.7 g were acclimated for at least three days before the study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22 ⁇ 2° C.) with a relative humidity (55 ⁇ 15%). NCI-N87 cells were obtained from the ATCC (Cat No. CRL-5822). The cells were grown in RPMI medium 1640 with L-glutamine (Gibco, Cat No.
  • NCI-H1975 (NSCLC) Xenograft Model Female NSG mice (Jackson Laboratory, Bar Harbor, Me.), 10-weeks-old and weighed between 18.8 g and 26.0 g were allowed to acclimate for at least three days before study enrollment. Mice had free access to food (Harlan 2916 rodent diet, Massachusetts, USA), sterile water and were housed on 12 hours light/dark cycle at (22 ⁇ 2° C.) with a relative humidity (55 ⁇ 15%). NCI-H1975 cells were obtained from the ATCC (Cat No. CRL-5908) and cultured in RPMI medium 1640 with L-glutamine (Gibco, Cat No.
  • mice Female C57BL/6J mice were implanted with B16F10 cells as described above. Mice were treated with 4 intratumoral injections (80 ⁇ g mRNA/20 ⁇ g per target) on days 11, 13, 15, and 17 with ModB cytokine mRNA mixture (IL-15sushi, IL-12sc, GM-CSF, IFN ⁇ ). After cytokine mRNA treatment 8 mice were tumor free. Four weeks after the last cytokine mRNA treatment tumor free mice were re-challenged with 0.5 ⁇ 10 ⁇ circumflex over ( ) ⁇ 6 B16F10 cells/200 ⁇ l per mouse by SC injection and tumor growth was monitored.
  • ModB cytokine mRNA mixture IL-15sushi, IL-12sc, GM-CSF, IFN ⁇
  • Tumors were measured with a caliper twice weekly until final sacrifice. When a tumor size reached approximately 2000 mm 3 or there are animal health issues (20% area of a tumor ulcerated), animals were euthanized. Tumor regression was defined as i) tumor volume ⁇ 20 mm3 at the end of the study or ii) T F /T 0 ⁇ 1, where the T F equals the final tumor volume and T 0 equals tumor volume on the day of the first intratumoral mRNA injection.
  • ModA mRNA Modification a
  • Synthetic DNA fragments coding for the gene of interest were cloned into a common starting vector, comprising a 5′-UTR (corresponding in some cases to SEQ ID NO: 1), a 3′ UTR consisting of two elements called F and I (corresponding in some cases to SEQ ID NO: 7), and a poly(A)-tail of 110 nucleotides in total (A30-linker-A70 structure; corresponding in some cases to SEQ ID NO: 78).
  • Linearization of plasmid DNA was performed downstream of the poly(dA:dT) with a classIIS restriction enzyme to generate a template with no additional nucleotide beyond the poly(dA:dT) (see Holtkamp et al., Blood 108(13):4009-172006 (2006)).
  • Linearized plasmid DNA was subjected to in vitro transcription with T7 RNA polymerase (Thermo Fisher) as previously described (see Grudzien-Nogalska E et al., Methods Mol Biol.
  • ModB mRNA Modification B
  • Synthetic DNA fragments coding for the gene of interest were cloned into a common starting vector, comprising a 5′-UTR (corresponding in some cases to the Tobacco Etch Viral leader sequences TEV, SEQ ID NO: 3), a 3′ UTR consisting of two elements called F and I (corresponding is some cases to SEQ ID NO: 7), and a poly(A)-tail of 110 nucleotides in total (A30-Linker-A70 structure).
  • a 5′-UTR corresponding in some cases to the Tobacco Etch Viral leader sequences TEV, SEQ ID NO: 3
  • a 3′ UTR consisting of two elements called F and I (corresponding is some cases to SEQ ID NO: 7)
  • a poly(A)-tail of 110 nucleotides in total A30-Linker-A70 structure
  • RNA was then purified using magnetic particles (Berensmeier 2006), and subsequently Cap1 structure was enzymatically introduced using a commercially available system based on the Vaccinia virus capping enzyme (NEB) and addition of mRNA Cap 2′-O-methyltransferase (NEB).
  • NEB Vaccinia virus capping enzyme
  • RNA concentration and quality were assessed spectrophotometry and analyzed by capillary gel electrophoresis systems, respectively. Presence of dsRNA was assessed in a Northwestern dot-blot assay using dsRNA-specific J2 mAb (English & Scientific Consulting) as described in Karikó et al. Nucleic Acids Res. 39(21):e142 (2011).
  • the coding sequence of a protein may influence the efficiency as well as the accuracy of protein translation (see Bossi L et al., Nature. 286(5769):123-7 (1980) and Irwin et al., J Biol Chem. 270(39):22801-6 (1995)).
  • codon variants of each target were designed and tested.
  • the design of the different codon variants for each target utilized publicly available software from Life Technologies GmbH GeneArt® (Regensburg, Germany) (see Raab D et al., Syst Synth Biol. 4(3):215-25 (2010)) and Eurofins MWG Operon (Ebersberg, Germany).
  • codon optimization was performed manually editing each codon separately. A GC-content comparable to the wild type sequence was maintained during the optimization process.
  • HEK293T/17 cells Forty thousand (40,000) HEK293T/17 cells (ATCC® CRL-11268TM) were seeded in flat bottom 96-well plates (VWR International, Cat No. 734-1794) in DMEM, high glucose, GlutaMAXTM (Life technologies, Cat No. 31966047) containing 0.5% FBS (Biochrom, Cat No. S 0115). Seeded cells in 96-well plates were incubated at 37° C., 7.5% CO2 for 16-18 hours. Adherent HEK293T/17 cells were transfected with RNA using Lipofectamine Messenger MAX Reagent (Invitrogen, Cat No.
  • LMRNA LMRNA
  • OptiMEM Thermo Fisher, Cat No. 31985070
  • RNAse-free 1.5 ml Safe-Lock tube biopur Eppendorf, Germany, Cat No. 0030121589
  • the tube containing the RNA was diluted into the tube containing the Lipofectamine Messenger MAX and incubated an additional 5 minutes prior to adding 10 ⁇ l of the RNA-lipid-complex drop wise to one well of the 96-well plate containing the HEK293T/17 cell layer in 100 ⁇ l medium.
  • the 10 ⁇ l RNA-lipid-complex contained 5 ng, 25 ng and 100 ng of target RNA respectively.
  • the plates were placed into the incubator for 3 h before an additional 140 ⁇ l of fresh medium (DMEM+0.5% FBS) was added.
  • the transfected cells were incubated for 15-18 hours and the supernatants were collected and analyzed for protein content by ELISA as described herein.
  • K562 a human cell line derived from chronic myeloid leukemia (ATCC® CCL-243TM) was cultivated in RPMI 1640 Medium, GlutaMAXTM (Life technologies, Cat No. 61870-044) supplemented with 5% FBS. K562 were electroporated in a 96-well plate system as follows. Cells were washed once in X-VIVO15 medium (Lonza, Cat No. BE02-060Q) and suspended to a final concentration of two hundred fifty thousand (250,000) cells/150 ⁇ l in X-VIVO15. A 150 ⁇ l of cell suspension was added per well of a 96-well plate containing 5 ng, 25 ng, or 100 ng of RNA.
  • Protein concentrations were determined by ELISAs specific for the RNA encoded cytokine according to the manufacturer's protocol.
  • Human IL-15 sushi/IL-15 sushi R alpha Complex DuoSet ELISA ii) Mouse IL-12sc Duo Set Development System (DY419-05)
  • the protein expression was evaluated for the wt-sequence and the different codon-optimized variants. Both data sets from lipofection of HEK293T/17 and electroporation of K562 each tested with the three different amounts of modified RNA were considered for selection of the protein coding sequence. A codon-optimized sequence was selected if an at least 1.5-fold increase of protein expression compared to WT sequence was measured. If this was not the case, the WT sequence was selected. For all constructs Earl-restriction sequences were eliminated by mutating the DNA recognition sequence (5′-CTCTTC-3′), while preserving the WT amino acid.
  • DMEM ThermoFisher Scientific, Cat 11885-084
  • Cells were transfected using Lipofectamine MessengerMAX Reagent (Invitrogen, Cat # LMRNA001) according to the manufacturer protocol. Briefly, for each well 0.3 ⁇ l of the transfection reagent was diluted with 5 ⁇ l of the Opti-MEM media (Life Technologies, Cat. 31985062) and incubated for 10 min at room temperature; mRNA mixtures were diluted with Opti-MEM media (5 ⁇ l per well) and mixed with diluted MessengerMax reagent, incubated for 5 min at room temperature and aliquoted to the 96-well plate. Cells were diluted in complete growth media and 40,000 cells per well were added to the transfection mixtures. Cells were incubated for 24 hours at 37° C. in a CO2 incubator then media was collected and cytokine concentration was determined by Meso Scale Discovery (MSD) assay.
  • MSD Meso Scale Discovery
  • Cytokine concentration was determine using MSD assays: Proinflammatory Panel 1 (human) MSD kit (catalog N05049A-1) for IL12p70, Cytokine Panel 1 (human) MSD kit (catalog N05050A-1) for GM-CSF and IL-15 sushi, and Human IFN- ⁇ 2a Ultra-sensitive Kit (catalog N05050A-1) for IFN ⁇ . Data were analyzed using MSD Discovery Workbench V. 4.0.12 software and GraphPad Prism V.6.00 software.
  • PBMCs Peripheral Blood Mononuclear Cells
  • PBMCs Peripheral Blood Mononuclear Cells
  • B16F10 tumor bearing mice received a single intratumoral injection of Immuno mRNA (IFN ⁇ , IL-15 sushi, GM-CSF, and IL-12sc, ModB) or control luciferase mRNA (Placebo). Seven days after intratumoral mRNA injection tumors were excised, processed for immunofluorescence and stained with an antibody for CD8 (gray). As shown in FIG. 25 , CD8 cells were present after cytokine mRNA intratumoral injection.
  • IFN ⁇ Immuno mRNA
  • IL-15 sushi IL-15 sushi
  • GM-CSF GM-CSF
  • IL-12sc, ModB control luciferase mRNA
  • the respective mRNA mixtures were prepared for in vivo studies by mixing equal quantities (micrograms) of mRNA in water at 2 ⁇ the intended dose.
  • the mRNA mixture was frozen at ⁇ 80 C until the day of intratumoral injection.
  • mRNA was thawed and mixed with an equal volume of 2 ⁇ sterile Ringer's solution.
  • the resulting 1 ⁇ mRNA/Ringer solution was used for intratumoral injection.
  • Example 2 Combinations of Three mRNAs Reduce Tumor Volume In Vivo
  • a mixture of modified mRNAs encoding GM-CSF, IL-2, and IL-12sc was injected into B16F10 tumor bearing mice and tumor growth was monitored to day 41.
  • intratumoral injection of a combination of three mRNAs encoding GM-CSF, IL-2, and IL-12sc having ModA SEQ ID NOs: 32, 38, and 56; FIG. 1A
  • a combination of three mRNAs encoding GM-CSF, IL-2, and IL-12sc having ModB SEQ ID NOs: 35, 41, and 59; FIG.
  • FIG. 1B mice treated with a control mRNA encoding luciferase (ModA) displayed tumor regression in 1 of 10 animals
  • FIG. 1C mice treated with a control mRNA encoding luciferase
  • FIG. 1D-1G mice treated with a control mRNA encoding luciferase
  • FIG. 1D-1G mice treated with a control mRNA encoding luciferase
  • FIG. 1D-1G a control mRNA encoding luciferase
  • cytokine mRNA treatment was evaluated in CT26 tumors.
  • Mice with established CT26 tumors were injected with a cytokine mRNA mixture encoding GM-CSF, IL-2, and IL-12sc in ModA and ModB formats, respectively.
  • Two control groups were included: i) mRNA Ringer's diluent and ii) ModA mRNA encoding firefly luciferase.
  • a total of 6 intratumoral injections were administered on days 19, 21, 24, 26, 28 and 31.
  • both GM-CSF, IL-2, and IL-12sc mRNA ModA SEQ ID NOs: 56, 32, and 38; FIG.
  • FIG. 2A and ModB (SEQ ID NOs: 59, 35, and 41; FIG. 2B ) resulted in tumor regression in 5 and 6 out of 8 mice, respectively, while no tumors treated with control mRNA in ModA ( FIG. 2C ) or Ringer's solution ( FIG. 2D ) displayed tumor regression.
  • the cytokine mRNA mixture encoding IL-15 sushi, GM-CSF and IL-12sc (ModB; SEQ ID NOs: 53, 59, and 41) and IL-2, GM-CSF and IL-12sc (ModB; SEQ ID NOs: 35, 59, and 41) were evaluated for anti-tumor activity in the CT26 tumor model.
  • Tumors received intratumoral mRNA injections on days 13, 15, 18, 20 and 22 after tumor inoculation.
  • intratumoral injection of either the IL-2 mixture ( FIG. 3A ) or IL-15 sushi mixture ( FIG. 3B ) resulted in tumor regression in 5 out of 10 or 11 tumor-bearing animals, respectively ( FIGS. 3A and B), whereas in the control group injected with luciferase mRNA (ModB) no tumor regression was observed ( FIG. 3C ).
  • FIG. 4A -ModA [SEQ ID NOs: 32, 38, and 56], 4B-ModB [SEQ ID NOs: 35, 41, and 59]), or IL-15 sushi, IL-12sc, and GM-CSF or ( FIG. 4C -ModA [SEQ ID NOs: 50, 38, and 56], 4D-ModB [SEQ ID NOs: 53, 41, and 59) was further evaluated in the B16F10 tumor model. Mice with B16F10 tumors were injected intratumorally with mRNA on days 11, 13, 15, and 17.
  • Example 3 Combinations of Four mRNAs Reduce Tumor Volume In Vivo
  • B16F10 tumor bearing mice received four intratumoral injections of ModB cytokine mRNA mixture encoding: i) GM-CSF, IL-2, IL-12sc (SEQ ID NOs: 59, 35, and 41; FIG. 5A ), ii) GM-CSF, IL-15 sushi, IL-12sc (SEQ ID NOs: 59, 53, and 41; FIG. 5B ) and iii) GM-CSF, IL-15 sushi, IL-12sc, IFN ⁇ (SEQ ID NOs: 59, 53, 41, and 47; FIG. 5C ), and tumor growth was monitored to day 45.
  • ModB cytokine mRNA mixture encoding: i) GM-CSF, IL-2, IL-12sc (SEQ ID NOs: 59, 35, and 41; FIG. 5A ), ii) GM-CSF, IL-15 sushi, IL-12sc (SEQ ID NOs: 59, 53,
  • cytokine mRNA mixtures had an anti-tumor effect with 4 out of 8 tumors regressing following intratumoral injection of cytokine mRNA mixtures of GM-CSF, IL-2, and IL-12sc or GM-CSF, IL-15 sushi, and IL-12sc, and 7 out of 8 tumors regressed upon treatment with GM-CSF, IL-15 sushi, IL-12sc, and IFN ⁇ .
  • Mice treated with control mRNA (ModB) exhibited no tumor regression ( FIG. 5D ).
  • the anti-tumor activity of GM-CSF, IL-2, IL-12sc, and IFN ⁇ was examined in three different murine in vivo tumor models, CT26, B16F10 and MC38.
  • Tumor bearing mice received 4-6 intratumoral injections of ModB cytokine mRNA encoding IL-2, IL-12sc, GM-CSF and IFN ⁇ (SEQ ID NOs: 35, 41, 59, and 47) or a control ModB mRNA encoding firefly luciferase.
  • Anti-tumor activity was assessed in each tumor model. Mice treated with this combination of cytokine mRNA had 4/8, 7/8 and 5/5 regressing tumors in the CT26 ( FIG.
  • CT26 tumor model was conducted in which individual components were systematically removed from the mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB, SEQ ID NOs: 53, 41, 59, and 47).
  • CT26 tumors were injected with cytokine mRNA on days 12, 15, 19 and 22 after inoculation.
  • T/C Tumor growth repression T/C (Tumor/Control based on mean tumor volume) was plotted to day 19 ( FIG. 8H ) for each of the treatment groups.
  • Tumors treated with the ModB 3-component mRNA mixtures of i) IL-2, IL-12sc, and IFN ⁇ , ii) IL-2, GM-CSF and IFN ⁇ , iii) IL-12sc, GM-CSF and IFN ⁇ , and iv) IL-12sc, GM-CSF and IL-2 resulted in regression of 2, 3, 3 and 4 tumors, respectively ( FIG. 9B-E ) and CT26 tumors treated with a control luciferase mRNA displayed no tumor regression ( FIG. 9F ).
  • mean tumor volumes were calculated for each treatment group up to day 36.
  • the smallest mean tumor volume was observed for mice treated with the mixture of IL-2, IL-12sc, GM-CSF and IFN ⁇ , while the largest mean tumor volume was observed in the luciferase treated animals ( FIG. 10 A).
  • Tumor growth repression T/C (Tumor/Control based on mean tumor volume) was plotted to day 30 ( FIG. 10B ) for each of the treatment groups.
  • the four-component mixture of IL-2, IL-12sc, GM-CSF and IFN ⁇ exhibited the largest T/C.
  • IL-2, IL-12sc, GM-CSF and IFN ⁇ mice treated with 4 component cytokine mRNA mixture
  • mice treated with 3 component mixture missing one of the four cytokines mice treated with control luciferase mRNA.
  • mice ii) 2 of 8 mice for IL-2, GM-CSF and IFN ⁇ (ModB), iii) 3 of 8 for IL-12sc, GM-CSF and IFN ⁇ and IL-2, IL-12sc, and IFN ⁇ and iv) 4 of 8 mice treated with the cytokine mRNA mixture of IL-2, IL-12sc, and GM-CSF (ModB) ( FIG. 11 ).
  • mice Female C57BL/6J mice were implanted with B16F10 cells as described above. Mice were treated with 4 intratumoral injection (8 ⁇ g mRNA/2 ⁇ g per target) on days 11, 15, 19, and 23 with ModB cytokine mRNA mixture (IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇ ) or control luciferase mRNA. Treatment with the 4-component mixture of cytokine mRNA resulted in tumor rejection in 6/10 treated mice. See, FIG. 24B . In comparison, no tumor free mice were observed in any of the groups treated with a single mRNA ( FIGS. 24C-F ).
  • ModB cytokine mRNA mixture IL-15 sushi, IL-12sc, GM-CSF, IFN ⁇
  • Treatment with the 4-component mixture of cytokine mRNA resulted in tumor rejection in 6/10 treated mice. See, FIG. 24B . In comparison, no tumor free mice were observed in any of the groups treated with a single m
  • B16F10 tumor bearing mice were treated with a cytokine mRNA mixture of IL-15sushi, IL-12sc, GM-CSF, and IFN ⁇ (Mod B; SEQ ID NOs: 53, 41, 59, and 47).
  • a portion of the cytokine mRNA treatment B16F10 tumors completely regressed leading to tumor free animals.
  • These tumor free animals were then re-challenged with B16F10 cells as a way to assess adaptive immune memory and 9 na ⁇ ve mice were implanted with B16F10 tumor cells as a positive control for tumor engraftment ( FIG.
  • mice 12A All 9 na ⁇ ve mice engrafted with B16F10 cells developed tumors, whereas all eight tumor-free mice rejected the B16F10 cells and did not exhibit growth of B16F10 tumors ( FIG. 12B ). A portion of mice previously treated with cytokine mRNA develop localized vitiligo at the original site of the tumor ( FIG. 12C ). This experiment was essentially repeated and results are shown in FIG. 33 .
  • CT26 tumor bearing mice were treated with a cytokine mRNA mixture of IL-15sushi, IL-12sc, GM-CSF, and IFN ⁇ (Mod B; SEQ ID NOs: 53, 41, 59, and 47).
  • a portion of the cytokine mRNA treatment CT26 tumors completely regressed leading to tumor free animals.
  • CT26- ⁇ gp70 gp70 epitope
  • mice were engrafted with B16F10 tumor cells on both the left and right flanks ( FIG. 13A ).
  • Mice bearing bilateral B16F10 tumors received four intratumoral injections with control mRNA encoding luciferase or a cytokine mRNA mixture encoding IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47).
  • the right tumor was injected with mRNA at three different dose levels (80 ⁇ g, 8 ⁇ g, and 0.8 ⁇ g mRNA corresponding to 20 ⁇ g, 2 ⁇ g and 0.2 ⁇ g mRNA/target), while tumors on the left flank were untreated.
  • Dose dependent anti-tumor activity was observed in both the injected ( FIG. 13B ) and uninjected ( FIG. 13C ) tumors with tumor growth inhibition ranging from 88% in the uninjected tumor to 96% in the injected tumor.
  • Groups treated with cytokine mRNA treatment had increased median survival compared to groups treated with the Luciferase control mRNA ( FIG. 13D ).
  • mice were engrafted with B16F10 tumor cells on the right flank and received an IV injection of Luciferase-expressing B16F10 cells for induction of tumors in the lung ( FIG. 19A ).
  • mice bearing B16F10 tumors received in total three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) into the flank tumor only, while tumors in the lung were untreated.
  • FIG. 19B shows exemplarily bioluminescence measurements of four animals and pictures of the according lungs taken out in order to visualize the dark tumor nodes.
  • Tumor growth of SC tumors was strongly suppressed by injection of cytokine mRNA mixture, whereas tumors injected with control mRNA grew progressively as depicted in FIG. 19C showing mean tumor volume of 15 mice in each group. Lung tumor growth was suppressed in animals which received intratumoral cytokine mRNA injection in SC tumors when compared to animals treated with control mRNA;
  • FIG. 19C shows exemplarily bioluminescence measurements of four animals and pictures of the according lungs taken out in order to visualize the dark tumor nodes.
  • Tumor growth of SC tumors was strongly suppressed by injection of cytokine mRNA mixture, whereas tumors injected with control mRNA grew progressively as depicted in FIG. 19C showing mean tumor volume of 15 mice in each group.
  • Lung tumor growth was suppressed in animals which received intratumoral cytokine
  • FIG. 19D shows total flux analysis of bioluminescence measurements of all 15 animals per group on day 20, which is a correlate for tumor burden due to Luciferase-expressing tumor cells; line indicates median and asterisk indicates p ⁇ 0.05 analyzed by T-test. Additionally, lungs of animals treated with cytokine mRNA had significantly less weight ( FIG. 19E , line indicates median). Higher weight of lungs of animals treated with control RNA resulted from higher tumor burden.
  • an mRNA mixture encoding the human cytokines IL-15 sushi, IL-12sc, GM-CSF, and IFN ⁇ 2b (SEQ ID Nos: 26, 18, 29, and 23) (ModB) were transfected into the HEK293 cell line along with four melanoma tumor cell lines (A375, A101D, A2058 and Hs294T) ( FIG. 14A ).
  • the cytokine mRNA mixture exhibited dose dependent expression and secretion across a panel of five human cell lines ( FIG. 14B-F ).
  • human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b were evaluated in vitro with human peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • human cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b (ModB) or the individual cytokine mRNAs encoding IL-12sc, IFN ⁇ 2b, IL-15 sushi or GM-CSF (ModB) were transfected in HEK293 cells and the conditioned media was collected at 24 hrs, diluted and added to human PBMC ( FIG. 15A ).
  • the median IFN ⁇ levels from 6 donors treated with the cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b was 5623 pg/mL, while treatment with the individual cytokine mRNA for IL-12sc, IFN ⁇ 2b, IL-15 sushi or GM-CSF induced median IFN ⁇ levels of 534, 67, 17, and 4 pg/mL, respectively ( FIG. 15B ).
  • ISG15, ISG54 and MX1 were monitored in the A375 tumors as a pharmacodynamics marker at 2 h, 4 h, 8 h, 24 h, 48 h and 72 h following mRNA injection of the cytokine mRNA mixtures of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ 2b (ModB) and IL-2, IL-12sc, GM-CSF and IFN ⁇ 2b (ModB).
  • A375 tumors treated with cytokine mRNA displayed greater than 100-fold induction of ISG15 ( FIG. 17A ), ISG54 ( FIG. 17B ) and MX1 ( FIG. 17C ) with peak induction occurring by 8 hrs after intratumoral mRNA injection.
  • B16F10-tumor-bearing mice received intratumoral injections of ModA (“standard”) cytokine mRNA encoding IL-2, Flt3 ligand (FLT3L), 41BBL (also known as CD137L or tumor necrosis factor superfamily member 9), and CD27L-CD40L (this comprises a fusion protein of the soluble domain of CD27L also known as CD70, and CD40L; both the CD27L and the CD40L is comprised of three soluble domains of either CD27L or CD40L, all separated by GS-Linker sequences ( FIG.
  • ModA SEQ ID NOs: 32, 62, 68, and 74
  • ModB ModB (“modified”) cytokine mRNA encoding IL-2, FLT3L, 41BBL, and CD27L-CD40L
  • FIG. 18B SEQ ID NOs: 35, 65, 71, and 77
  • ModA mRNA encoding IFN ⁇ SEQ ID NO: 44
  • ModB mRNA encoding IFN ⁇ SEQ ID No: 47
  • mice treated with this combination of ModA mRNA had 4/9 mice tumor-free without IFN ⁇ ( FIG. 18B ) and 3/9 mice tumor-free with IFN ⁇ ( FIG. 18D ). Therefore, treatment with IFN ⁇ mRNA did not appear to increase the response to the cytokines when mRNA was dosed in the ModA form.
  • mice treated with the combination of ModB mRNA had 1/9 mice tumor-free without IFN ⁇ ( FIG. 18C ) and 7/9 mice tumor-free with IFN ⁇ ( FIG. 18E ).
  • treatment with IFN ⁇ mRNA increased the response to the mixture of cytokines when mRNA was dosed in the ModB form.
  • mice were engrafted with B16F10 or MC38 tumor cells on both the left and right flanks.
  • Mice received four intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) into only one of the flank tumors on Days 11, 15, 19, 23, while the other flank tumor was left untreated.
  • mice also received intraperitoneal injection anti-PD1 antibody (Sanofi murinized version of rat IgG2a anti-mouse PD-1 clone RMP1-14 at 5 mg/kg) on Days 10, 13, 16, 19, 22, 25. Groups were as follows: 1) control mRNA (80 ⁇ g total mRNA; 50 ⁇ L intratumoral injection at 1.6 mg/mL plus control isotype antibody (clone MOPC-21 (BioLegend); 5 mg/kg): 2) control mRNA plus anti-PD1 antibody; 3) cytokine mRNA plus control isotype antibody; and 4) cytokine mRNA plus anti-PD1. Overall survival was monitored in both the B16F10 ( FIG. 20A ) and MC38 ( FIG.
  • mice 20B tumor models.
  • the highest overall survival was observed with the combination of cytokine mRNA and anti-PD-1 treatment with 60% of mice bearing B16F10 and 80% of MC38 bearing mice tumor free at the end of the study.
  • the B16F10 tumor model 10% of mice treated with anti-PD-1 or cytokine mRNA alone were tumor free, while in the MC38 model 40% of mice treated with anti-PD-1 and 30% of mice treated with cytokine alone were tumor free.
  • the results indicate strong antitumor activity associated with cytokine mRNA and PD-1 combination.
  • mice were engrafted with B16F10 tumor cells on the right flank and received one day later an IV injection of Luciferase-expressing B16F10 cells for induction of lung metastasis.
  • mice bearing B16F10 tumors received in total three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) into the flank tumor only, while tumors in the lung were untreated.
  • mice On the same day mice also received intraperitoneal (IP) injections of PD-1 antibody (Sanofi murinized version of rat IgG2a anti-mouse PD-1 clone RMP1-14 at 10 mg/kg). Groups were as follows: 1) control mRNA (40 ⁇ g total mRNA; 50 ⁇ L intratumoral injection of control isotype antibody (clone MOPC-21 (BioLegend); 10 mg/kg) ( FIG. 20C ); 2) control mRNA plus anti-PD1 antibody ( FIG. 20D ); 3) cytokine mRNA plus control isotype antibody ( FIG. 20E ); and 4) cytokine mRNA plus anti-PD1 ( FIG. 20F ).
  • control mRNA 40 ⁇ g total mRNA; 50 ⁇ L intratumoral injection of control isotype antibody (clone MOPC-21 (BioLegend); 10 mg/kg)
  • FIG. 20D 2) control mRNA plus anti-PD1 antibody
  • FIG. 20E
  • FIGS. 20C-F Tumor growth of SC tumors was monitored ( FIGS. 20C-F ) as well as survival ( FIG. 20G ).
  • Overall survival in this model was determined by tumor burden due to SC tumors as well as lung pseudometastasis tumor (not shown in this Figure); in some mice the SC tumor was rejected, while lung metastasis grew progressively.
  • the highest overall survival was observed with the combination of cytokine mRNA and anti-PD-1 treatment. 6-7% of mice treated with cytokine mRNA alone were tumor free, while mice that had received anti-PD-1 alone or control mRNA+isotype antibody were all sacrificed at day 22 due to high tumor burden.
  • the results indicate strong antitumor activity associated with cytokine mRNA and PD-1 combination in this B16F10 tumor model with lung pseudo-metastasis, while anti-PD-1 antibody alone did not show any anti-tumor activity.
  • mice were engrafted with CT26 tumor cells on right flanks. Mice received four intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) on day 11, 14, 18 and 21 after tumor inoculation.
  • cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) on day 11, 14, 18 and 21 after tumor inoculation.
  • mice On the same day mice also received intraperitoneal (IP) injections of an anti-CTLA-4 antibody (100 ⁇ g/200 ⁇ L per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 ⁇ g/200 ⁇ L per mouse; Armenian hamster IgG from BioXCell). Groups were as follows: 1) cytokine mRNA plus anti-CTLA-4 antibody ( FIG. 21A ); 2) cytokine mRNA plus isotype control antibody ( FIG. 21B ); 3) control mRNA plus anti-CTLA-4 antibody ( FIG. 21C ) and 4) control mRNA plus isotype control antibody ( FIG. 21D ).
  • IP intraperitoneal
  • FIG. 21A Combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 12 tumor-free mice out of 16 mice on day 55 after tumor inoculation ( FIG. 21A ).
  • FIG. 21D In comparison, in the group that received control mRNA plus isotype control antibody ( FIG. 21D ), only one tumor-free mouse remained at the conclusion of the study.
  • mice were engrafted with B16F10 tumor cells on right flanks. Mice received three intratumoral injections with cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47) on day 13, 17 and 20 after tumor inoculation.
  • cytokine mRNA mixture of IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 53, 41, 59, and 47)
  • mice On day 13, 17, 20 and 24 after tumor inoculation mice also received intraperitoneal (IP) injections of an anti-CTLA-4 antibody (100 ⁇ g/200 ⁇ L per mouse; clone 9H10 from InVivoMAb) or the isotype control antibody (100 ⁇ g/200 ⁇ L per mouse; Armenian hamster IgG from BioXCell). Tumor growth of SC tumors as well as survival was monitored. Groups were as follows: 1) cytokine mRNA plus anti-CTLA-4 antibody ( FIG. 21 E); 2) cytokine mRNA plus isotype control antibody ( FIG. 21F ); 3) control mRNA plus anti-CTLA-4 antibody ( FIG. 21G ) and 4) control mRNA plus isotype control antibody ( FIG.
  • FIG. 21H Combination therapy of intratumoral cytokine mRNA and IP-injected anti-CTLA-4 resulted in strongest anti-tumoral activity with 6 tumor-free mice out of 9 mice on day 60 after tumor inoculation ( FIG. 21E ).
  • FIG. 21G In comparison, in the two groups that either received control mRNA plus anti-CTLA-4 antibody ( FIG. 21G ) or control mRNA plus isotype control antibody ( FIG. 21H ), no tumor-free mouse remained at the conclusion of the study. Percent survival is depicted in FIG.
  • Expression of each of the 4 cytokines of IL-15 sushi FIG. 22D
  • IL-12sc FIG. 22A
  • GM-CSF FIG.
  • mice received a single intratumoral injection of a cytokine mRNA mixture of human IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 26, 18, 29, and 23).
  • cytokine mRNA mixture of human IL-15 sushi, IL-12sc, GM-CSF and IFN ⁇ (ModB; SEQ ID NOs: 26, 18, 29, and 23).
  • serum was collected and cytokine expression was analyzed by Meso Scale Discovery assay.
  • mice bearing a single CT26 tumor on one flank received a four intratumoral injections of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (ModB; SEQ ID NOs: 53, 41, 59, and 47).
  • Blood was collected 13 days after first intratumoral mRNA administration and T cells specific for the gp70 tumor antigen were quantified by flow cytometry. Frequency of T cells specific for the gp70 tumor antigen in blood were strongly increased in mice upon intratumoral injection of mRNA cytokines compared to mice that had received control RNA.
  • Example 12 Cytokine mRNA Induces Multiple Pro-Inflammatory Pathways and Increases Immune Infiltrate in Both Treated and Untreated Tumors
  • treatment with a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc upregulated multiple proinflammatory pathways including a range of IFNgamma response genes.
  • the upregulation of proinflammatory/IFNgamma related pathways occurred in both the treated and untreated tumors, supporting the notion that local intratumoral treatment has systemic immune modulatory effects.
  • Relative abundance of infiltrated immune cells is determined by calculating the average expression of immune cell-type specific gene signatures.
  • Example 13 Cytokine mRNA Increases CD4+ and CD8+ T Cells in Both Treated and Untreated Tumors
  • FIGS. 28C and D were treated with control mRNA. Panels A and C are from the tumors injected with mRNA, while panels B and D are from the corresponding contralateral tumor not injected. ( FIGS. 28A-D ). For both the cytokine mRNA treated and control mRNA treated groups 5 tumors injected with RNA and the corresponding 5 contralateral tumors uninjected were subjected to immunofluorescent staining for CD4+, CD8+, FOXP3+ cells. The relative frequency and ratio of cells are plotted in FIGS. 28E , F, G.
  • cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc increases CD8+ and CD4+ T cells infiltration leading to altering the CD8+/Treg ratio.
  • An increase in immune infiltration occurred in both the treated and untreated tumors, supporting the notion that local intratumoral treatment has systemic immune modulatory effects.
  • mice bearing B16F10 tumors received a single mRNA injection with 80, 8 or 0.8 ⁇ g of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47). Approximately 6 hrs after the intratumoral injection, the tumor was removed and lysed, and levels of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc, IFNgamma and IP-10 were quantified in the tumor lysate.
  • FIGS. 30A-F show that the cytokine mRNA was expressed intratumorally in a dose-dependent manner.
  • mice bearing B16F10 tumors received a single mRNA injection with 80, 8 or 0.8ug of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47).
  • a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc SEQ ID Nos: 59, 53, 41, and 47.
  • the tumors were dissociated, stained with a panel of antibodies, and analyzed by flow cytometry.
  • the antibodies used were against murine: CD45, CD4, CD3, CD8, CD279, IFNgamma, TNFalpha, FOXP3, Granzyme B).
  • the results indicate that treatment with the cytokine mRNA mixture altered the CD8+/Treg ratio ( FIG.
  • FIG. 31A-B led to increased frequency of polyfunctional CD8+ T cells in the tumor microenvironment ( FIG. 31C-D ), increased PD-L1 on infiltrating myeloid cells ( FIG. 31E ), and increased levels of PD-1 on infiltrating CD8+ T cells ( FIG. 31F ).
  • mice bearing B16F10 tumors on the left and right flank received a single intratumoral injection of a cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (SEQ ID Nos: SEQ ID NOs: 59, 53, 41, and 47) or control mRNA into only one of the tumors (treated), while the other tumor remained untreated.
  • the injected tumor was collected and processed for flow cytometry staining with antibodies for CD45+, CD8+, CD3+, and Granzyme B.
  • the results indicate that the cytokine mRNA mixture increased the frequency of intratumoral Granzyme B CD8+ T cells in the tumor ( FIG. 31G-H ).
  • mice bearing B16F10 tumors received a single intratumoral injection of 50 ⁇ g mRNA encoding firefly luciferase.
  • 3 mice were sacrificed and tumor, liver, spleen, tumor draining lymph node (TDLN) and non-tumor draining lymph node (NDLN) were analyzed ex vivo for luciferase expression.
  • FIGS. 32A-B show that luciferase expression was highest in the tumor, in which expression was greater than 100-fold above any other tissue.
  • mice bearing B16F10 tumors were treated with 100 ⁇ g of depleting antibodies (anti-CD4, anti-CD8, anti-NK1.1) by intraperitoneal injection once a week for 4 weeks total.
  • Antibody mediated cellular depletion was initiated one day prior to treatment with an 80 ⁇ g cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47). The effect of antibody depletion on overall survival was monitored.
  • the results, shown in FIG. 34 indicate that individual depletion CD8+, CD4+, or NK cells reduced, to varying degrees, the anti-tumor activity and overall survival of the cytokine mRNA.
  • mice and C57BL6J mice deficient for the murine IFN ⁇ were implanted with B16F10 tumor cells as described in Example 1.
  • Mice were treated by intratumoral injection with 80 ⁇ g (20 ⁇ g/target) cytokine mRNA mixture of IL-15 sushi, GM-CSF, IFN ⁇ , and IL-12sc (SEQ ID Nos: 59, 53, 41, and 47) or 80 ⁇ g control mRNA, and overall survival was monitored.
  • the results, depicted in FIG. 35 indicate that mice lacking IFN ⁇ did not exhibit a detectable antitumor response when treated with the cytokine mRNA.

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