Polynucleotides and uses thereof

Abstract

The present disclosure relates to a polynucleotide or set of polynucleotides comprising a first nucleic acid molecule encoding an innate immune inhibitor, such as influenza nonstructural (NS1) protein, and a second nucleic acid molecule encoding a heterologous target mRNA. The present disclosure also includes a method of making the polynucleotide, a method of modifying a cell, and a method of using it to treat a subject. The present disclosure provides an isolated polynucleotide (e.g., a replicon) or set of polynucleotides comprising a first nucleotide sequence encoding an innate immune inhibitor, such as influenza nonstructural (NS1) protein, and a second nucleotide sequence encoding a heterologous target mRNA. Such polynucleotides can drive enhanced and sustained expression of the heterologous target mRNA in cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 228,892, filed August 3, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] References to sequence listings submitted electronically via EFS WEB The contents of the electronically submitted Sequence Listing (4597_008PC01_Seqlisting_st26.xml; size: 96,785 bytes; creation date: August 3, 2022) submitted in this application are incorporated herein by reference in their entirety.

[0003] Field of the Disclosure The present disclosure provides an isolated polynucleotide (e.g., a replicon) or set of polynucleotides comprising a first nucleotide sequence encoding an innate immune inhibitor, e.g., influenza nonstructural (NS1) protein, and a second nucleotide sequence encoding a heterologous target mRNA. Such polynucleotides are capable of driving enhanced and sustained expression of the heterologous target mRNA in a cell. [Background technology]

[0004] Background of the Disclosure Nucleic acid therapeutics have emerged as promising and rapidly developing treatments for a wide variety of diseases. These therapies rely on cells to produce biologically active molecules, such as functional RNA and / or therapeutic polypeptides, in vitro, ex vivo, or in vivo, with retention of native conformation and post-translational modifications that are often difficult to achieve with recombinant proteins. Synthetic mRNA has proven to be a valuable tool, with an improved safety profile compared to virus- or DNA-based therapies. However, the human immune system naturally degrades RNA, limiting the efficacy and persistence of administered synthetic RNA (e.g., circular RNA). Thus, there remains a need in the art for RNA therapeutics that provide potent and durable effects in vitro and in vivo. Summary of the Invention

[0005] BRIEF SUMMARY OF THE DISCLOSURE Some embodiments of the present disclosure relate to a polynucleotide or set of polynucleotides comprising a first nucleic acid molecule encoding an influenza nonstructural (NS1) protein and a second nucleic acid molecule encoding a heterologous target mRNA.

[0006] In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are present on a first vector. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein is present on a first vector and the second nucleic acid molecule encoding the target mRNA is present on a second vector.

[0007] In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein is expressed under the control of a first promoter. In some embodiments, the second nucleic acid molecule encoding the target mRNA is expressed under the control of a second promoter. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are different.

[0008] In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are expressed under the control of a first promoter, which drives the expression of both the influenza NS1 protein and the target mRNA. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are linked by an IRES sequence. In some embodiments, the first vector, the second vector, or both, comprise one or more regulatory elements.

[0009] In some embodiments, expression of the target mRNA is increased compared to expression of the target mRNA in the absence of the first nucleic acid molecule encoding an influenza NS1 protein. In some embodiments, expression of the target mRNA is increased by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% compared to expression of the target mRNA in the absence of the first nucleic acid molecule encoding an influenza NS1 protein. In some embodiments, the increased expression of the target mRNA persists for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, or at least about 48 hours.

[0010] In some embodiments, the target mRNA is a set that encodes a biologically active polypeptide. In some embodiments, the biologically active polypeptide comprises a cytokine, a chemokine, a growth factor, a clotting factor, an enzyme, or any combination thereof. In some embodiments, the cytokine is IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD15 4, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, a fragment thereof, a variant thereof, or any combination thereof.

[0011] In some embodiments, the target mRNA encodes an IL-12 polypeptide or a fragment or variant thereof. In some embodiments, the target mRNA encodes the p35 subunit of IL-12 and the p40 subunit of IL-12. In some embodiments, the p35 subunit and the p40 subunit are expressed from a single promoter. In some embodiments, the p35 subunit and the p40 subunit are expressed as a single contiguous polypeptide. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more covalent bonds. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more peptide bonds. In some embodiments, the portion of the mRNA encoding the p35 subunit is separated from the portion of the mRNA encoding the p40 subunit by an IRES.

[0012] In some embodiments, the target mRNA encodes a miRNA, siRNA, shRNA, dsRNA, antisense oligonucleotide, guide RNA, or any combination thereof.

[0013] In some embodiments, the first promoter is an inducible promoter, a tissue-specific promoter, or a constitutively active promoter. In some embodiments, the second promoter is an inducible promoter, a tissue-specific promoter, or a constitutively active promoter.

[0014] In some embodiments, the influenza NS1 is influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, or a mutant thereof. In some embodiments, the influenza NS1 is H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, or any combination thereof. In some embodiments, the influenza NS1 is H5N1 NS1. In some embodiments, the influenza NS1 is H1N1 NS1. In some embodiments, the H1N1 NS1 is H1N1 TX91 mutant NS1.

[0015] In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2, wherein the nucleotide sequence encodes an influenza NS1 protein. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 1 or 2, wherein the nucleotide sequence encodes an influenza NS1 protein. In some embodiments, (i) the first nucleic acid molecule, (ii) the second nucleic acid molecule, or (iii) both (i) and (ii) are circular RNA.

[0016] In some embodiments, the polynucleotide or set of polynucleotides comprises one or more modified nucleic acid molecules.

[0017] Some aspects of the present disclosure relate to a polynucleotide or a set of polynucleotides comprising a self-replicating target mRNA, wherein the self-replicating target mRNA comprises one or more modified nucleic acid molecules. In some aspects, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some aspects, about 25% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some aspects, the one or more modified nucleic acid molecules are modified rNTPs. In some aspects, the one or more modified nucleic acid molecules comprise N1-methylpseudouracil, 5-methylcytosine, N6-methyladenosine, or a combination thereof.

[0018] Some aspects of the disclosure relate to a vector or a set of vectors comprising a polynucleotide or a set of polynucleotides disclosed herein. In some aspects, the vector is a replicon. In some aspects, the vector is a Venezuelan Equine Encephalitis (VEE) replicon or a derivative or portion thereof. In some aspects, the vector is a Venezuelan Equine Encephalitis (VEE) replicon comprising a nucleotide sequence encoding a lysine at residue 739 according to the wild type amino acid sequence VEE.

[0019] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA; and (v) a VEE 3'UTR or a derivative thereof.

[0020] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; and (vi) a VEE 3'UTR or a derivative thereof.

[0021] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA; (v) an E1 sequence; and (vi) a VEE 3'UTR or a derivative thereof.

[0022] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; (vi) an E1 sequence; and (vii) a VEE 3'UTR or a derivative thereof.

[0023] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0024] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a VEE 3'UTR or a derivative thereof.

[0025] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a second nucleic acid molecule encoding a target mRNA; and (v) a VEE 3'UTR or a derivative thereof.

[0026] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; (vi) an E1 sequence; and (vii) a VEE 3'UTR or a derivative thereof.

[0027] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0028] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a VEE 3'UTR or a derivative thereof.

[0029] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0030] In some embodiments, the vector comprises: (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0031] In some embodiments, the vector comprises: (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0032] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule encoding an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a VEE 3'UTR or a derivative thereof.

[0033] In some embodiments, the vector comprises: (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:2; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a VEE 3'UTR or a derivative thereof.

[0034] In some embodiments, the vector comprises (i) a VEE 5'UTR or a derivative thereof; (ii) one or more nonstructural proteins (nsPs); (iii) a first nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:2; (iv) a second nucleic acid molecule encoding a target mRNA, the target mRNA encoding a human IL-12 polypeptide; and (v) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE. In some embodiments, the one or more nsPs comprise a VEE nsP or a derivative thereof. In some embodiments, the VEE nsPs are selected from nsP2, nsP3, nsP4, and any combination thereof.

[0035] Some aspects of the disclosure relate to a cell comprising a polynucleotide or set of polynucleotides disclosed herein, or a vector or set of vectors disclosed herein. In some aspects, the cell is a mammalian cell. In some aspects, the cell is a human cell. In some aspects, the cell is an immune cell.

[0036] Some aspects of the present disclosure relate to a pharmaceutical composition comprising a polynucleotide or set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, or a cell disclosed herein, and a pharma- ceutical acceptable carrier.

[0037] Some aspects of the present disclosure relate to a method for expressing a target mRNA in a cell, comprising transfecting said cell with a polynucleotide or set of polynucleotides disclosed herein or a vector or set of vectors disclosed herein. In some aspects, the cell is a human cell. In some aspects, the cell is an ex vivo human cell. In some aspects, the cell is a human immune cell.

[0038] Some aspects of the present disclosure relate to methods of treating a subject in need of treatment comprising administering to the subject a polynucleotide or set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0039] Some aspects of the present disclosure relate to a method of expressing a target mRNA in a subject in need thereof, comprising administering to the subject a polynucleotide or set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0040] In some embodiments, the subject suffers from cancer.In some embodiments, the cancer is selected from the group consisting of melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, and various types of head and neck cancer, including head and neck squamous cell carcinoma.In some embodiments, the cancer can be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, and any combination thereof.

[0041] Some embodiments of the present disclosure relate to a method of expressing a target mRNA in a cell, comprising co-expressing the target mRNA and an influenza NS1 protein in the cell, wherein the target mRNA is not an influenza mRNA, hi some embodiments, the influenza NS1 protein is encoded by a first nucleic acid molecule and the target mRNA is encoded by a second nucleic acid molecule.

[0042] In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are present on a first vector. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein is present on a first vector and the second nucleic acid molecule encoding the target mRNA is present on a second vector.

[0043] In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein is expressed under the control of a first promoter. In some embodiments, the second nucleic acid molecule encoding the target mRNA is expressed under the control of a second promoter. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are different. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are expressed under the control of a first promoter, which drives the expression of both the influenza NS1 protein and the target mRNA. In some embodiments, the first nucleic acid molecule encoding the influenza NS1 protein and the second nucleic acid molecule encoding the target mRNA are linked by an IRES sequence. In some embodiments, the first vector, the second vector, or both, comprise one or more regulatory elements. In some embodiments, the expression of the target mRNA is increased compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, (i) the first nucleic acid molecule, (ii) the second nucleic acid molecule, or (iii) both (i) and (ii) are circular RNA.

[0044] Some embodiments of the present disclosure relate to a method for expressing target mRNA in a cell, comprising transfecting the cell with a polynucleotide or a set of polynucleotides comprising a self-replicating target mRNA comprising one or more modified nucleic acid molecules. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some embodiments, about 25% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some embodiments, the one or more modified nucleic acid molecules are modified rNTPs. In some embodiments, the one or more modified nucleic acid molecules comprise N1-methylpseudouracil, 5-methylcytosine, N6-methyladenosine, or a combination thereof.

[0045] In some embodiments, expression of the target mRNA is increased compared to expression of the target mRNA from a self-replicating target mRNA that does not include one or more modified nucleic acid molecules. In some embodiments, expression of the target mRNA is increased by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%.

[0046] In some embodiments, the increased expression of the target mRNA persists for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, or at least about 48 hours.

[0047] In some embodiments, the target mRNA encodes a biologically active polypeptide. In some embodiments, the biologically active polypeptide comprises a cytokine, a chemokine, a growth factor, a clotting factor, an enzyme, or any combination thereof. In some embodiments, the cytokine is IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-CSF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD15 4, LT-β, ​​TNF-α, TNF-β, 4-1BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, TGF-β1, TGF-β2, TGF-β3, Epo, Tpo, Flt-3L, SCF, M-CSF, MSP, a fragment thereof, a variant thereof, or any combination thereof.

[0048] In some embodiments, the target mRNA encodes an IL-12 polypeptide or a fragment or variant thereof. In some embodiments, the target mRNA encodes the p35 subunit of IL-12 and the p40 subunit of IL-12. In some embodiments, the p35 subunit and the p40 subunit are expressed from a single promoter. In some embodiments, the p35 subunit and the p40 subunit are expressed as a single contiguous polypeptide. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more covalent bonds. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more peptide bonds. In some embodiments, the portion of the mRNA encoding the p35 subunit is separated from the portion of the mRNA encoding the p40 subunit by an IRES.

[0049] In some embodiments, the target mRNA comprises miRNA, siRNA, shRNA, dsRNA, antisense oligonucleotide, guide RNA, circular RNA, or any combination thereof.

[0050] In some embodiments, the first promoter is an inducible promoter, a tissue-specific promoter, or a constitutively active promoter. In some embodiments, the second promoter is an inducible promoter, a tissue-specific promoter, or a constitutively active promoter.

[0051] In some embodiments, the influenza NS1 is influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, or a mutant thereof. In some embodiments, the influenza NS1 is H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, or a combination thereof. In some embodiments, the influenza NS1 is H5N1 NS1. In some embodiments, the influenza NS1 is H1N1 NS1. In some embodiments, the H1N1 NS1 is H1N1 TX91 mutant NS1.

[0052] In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 2. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 2, wherein the nucleotide sequence encodes an influenza NS1 protein. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence set forth in SEQ ID NO: 1 or 2, wherein the nucleotide sequence encodes an influenza NS1 protein. [Brief description of the drawings]

[0053] [Figure 1A] 1A-1B are diagrams of sample non-cytopathic EGFP vector (FIG. 1A) and non-cytopathic NS1-EGFP vector (FIG. 1B). [Figure 1B] 1A-1B are diagrams of sample non-cytopathic EGFP vector (FIG. 1A) and non-cytopathic NS1-EGFP vector (FIG. 1B).

[0054] [Figure 2A-D] Figures 2A-2D are images of 4T1 cells 24 hours after transfection with Strand-mCherry (Figure 2A), mCherry modRNA (Figure 2B), and Strand-mCherry+NS1 modRNA mRNA (Figures 2C-2D) using Lipofectamine MessengerMax.

[0055] [Figure 2E] FIG. 2E summarizes the mean fluorescence intensity observed for each mRNA transfection tested in FIGS. 2A-2D.

[0056] [Figure 3A-D] Figures 3A-3D are images of 4T1 cells 24 hours after transfection with Strand-mCherry (Figure 3A), mCherry modRNA (Figure 3B), and Strand-mCherry+NS1 modRNA mRNA (Figures 3C-3D) using TT3 lipid nanoparticles.

[0057] [Figure 3E-F] Figures 3E–3F summarize the mean fluorescence intensity observed for each mRNA transfected by tandem NS1 expression (Figure 3E) or NS1 cotransfection (Figure 3F).

[0058] [Figure 4A-B] Figures 4A-4H are graphs of flow cytometry data showing the number of cells expressing mCherry 24 hours (Figures 4A-4D) and 48 hours (Figures 4E-4H) after transfection of Hcc38 tumor cells with NS1 modRNA (Figures 4B and 4F), NS1 repRNA (Figures 4C and 4G), or NS1-P2A-mCherry mRNA (Figures 4D and 4H) using Lipofectamine MessengerMax. [Figure 4C-D] Figures 4A-4H are graphs of flow cytometry data showing the number of cells expressing mCherry 24 hours (Figures 4A-4D) and 48 hours (Figures 4E-4H) after transfection of Hcc38 tumor cells with NS1 modRNA (Figures 4B and 4F), NS1 repRNA (Figures 4C and 4G), or NS1-P2A-mCherry mRNA (Figures 4D and 4H) using Lipofectamine MessengerMax. [Figure 4E-F] Figures 4A-4H are graphs of flow cytometry data showing the number of cells expressing mCherry 24 hours (Figures 4A-4D) and 48 hours (Figures 4E-4H) after transfection of Hcc38 tumor cells with NS1 modRNA (Figures 4B and 4F), NS1 repRNA (Figures 4C and 4G), or NS1-P2A-mCherry mRNA (Figures 4D and 4H) using Lipofectamine MessengerMax. [Figure 4G-H] Figures 4A-4H are graphs of flow cytometry data showing the number of cells expressing mCherry 24 hours (Figures 4A-4D) and 48 hours (Figures 4E-4H) after transfection of Hcc38 tumor cells with NS1 modRNA (Figures 4B and 4F), NS1 repRNA (Figures 4C and 4G), or NS1-P2A-mCherry mRNA (Figures 4D and 4H) using Lipofectamine MessengerMax.

[0059] [Figure 4I] Figures 4I-4K show the median fluorescence intensity (MFI) of Scc9 HNSCC cells (Figure 4I) and FaDu HNSCC cells (Figures 4J-4K). [Fig. 4J-K] Figures 4I-4K show the median fluorescence intensity (MFI) of Scc9 HNSCC cells (Figure 4I) and FaDu HNSCC cells (Figures 4J-4K).

[0060] [Figure 5A-D] 5A-5D are graphical representations of flow cytometry data showing the number of cells expressing EGFP 24 hours after transfection of BT20 cancer with TT3 LNPs containing a replicon vector encoding EGFP containing either the original (Strand) backbone (FIGS. 5A-5B) or one containing the Q739L mutation (noncytopathic; FIGS. 5C-5D).

[0061] [Figure 5E-F]Figures 5E-5F are graphical representations of the median fluorescence intensity (MFI; Figure 5E) as measured by the percentage of GFP positive cells and transfection efficiency (Figure 5F).

[0062] [Figure 6A-F] Figures 6A-6F are graphs of flow cytometry data showing the number of B16.F10 cells (Figures 6A-6C) and 4T1 cells (Figures 6D-6F) expressing mCherry after electroporation with replicons made using unmodified rNTPs (Unmodified Rep; Figures 6A and 6D) or a 1:1 (50%; Figures 6B and 6E) or 1:3 (25%; Figures 6C and 6F) ratio of UTP to N1-methyl-pseudo-UTP (indicated in psi).

[0063] [Figure 6G] FIG. 6G is a graphical representation of the median fluorescence intensity (MFI) of the cells described in FIGS. 6A-6G.

[0064] [Figure 7] FIG. 7 shows the specific rNTPs using unmodified rNTPs (unmod) or a 1:3 ratio. [ka] 1 is a bar graph showing the relative luminescence of 4T1 cells transfected with a replicon encoding firefly luciferase (Fluc) made using ;1:3 C:5me-C(M2), where C refers to cytidine and 5me-C refers to 5-methyl-cytidine. Luminescence was measured 24 and 48 hours after transfection by electroporation.

[0065] [Figure 8A-B] 8A-8B show the results of using unmodified rNTPs (unmod) or a specific 1:3 ratio. [ka] 8A and 8B are bar graphs showing relative luminescence (FIG. 8A) and type I IFN activity measured using a colorimetric SEAP reporter assay (FIG. 8B) of B16-ISG cells, an interferon-inducible cell line, transfected with a replicon encoding firefly luciferase (Fluc) made using either 1:3 C:5me-C(M2) or 1:3 C:5me-C(M3) (where C refers to cytidine and 5me-C refers to 5-methyl-cytidine).

[0066] [Figure 9A-B] Figures 9A-9C are graphical representations of payload expression (Figure 9A), signal intensity (Figure 9B), and type I IFN activity (Figure 9C) of B16-ISG cells transfected with the Q739L replicon expressing NS1-EGFP (P2A linker) generated using unmodified or single (M1) or doubly modified rNTPs. [Figure 9C] Figures 9A-9C are graphical representations of payload expression (Figure 9A), signal intensity (Figure 9B), and type I IFN activity (Figure 9C) of B16-ISG cells transfected with the Q739L replicon expressing NS1-EGFP (P2A linker) generated using unmodified or single (M1) or doubly modified rNTPs.

[0067] [Figure 10A-F] Figures 10A-10F are images of GFP expression in T cells activated for 2 days with anti-CD3 / CD28 / CD2 cocktail containing high dose IL-2 with (Figures 10D-10F) or without (Figures 10A-10C) recombinant protein (enhancer) added to the medium. T cells were transfected with lipid 1 (Figures 10A and 10D), lipid 2-cholesterol (Figures 10B and 10E), or lipid 2-β-sitosterol (Figures 10C and 10F).

[0068] [Figure 11A-C]11A-11C are graphical representations of transfection efficiency (% GFP positive cells; FIG. 11A), median fluorescence intensity (FIG. 11B), and IFN-γ levels (FIG. 11C) in primary human T cells activated 2 days post-thaw using IL-2 and anti-CD3 / CD28 / CD2, transfected using the Q739L replicon driving NS1-EGFP as used above, and generated using unmodified or single (M1) or double (M2) modified mRNA.

[0069] [Figure 12A-B] Figures 12A-12B are graphical representations of transfection efficiency (% GFP positive cells; Figure 12A) and IFN-α activation (Figure 12B) in human PBMCs isolated from three healthy donors and delivered mRNA:lipid with NS1-EGFP or M2-modified Q739L replicon driving EGFP, or conventional EGFP mRNA (EGFP-mod) after growth for 2 days with either low dose IL-2 (resting) or high dose IL-2 in the presence of an anti-CD3 / CD28 / CD2 cocktail (activating). Measurements were performed 24 hours after transfection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description of the Disclosure Some aspects of the present disclosure relate to a polynucleotide or set of polynucleotides comprising a first nucleic acid molecule encoding an influenza nonstructural (NS1) protein and a second nucleic acid molecule encoding a heterologous target mRNA. The present disclosure provides that expression of a target mRNA encoded by a polynucleotide, e.g., a replicon, can be enhanced in the presence of influenza NS1. In particular, co-expression of influenza NS1 and the target mRNA increases the amount of expression of the target mRNA in a cell and the persistence of expression of the target mRNA. Thus, some aspects of the present disclosure relate to a method of expressing a target mRNA in a cell, e.g., a human cell, comprising transfecting the cell with a polynucleotide (e.g., a circular RNA) or set of polynucleotides (e.g., a set of circular RNAs) comprising a first nucleic acid molecule encoding an influenza nonstructural (NS1) protein and a second nucleic acid molecule encoding a heterologous target mRNA. In some aspects, the target mRNA encodes an IL-12 polypeptide or a fragment or variant thereof.

[0071] Further aspects of the disclosure are provided throughout the application.

[0072] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0073] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended as a precedent for using exclusive language such as "solely," "only," and the like, or for using "negative" limitations in connection with the recitation of elements in the claims.

[0074] The term "and / or" as used herein should be construed as specifically disclosing each of the two specified features or components with or without the other. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0075] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0076] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value that is similar to a stated reference value and, unless otherwise stated or clear from the context, falls within a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater or less) of the stated reference value (except where such number exceeds 100% of possible values). When the term "approximately" or "about" is applied to a particular value herein, the value without the term "approximately" or "about" is also disclosed herein.

[0077] As described herein, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the recited range, and fractions thereof, where appropriate (such as tenths and hundredths of integers), unless otherwise stated.

[0078] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM," respectively.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many terms used in this disclosure.

[0080] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers that define the range. When a range of values ​​is described, it is understood that each intervening integer value and each fraction between the stated upper and lower limits of the range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can be independently included or excluded from the range, and each range that includes either limit, neither limit, or both limits is also encompassed by the present disclosure. Thus, ranges recited herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0081] When a value is explicitly recited, it is understood that values ​​that are approximately the same amount or amount as the recited value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed separately, their combinations are also disclosed. Where any element of this disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded, alone or in any combination with other alternatives, are also hereby disclosed. Two or more elements of this disclosure may have such an exclusion, and all combinations of elements with such exclusions are disclosed herein.

[0082] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. Nucleotides are referred to herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "a" stands for adenine, "c" stands for cytosine, "g" stands for guanine, "t" stands for thymine, and "u" stands for uracil.

[0083] Amino acid sequences are written left to right in amino to carboxy orientation. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0084] The terms "administration", "administering" and grammatical variations thereof refer to the introduction of a composition (such as an isolated polynucleotide described herein) into a subject via a pharma- ceutically acceptable route. The introduction of a composition into a subject can be by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular or topical. Administration includes self-administration and administration by another. A suitable route of administration allows the composition to perform its intended function. For example, if a suitable route is intravenous, the composition can be administered by introducing the composition into a subject's vein.

[0085] As used herein, the term "cancer" refers to a broad group of various diseases characterized by uncontrolled proliferation of abnormal cells in the body. Uncontrolled cell division and proliferation division and proliferation can lead to the formation of malignant tumors that can invade nearby tissues and metastasize to distant parts of the body through the lymphatic system or bloodstream. The compositions disclosed herein can be used to treat any cancer, including, but not limited to, melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, and various types of head and neck cancer, including head and neck squamous cell carcinoma. In some aspects, the cancer can be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, Hodgkin's lymphoma, and any combination thereof.

[0086] The term "coding sequence" or "encoding" sequence is used herein to mean a DNA or RNA region (transcribed region) that "encodes" a particular protein, e.g., influenza NS1 protein or a target heterologous protein. A coding sequence, when placed under the control of an appropriate regulatory region, such as a promoter, is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic sources, genomic DNA from prokaryotic or eukaryotic sources, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0087] The term "downstream" refers to a nucleotide sequence located 3' of a reference nucleotide sequence. In some embodiments, the downstream nucleotide sequence relates to a sequence following the start of transcription. For example, the translation start codon of a gene is located downstream of the start site of transcription.

[0088] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.

[0089] The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product, such as an RNA or a polypeptide (e.g., a therapeutic protein, such as influenza NS1 nonstructural protein). It includes, but is not limited to, the transcription of a polynucleotide into a microRNA binding site, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), or any other RNA product. It includes, but is not limited to, the transcription of a polynucleotide into a messenger RNA (mRNA) and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be a nucleic acid, such as, for example, an RNA produced by transcription of a gene. As used herein, a gene product can be either a nucleic acid, an RNA (e.g., a circular RNA) or an miRNA produced by transcription of a gene, or a polypeptide translated from a transcription product. Gene products as described herein further include nucleic acids that have post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides that have post-translational modifications, such as phosphorylation, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0090] As used herein, the term "heterologous target mRNA" refers to any mRNA (linear or circular) that does not naturally occur in a target cell that can be expressed in a target cell using the polynucleotides described herein. Unless otherwise indicated, the heterologous target mRNA can code for a polypeptide or RNA molecule (e.g., miRNA, dsDNA, lncRNA, siRNA, antisense oligonucleotide, phosphorodiamidate morpholino oligomer (PMO), peptide-linked phosphorodiamidate morpholino oligomer (PPMO), or combinations thereof) that has a regulatory function. Thus, as used herein, the term "encode" refers to the production of a portion of interest (e.g., an RNA molecule such as a polypeptide or circular RNA) from a nucleic acid molecule (e.g., a heterologous target mRNA). In some embodiments, the heterologous target mRNA codes for a biologically active polypeptide, including, but not limited to, a cytokine, a chemokine, a growth factor, a clotting factor, an enzyme, or any combination thereof. In some embodiments, the heterologous target mRNA is referred to herein as a "payload". Unless otherwise indicated, the terms "target mRNA" and "heterologous target mRNA" are used interchangeably.

[0091] In some embodiments, two or more sequences are said to be "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least about 30% identical, at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0092] As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polypeptide or polynucleotide molecules (e.g., DNA and / or RNA molecules). The term "identical" without additional modifiers, e.g., protein A is identical to protein B, means that the sequences are 100% identical (sequence identity is 100%). Describing two sequences as, e.g., "70% identical" is equivalent to describing them as having, e.g., "70% sequence identity."

[0093] Calculation of percent identity of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second polypeptide or polynucleotide sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the aligned sequence for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or about 100% of the length of the reference sequence. The amino acids, or in the case of polynucleotides, bases at corresponding amino acid positions are then compared.

[0094] If a position of the first sequence is occupied by the same amino acid as the amino acid at the corresponding position of the second sequence, the molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap.Comparing sequences and determining the percent identity between two sequences can be accomplished using a mathematical algorithm.

[0095] Suitable software programs for alignment of both protein and nucleotide sequences are available from a variety of sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the suite of BLAST programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher. These are part of the EMBOSS suite of bioinformatics programs, also available from the European Bioinformatics Institute (EBI) at worldwideweb.ebi.ac.uk / Tools / psa.

[0096] Sequence alignment can be performed using methods known in the art such as MAFFT, Clustal (ClustalW, ClustalX or Clustal Omega), MUSCLE, etc.

[0097] Different regions in a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own sequence identity percentage.Please note that the sequence identity percentage value is rounded off to the nearest tenth.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.Please also note that the length value is always an integer.

[0098] In some embodiments, the percent identity (%ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID=100x(Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) scored as perfect matches in an alignment of the first and second sequences (aligned by visual inspection or by a specific sequence alignment program) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0099] Those skilled in the art will understand that the generation of sequence alignment for calculating percent sequence identity is not limited to binary sequence-sequence comparison driven only by primary sequence data. It will also be understood that sequence alignment can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., protein crystal structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignment is T-Coffee, available at www.tcoffee.org, or from, for example, EBI. It will also be understood that the final alignment used to calculate percent sequence identity can be curated automatically or manually.

[0100] As used herein, the terms "isolated" and "purified" and grammatical variations thereof are used interchangeably and refer to the state of preparation of the desired composition of the present disclosure that has undergone one or more purification processes. In some embodiments, isolation or purification as used herein is a process of removing, partially removing (e.g., fractionating) the composition of the present disclosure from a sample containing contaminants. In some embodiments, the isolated composition has no detectable undesirable activity or has a lower level or amount of undesirable activity than an acceptable level or amount. In some embodiments, the isolated composition has an acceptable amount and / or concentration and / or activity or greater amount and / or concentration of the desired composition of the present disclosure. In some embodiments, the isolated composition is concentrated compared to the starting material from which the composition is obtained. This enrichment can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% relative to the starting material. In some embodiments, the isolated preparation is substantially free of residual biological products. In some embodiments, an isolated preparation is 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of contaminating biological material. Residual biological products may include abiotic material (including chemicals) or undesirable nucleic acids, proteins, lipids, or metabolites.

[0101] The term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently linked to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid sequence or polynucleotide sequence can be directly linked or juxtaposed to the second amino acid sequence or polynucleotide sequence, or an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" not only refers to the fusion of the first polynucleotide sequence with the second polynucleotide sequence at the 5' or 3' end, but also includes the insertion of any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively) over the entire first polynucleotide sequence (or the second polynucleotide sequence). The first polynucleotide sequence can be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker can be, for example, a polynucleotide.

[0102] As used herein, the terms "modulate", "modify" and grammatical variations thereof, when applied to a particular concentration, level, expression, function or behavior, generally refer to the ability to change a particular concentration, level, expression, function or behavior by increasing or decreasing, for example, by directly or indirectly promoting / stimulating / upregulating or interfering / inhibiting / downregulating, for example, acting as an antagonist or agonist. In some cases, a modulator can increase and / or decrease a particular concentration, level, activity or function compared to a control, or compared to a generally expected average activity level, or compared to a control activity level.

[0103] As used herein, the term "nonstructural protein" refers to a protein that is encoded by a virus but is not part of a virus particle. More specifically, the nonstructural proteins described herein include influenza NS1 proteins, including, but not limited to, influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, combinations thereof, or variants thereof. Further disclosure regarding such nonstructural proteins is provided elsewhere in this disclosure.

[0104] "Nucleic acid", "nucleic acid molecule", "nucleotide sequence", and grammatical variations thereof are used interchangeably and refer to the phosphate polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecule") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine or deoxycytidine; "DNA molecule"), or their phosphate analogs such as phosphorothioates and thioesters, in either single-stranded or double-stranded helical form. Furthermore, as will be apparent from the present disclosure, in some embodiments, the nucleic acid molecule may be in a circular form (e.g., circular RNA). A single-stranded nucleic acid sequence refers to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, particularly DNA or RNA molecule, refers only to the primary and secondary structure of the molecule and does not limit it to any particular tertiary form. Thus, the term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. When discussing the structure of a particular double-stranded DNA molecule, the sequence may be described herein according to the usual convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.

[0105] As used herein, the term "circular RNA" refers to a polyribonucleotide that forms a circular structure by covalent bonds. As is evident from the present disclosure, any of the polynucleotides, sets of polynucleotides, first nucleic acid molecules, and second nucleic acid molecules may be circular in structure. For example, in some embodiments, the polynucleotides described herein (e.g., comprising a first nucleic acid molecule encoding an influenza NS1 protein and a second nucleic acid encoding a heterologous target mRNA) comprise circular RNA. In some embodiments, the first nucleic acid molecule provided herein (e.g., encoding an influenza NS1 protein) comprises circular RNA. In some embodiments, the second nucleic acid molecule provided herein (e.g., encoding a heterologous target mRNA) comprises circular RNA. In some embodiments, the polynucleotides provided herein comprise circular RNA comprising a first nucleic acid molecule and a second nucleic acid molecule.

[0106] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", and grammatical variations thereof, include any agent approved by a regulatory agency of the U.S. Federal government or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to an extent that would prohibit administration of the composition to a subject and does not abolish the biological activity and properties of the administered compound. Included are excipients and carriers that are generally safe, non-toxic, and desirable, and are useful in the preparation of pharmaceutical compositions.

[0107] As used herein, the term "pharmaceutical composition" refers to one or more polynucleotides described herein mixed, admixed, or suspended with one or more other chemical components, such as pharma- ceutically acceptable carriers and excipients. In some embodiments, the purpose of a pharmaceutical composition is to facilitate administration of a preparation of a polynucleotide to a subject.

[0108] The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. In some embodiments, polynucleotides useful in the present disclosure can be linear. In some embodiments, polynucleotides are circular (e.g., circular RNA). The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified and unmodified forms of polynucleotides, for example, by alkylation and / or by capping.

[0109] More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), whether spliced ​​or not, including tRNA, rRNA, hRNA, siRNA, and mRNA (including circular RNA), any other type of polynucleotide that is an N- or C-glycoside of purine or pyrimidine bases, and other polymers that contain normucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers that contain nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA.

[0110] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids and creatine, etc.), as well as other modifications known in the art, are also included in this definition. The term "polypeptide" as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the above. A polypeptide can be a single polypeptide or a multi-molecular complex, such as a dimer, trimer, or tetramer. They can also include single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0111] As used herein, the terms "prevent", "preventing" and variations thereof refer to partially or completely delaying the onset of a disease, disorder and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics or clinical symptoms of a particular disease, disorder and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics or signs of a particular disease, disorder and / or condition; partially or completely delaying the progression from a particular disease, disorder and / or condition; and / or reducing the risk of developing a condition associated with a disease, disorder and / or condition. In some embodiments, prevention of an outcome is achieved by prophylactic treatment.

[0112] As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, for example between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent similarity of polymer molecules to each other can be performed in the same manner as calculation of percent identity, except that the calculation of percent similarity takes into account conservative substitutions, as understood in the art. It is understood that the percentage of similarity depends on the comparison measure used, i.e., whether amino acids are compared according to, for example, their evolutionary proximity, charge, volume, flexibility, polarity, hydrophobicity, aromaticity, isoelectric point, antigenicity, or a combination thereof.

[0113] The terms "subject," "patient," "individual," and "host," and variations thereof, are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, including, but not limited to, humans, domestic animals (e.g., dogs, cats, etc.), livestock (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.). The disclosure provided herein is applicable to both human therapy and veterinary applications.

[0114] The term "treat", "treatment" or "treating" as used herein refers to, for example, reducing the severity of a disease or condition; shortening the disease course; improving or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject having a disease or condition without necessarily curing the disease or condition. This term also includes prevention or prophylaxis of a disease or condition or a symptom thereof. In some embodiments, the term "treat" or "treatment" refers to inducing an immune response in a subject against an antigen (e.g., a heterologous payload disclosed herein).

[0115] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence.

[0116] II. Compositions of the Disclosure Some embodiments of the present disclosure relate to a polynucleotide or a set of polynucleotides comprising a first nucleic acid molecule encoding an innate immunity inhibitor and a second nucleic acid molecule encoding a heterologous target mRNA. In some embodiments, the first nucleic acid molecule is circular. In some embodiments, the second nucleic acid molecule is circular. In some embodiments, both the first nucleic acid molecule and the second nucleic acid molecule are circular. As described herein, in some embodiments, the polynucleotide or set of polynucleotides comprises a circular RNA. Thus, in some embodiments, a circular RNA is provided herein comprising a first nucleic acid molecule encoding an innate immunity inhibitor and a second nucleic acid molecule encoding a heterologous target mRNA. Also provided herein is a set of circular RNA comprising a first nucleic acid molecule encoding an innate immunity inhibitor and a second nucleic acid molecule encoding a heterologous target mRNA.

[0117] Any natural immune inhibitor can be used in the compositions and methods disclosed herein. Examples of natural immune inhibitors include, but are not limited to, influenza NS1, African swine fever virus (ASFV) g5R, Coxsackievirus B3 (CVB3) 2A protease, CVB3 3C protease, Encephalomyocarditis virus (EMCV) 2A protein (e.g., without NLS), EMCV 3C protease, feline calicivirus (FCV) 3C-like protease, foot-and-mouth disease virus (FMDV) L protease, group A rotavirus (RVA) NSP3, hantavirus N, human adenovirus 5 (Ad5) 100K, human immunodeficiency virus 1 (HIV-1) protease, human rhinovirus (HRV) 2A protease, HRV3C protease, human herpesvirus 1 (HSV) vhs, human T-cell leukemia virus (HTLV-1) protease, influenza A virus (FluAv) Pol, human herpesvirus 8 (KSHV) SOX, MD145-12 3C-like protease, measles virus (MV) N, poliovirus (PV) 2A protease, PV3C protease, Moloney murine leukemia virus (MMLV) protease 3C, rabies virus (RV) M, SARS-CoV NSP1, SARS-CoV S, SARS-CoV spike, simian virus 40 (SV40) small T antigen, vaccinia virus (VV) D10, VV D9, mouse 4E-BP1 (e.g., constitutively active), mouse 4E-BP2 (e.g., constitutively active), mouse 4E-BP3 (e.g., constitutively active), mouse 4EHP, mouse Ago1, mouse Ago2, mouse Ago3, mouse Ago4, mouse CPEB2, mouse DDX6, mouse eIF4E, mouse eIF4E(S209A), mouse eIF4E(S209D), mouse eIF4E(S209E), mouse eIF4g(N-term), mouse FMRP, mouse GW182, mouse p54, mouse p56 mouse p60, mouse PABP (eIF4G binding domain), mouse PDCD4, mouse RNase L (NΔ385: constitutively active), mouse Upf1 (e.g., constitutively active), mouse (Me31B), EBFP2, any derivatives thereof, and any combinations thereof.In some embodiments, the innate immunity inhibitor is an innate immunity inhibitor disclosed in US Patent Publication No. 20180296702, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the innate immunity inhibitor comprises a viral nonstructural (NS) protein, such as an influenza NS1 protein. In some embodiments, the first nucleic acid molecule encodes a viral nonstructural (NS) protein. In some embodiments, the first nucleic acid molecule encodes an influenza NS1 protein or a derivative thereof.

[0118] In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, and the second nucleic acid molecule encoding the target mRNA are present in a first vector. In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, is present in a first vector, and the second nucleic acid molecule encoding the target mRNA is present in a second vector.

[0119] In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, and the second nucleic acid molecule encoding the target mRNA are expressed under the control of a single promoter. In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, and the second nucleic acid molecule encoding the target mRNA are transcribed as a polycistronic mRNA. In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, is expressed under the control of a first promoter, and the second nucleic acid molecule encoding the target mRNA is expressed under the control of a second promoter. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are different.

[0120] In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, and the second nucleic acid molecule encoding the target mRNA are expressed under the control of a first promoter, which drives the expression of both the innate immune inhibitor, e.g., influenza NS1 protein, and the target mRNA. In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, e.g., influenza NS1 protein, and the second nucleic acid molecule encoding the target mRNA are linked by an IRES sequence.

[0121] In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, for example influenza NS1 protein, the second nucleic acid molecule encoding the target mRNA, or both, are expressed under the control of an inducible promoter.In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, for example influenza NS1 protein, the second nucleic acid molecule encoding the target mRNA, or both, are expressed under the control of a tissue-specific promoter.In some embodiments, the first nucleic acid molecule encoding the innate immune inhibitor, for example influenza NS1 protein, the second nucleic acid molecule encoding the target mRNA, or both, are expressed under the control of a constitutively active promoter.

[0122] Some embodiments of the present disclosure relate to a polynucleotide or a set of polynucleotides comprising a self-replicating target mRNA, the self-replicating target mRNA comprising one or more modified nucleic acid molecules. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, or less than about 25% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some embodiments, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% of the nucleic acids in the polynucleotide or set of polynucleotides are modified nucleic acid molecules. In some embodiments, one or more modified nucleic acid molecules are modified rNTPs.

[0123] In some embodiments, the one or more modified nucleic acid molecules include N1-methylpseudouracil. In some embodiments, the one or more modified nucleic acid molecules include 5-methylcytosine. In some embodiments, the one or more modified nucleic acid molecules include N1-methylpseudouracil and 5-methylcytosine. Non-limiting examples of additional modified nucleic acid molecules that can be used in the present disclosure include: 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, cytidine, pyrrolo-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, 1,6-dimethyl-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-(1-propynyl)-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-(2-propynyl)-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-allyl-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-ethynyl-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-homoallyl-pseudo-uracil, 1-(optionally substituted C1-C6 alkyl)-6-vinyl-pseudo-uracil, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-uracil, 1-methyl-6-(4-morpholino)-pseudo-uracil, 1-methyl-6-(4-thiomorpholino)-pseudo-uracil, 1-methyl-6-(optionally substituted phenyl)pseudo-uracil, 1-methyl-6-amino-pseudo-uracil, 1-methyl-6-azido-pseudo-uracil, 1-methyl-6-bromo-pseudo-uracil, 1-methyl-6-butyl-pseudo-uracil, 1-methyl-6-chloro-pseudo-uracil, 1-methyl-6-cyano-pseudo-uracil, 1-methyl-6-dimethylamino-pseudo-uracil, 1-methyl-6-ethoxy-pseudo-uracil, 1-methyl-6-ethylcarboxylate-pseudo-uracil, 1-methyl-6-ethyl-cyano-pseudo-uracil, pseudouracil, 1-methyl-6-fluoro-pseudouracil, 1-methyl-6-formyl-pseudouracil, 1-methyl-6-hydroxyamino-pseudouracil, 1-methyl-6-hydroxy-pseudouracil, 1-methyl-6-iodo-pseudouracil, 1-methyl-6-isopropyl-pseudouracil, 1-methyl-6-methoxy-pseudouracil, 1-methyl-6-methylamino-pseudouracil, 1-methyl-6-phenyl-pseudouracil, 1-methyl-6-propyl-pseudouracil, 1-methyl-6-tert-butyl-pseudouracil, 1-methyl-6-trifluoromethoxy-pseudouracil, 1-methyl-6-trifluoromethyl-pseudouracil, 6-(2,2,2-trifluoroethyl)-pseudo-uracil, 6-(4-morpholino)-pseudo-uracil, 6-(4-thiomorpholino)-pseudo-uracil, 6-(optionally substituted phenyl)-pseudo-uracil, 6-amino-pseudo-uracil, 6-azido-pseudo-uracil, 6-bromo-pseudo-uracil, 6-butyl-pseudo-uracil, 6-chloro-pseudo-uracil, 6-cyano-pseudo-uracil, 6-dimethylamino-pseudo-uracil, 6-ethoxy-pseudo-uracil, 6-ethylcarboxylate-pseudo-uracil, 6-ethyl-pseudo-uracil, Uracil, 6-fluoro-pseudo-uracil, 6-formyl-pseudo-uracil, 6-hydroxyamino-pseudo-uracil, 6-hydroxy-pseudo-uracil, 6-iodo-pseudo-uracil, 6-iso-propyl-pseudo-uracil, 6-methoxy-pseudo-uracil, 6-methylamino-pseudo-uracil, 6-methyl-pseudo-uracil, 6-phenyl-pseudo-uracil, 6-propyl-pseudo-uracil, 6-tert-butyl-pseudo-uracil, 6-trifluoromethoxy-pseudo-uracil, 6-trifluoromethyl-pseudo-uracil, 1-(3-amino-3-carboxypropyl)pseudo-uracil, 1-(2,2,2-trifluoroethyl)-pseudo-uracil, 1-(2,4,6-trimethyl-benzyl)pseudo-uracil, 1-(2,4,6-trimethyl-phenyl)pseudo-uracil, 1-(2-amino-2-carboxyethyl)pseudo-uracil, 1-(2-amino-ethyl)pseudo-uracil, 1-(3-amino-propyl)pseudo-uracil, 1-(4-amino- 4-carboxybutyl)pseudo-uracil, 1-(4-amino-benzyl)pseudo-uracil, 1-(4-amino-butyl)pseudo-uracil, 1-(4-amino-phenyl)pseudo-uracil, 1-(4-methoxy-benzyl)pseudo-uracil, 1-(4-methoxy-phenyl)pseudo-uracil, 1-(4-methyl-benzyl)pseudo-uracil, 1-(4-nitro-benzyl)pseudo-uracil, 1(4-nitro-phenyl)pseudo-uracil -uracil, 1-(5-amino-pentyl)pseudo-uracil, 1-(6-amino-hexyl)pseudo-uracil, 1-aminomethyl-pseudo-uracil, 1-benzyl-pseudo-uracil, 1-butyl-pseudo-uracil, 1-cyclobutylmethyl-pseudo-uracil, 1-cyclobutyl-pseudo-uracil, 1-cycloheptylmethyl-pseudo-uracil, 1-cycloheptyl-pseudo-uracil, 1-cyclohexylmethyl-pseudo-uracil -uracil, 1-cyclohexyl-pseudo-uracil, 1-cyclooctylmethyl-pseudo-uracil, 1-cyclooctyl-pseudo-uracil, 1-cyclopentylmethyl-pseudo-uracil, 1-cyclopentyl-pseudo-uracil, 1-cyclopropylmethyl-pseudo-uracil, 1-cyclopropyl-pseudo-uracil, 1-ethyl-pseudo-uracil, 1-hexyl-pseudo-uracil, 1-isopropyl-pseudo-uracil1-pentyl-pseudo-uracil, 1-phenyl-pseudo-uracil, 1-propyl-pseudo-uracil, 1-p-tolyl-pseudo-uracil, 1-tert-butyl-pseudo-uracil, 1-trifluoromethyl-pseudo-uracil, 3-(optionally substituted C1-C6 alkyl)-pseudo-uracil, pseudo-uracil-N1-2-ethanoic acid, pseudo-uracil-N1-3-propionic acid, pseudo-uracil -N1-4-butanoic acid, pseudo-uracil-N1-5-pentanoic acid, pseudo-uracil-N1-6-hexanoic acid, pseudo-uracil-N1-7-heptanoic acid, pseudo-uracil-N1-methyl-p-benzoic acid, 6-phenyl-pseudo-uracil, 6-azido-pseudo-uracil, pseudo-uracil-N1-p-benzoic acid, N3-methyl-pseudo-uracil, 5-methyl-amino-methyl-uracil, 5-carboxy-methyl- Amino-methyl-uracil, 5-(carboxyhydroxymethyl)uracil methyl ester 5-(carboxyhydroxymethyl)uracil, 2-anhydro-cytosine, 2-anhydro-uracil, 5-methoxycarbonylmethyl-2-thio-uracil, 5-methylaminomethyl-2-seleno-uracil, 5-(iso-pentenylaminomethyl)-uracil, 5-(iso-pentenylaminomethyl)-2-thio-uracil, 5-(iso-pentenylaminomethyl)-uracil thenylaminomethyl)-uracil, 5-trideuteromethyl-6-deutero-uracil, 5-(2-chloro-phenyl)-2-thio-cytosine, 5-(4-amino-phenyl)-2-thio-cytosine, 5-(2-furanyl)-uracil, 8-trifluoromethyl-adenosine, 2-trifluoromethyl-adenosine, 3-deaza-3-fluoro-adenosine, 3-deaza-3-bromo-adenosine, 3-deaza-3-iodo-adenosine, 1-Hydroxymethyl-pseudo-uracil, 1-(2-hydroxyethyl)-pseudo-uracil, 1-methoxymethyl-pseudo-uracil, 1-(2-methoxyethyl)-pseudo-uracil, 1-(2,2-diethoxyethyl)-pseudo-uracil, 1-(2-hydroxypropyl)-pseudo-uracil, (2R)-1-(2-hydroxypropyl)-pseudo-uracil, (2S)-1-(2-hydroxypropyl)-pseudo-uracil, 1-cyanomethyl-pseudo-uracil, 1-morpholinomethyl-pseudo-uracil, 1-thiomorpholinomethyl-pseudo-uracil, 1-benzyloxymethyl-pseudo-uracil, 1-(2,2,3,3,3-pentafluoropropyl)-pseudo-uracil, 1-thiomethoxymethyl-pseudo-uracil, 1-methanesulfonate Nylmethyl-pseudo-uracil, 1-vinyl-pseudo-uracil, 1-allyl-pseudo-uracil, 1-homoallyl-pseudo-uracil, 1-propargyl-pseudo-uracil, 1-(4-fluorobenzyl)-pseudo-uracil, 1-(4-chlorobenzyl)-pseudo-uracil, 1-(4-bromobenzyl)-pseudo-uracil, 1-(4-iodobenzyl)-pseudo-uracil, 1-(4-methylbenzyl)-pseudo-uracil, 1-(4-trifluoromethylbenzyl)-pseudo-uracil, 1-(4-methoxybenzyl)-pseudo-uracil, 1-(4-trifluoromethoxybenzyl)-pseudo-uracil, 1-(4-thiomethoxybenzyl)-pseudo-uracil, 1-(4-methanesulfonylbenzyl)-pseudo-uracil, pseudouracil 1-(4-methylbenzoic acid), pseudouracil 1-(4-methylbenzenesulfonic acid), 1-(2,4,6-trimethylbenzyl)-pseudouracil, 1-(4-nitrobenzyl)-pseudouracil, 1-(4-azidobenzyl)-pseudouracil, 1-(3,4-dimethoxybenzyl)-pseudouracil, 1-(3,4-bis-trifluoromethoxybenzyl)-pseudouracil, 1-acetyl-pseudouracil, 1-trifluoroacetyl-pseudouracil, 1-benzoyl-pseudouracil, 1-pivaloyl-pseudouracil,1-(3-cyclopropyl-prop-2-ynyl)-pseudouracil, pseudouracil 1-methylphosphonic acid diethyl ester, pseudouracil 1-methylphosphonic acid, pseudouracil 1-[3-(2-ethoxy)]propionic acid, pseudouracil 1-[3-{2-(2-ethoxy)-ethoxy}]propionic acid, pseudouracil 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouracil 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid, pseudouracil 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic acid, 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl}pseudo-uracil, 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]-pseudo-uracil, 1-biotinyl-pseudo-uracil, 1-biotinyl-PEG2-pseudo-uracil, 5-(C3-8 cycloalkyl)-cytosine, 5-methyl-N6-acetyl-cytosine, 5-(carboxymethyl)-N6-trifluoroacetyl-cytosine trifluoromethyl ester, N6-propionyl-cytosine, 5-monofluoromethyl-cytosine, 5-trifluoromethoxy-cytosine, N6-(1,1,1-trifluoro-propionyl)-cytosine, 4-acetyl-pseudo-iso-cytosine, 1-ethyl-pseudo-iso-cytosine, 1-hydroxy-pseudo-iso-cytosine, or 1-(2,2,2-trifluoroethyl)-cytosine. pseudouracil, 1,6-dimethyl-pseudouracil, 1-(optionally substituted C1-C6 alkyl)-6-(1-propynyl)-pseudouracil, 1-(optionally substituted C1-C6 alkyl)-6-(2-propynyl)-pseudouracil, 1-(optionally substituted C1-C6 alkyl)-6-allyl-pseudouracil, 1-(optionally substituted C1-C6 alkyl)-6-ethynyl-pseudouracil, 1-(optionally substituted C1-C6 alkyl)-6-homoallyl-pseudouracil,1-(optionally substituted C1-C6 alkyl)-6-vinyl-pseudo-uracil, 1-methyl-6-(2,2,2-trifluoroethyl)pseudo-uracil, 1-methyl-6-(4-morpholino)-pseudo-uracil, 1-methyl-6-(4-thiomorpholino)-pseudo-uracil, 1-methyl-6-(optionally substituted phenyl)pseudo-uracil, 1-methyl-6-amino-pseudo-uracil, 1-methyl-6-azido-pseudo-uracil, 1-methyl-6-bromo-pseudo-uracil, 1-methyl-6-butyl-pseudo-uracil Do-uracil, 1-methyl-6-chloro-pseudo-uracil, 1-methyl-6-cyano-pseudo-uracil, 1-methyl-6-dimethylamino-pseudo-uracil, 1-methyl-6-ethoxy-pseudo-uracil, 1-methyl-6-ethylcarboxylate-pseudo-uracil, 1-methyl-6-ethyl-pseudo-uracil, 1-methyl-6-fluoro-pseudo-uracil, 1-methyl-6-formyl-pseudo-uracil, 1-methyl-6-hydroxyamino-pseudo-uracil, 1-methyl-6-hydroxy-pseudo-uracil , 1-methyl-6-iodo-pseudo-uracil, 1-methyl-6-iso-propyl-pseudo-uracil, 1-methyl-6-methoxy-pseudo-uracil, 1-methyl-6-methylamino-pseudo-uracil, 1-methyl-6-phenyl-pseudo-uracil, 1-methyl-6-propyl-pseudo-uracil, 1-methyl-6-tert-butyl-pseudo-uracil, 1-methyl-6-trifluoromethoxy-pseudo-uracil, 1-methyl-6-trifluoromethyl-pseudo-uracil, 6-(2,2,2-trifluoroethyl)-silicone pseudo-uracil, 6-(4-morpholino)-pseudo-uracil, 6-(4-thiomorpholino)-pseudo-uracil, 6-(substituted phenyl)-pseudo-uracil, 6-amino-pseudo-uracil, 6-azido-pseudo-uracil, 6-bromo-pseudo-uracil, 6-butyl-pseudo-uracil, 6-chloro-pseudo-uracil, 6-cyano-pseudo-uracil, 6-dimethylamino-pseudo-uracil, 6-ethoxy-pseudo-uracil, 6-ethylcarboxylate-pseudo-uracil, 6-ethyl-pseudo-uracil,6-Fluoro-pseudo-uracil, 6-formyl-pseudo-uracil, 6-hydroxyamino-pseudo-uracil, 6-hydroxy-pseudo-uracil, 6-iodo-pseudo-uracil, 6-iso-propyl-pseudo-uracil, 6-methoxy-pseudo-uracil, 6-methylamino-pseudo-uracil, 6-methyl-pseudo-uracil, 6-phenyl-pseudo-uracil, 6-phenyl-pseudo-uracil, 6-propyl-pseudo-uracil, 6-tert-butyl-pseudo-uracil, 6-trifluoromethoxy- pseudouracil, 6-trifluoromethyl-pseudouracil, 1-(3-amino-3-carboxypropyl)pseudouracil, 1-(2,2,2-trifluoroethyl)pseudouracil, 1-(2,4,6-trimethyl-benzyl)pseudouracil, 1-(2,4,6-trimethyl-phenyl)pseudouracil, 1-(2-amino-2-carboxyethyl)pseudouracil, 1-(2-amino-ethyl)pseudouracil, 1-(3-amino-propyl)pseudouracil, 1-(4-amino-4-carboxybutyl)pseudouracil )pseudo-uracil, 1-(4-amino-benzyl)pseudo-uracil, 1-(4-amino-butyl)pseudo-uracil, 1-(4-amino-phenyl)pseudo-uracil, 1-(4-methoxy-benzyl)pseudo-uracil, 1-(4-methoxy-phenyl)pseudo-uracil, 1-(4-methyl-benzyl)pseudo-uracil, 1-(4-nitro-benzyl)pseudo-uracil, 1(4-nitro-phenyl)pseudo-uracil, 1-(5-amino-pentyl)pseudo-uracil, 1-(6-amino-hexyl)pseudo-uracil pseudouracil, 1-aminomethyl-pseudouracil, 1-benzyl-pseudouracil, 1-butyl-pseudouracil, 1-cyclobutylmethyl-pseudouracil, 1-cyclobutyl-pseudouracil, 1-cycloheptylmethyl-pseudouracil, 1-cycloheptyl-pseudouracil, 1-cyclohexylmethyl-pseudouracil, 1-cyclohexyl-pseudouracil, 1-cyclooctylmethyl-pseudouracil, 1-cyclooctyl-pseudouracil, 1-cyclopentylmethyl-pseudouracil,1-Cyclopentyl-pseudo-uracil, 1-cyclopropylmethyl-pseudo-uracil, 1-cyclopropyl-pseudo-uracil, 1-ethyl-pseudo-uracil, 1-hexyl-pseudo-uracil, 1-isopropyl-pseudo-uracil, 1-pentyl-pseudo-uracil, 1-phenyl-pseudo-uracil, 1-propyl-pseudo-uracil, 1-p-tolyl-pseudo-uracil, 1-tert-butyl-pseudo-uracil, 1-trifluoromethyl-pseudo-uracil, 3-(optionally substituted C1-C6 alkyl)-pseudo-uracil, pseudo-uracil-N1-2-ethanoic acid, pseudo-uracil-N1-3-propanol Pionic acid, pseudouracil-N1-4-butanoic acid, pseudouracil-N1-5-pentanoic acid, pseudouracil-N1-6-hexanoic acid, pseudouracil-N1-7-heptanoic acid, pseudouracil-N1-methyl-p-benzoic acid, 6-phenyl-pseudo-uracil, 6-azido-pseudo-uracil, or pseudouracil-N1-p-benzoic acid, N3-methyl-pseudo-uracil, 5-methyl-amino-methyl-uracil, 5-carboxy-methyl-amino-methyl-uracil, 5-(carboxyhydroxymethyl)uracil methyl ester, or 5-(carboxyhydroxymethyl)uracil, and combinations thereof.

[0124] In some embodiments, the compositions described herein increase the expression of heterologous target mRNA in a host cell. In some embodiments, the expression of the target mRNA is increased compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased compared to the expression of the target mRNA expressed from a replicating RNA that does not contain one or more modified nucleic acid molecules. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 100%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 200%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 300%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 400%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, by at least about 500%, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein.

[0125] In some embodiments, the expression of the target mRNA is increased, for example, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, or at least about 6-fold, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, at least about 2-fold, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, at least about 3-fold, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, at least about 4-fold, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, the expression of the target mRNA is increased, for example, at least about 5-fold, compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding the influenza NS1 protein. In some embodiments, expression of the target mRNA is increased, for example, at least about 6-fold, compared to expression of the target mRNA in the absence of the first nucleic acid molecule encoding an influenza NS1 protein.

[0126] In some embodiments, the compositions described herein have increased heterologous target mRNA expression and / or increased heterologous target mRNA persistence in a host cell. In some embodiments, the increased expression of the target mRNA, e.g., compared to the expression of the target mRNA in the absence of the first nucleic acid molecule encoding an influenza NS1 protein, or compared to the expression of the target mRNA expressed from a replicating RNA that does not include one or more modified nucleic acid molecules, persists for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 42 hours, at least about 48 hours, at least about 60 hours, at least about 72 hours, at least about 84 hours, at least about 96 hours, at least about 108 hours, or at least about 120 hours after transfection of the cell. In some embodiments, the expression of the target mRNA persists for at least about 48 hours after transfection of the cell. In some embodiments, the expression of the target mRNA persists for at least about 48 hours after transfection of the cell. In some embodiments, the expression of the target mRNA persists for at least about 60 hours after transfection of the cell. In some embodiments, expression of the target mRNA persists for at least about 72 hours after transfection of the cell. In some embodiments, expression of the target mRNA persists for at least about 84 hours after transfection of the cell. In some embodiments, expression of the target mRNA persists for at least about 96 hours after transfection of the cell. In some embodiments, expression of the target mRNA persists for at least about 108 hours after transfection of the cell. In some embodiments, expression of the target mRNA persists for at least about 120 hours after transfection of the cell.

[0127] II.A. Heterologous target mRNA The heterologous target mRNA of the present disclosure can code for any polypeptide or functional RNA of interest.In some embodiments, the target mRNA codes for a biologically active polypeptide.In some embodiments, the biologically active polypeptide comprises cytokines, chemokines, growth factors, clotting factors, hormones, receptors, mitogens, immunoglobulins (e.g., antibodies), enzymes, or any combination thereof.

[0128] In some embodiments, the cytokine is IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, GM-C SF, IL-6, IL-11, G-CSF, IL-12, LIF, OSM, IL-10, IL-20, IL-14, IL-16, IL-17, IFN-α, IFN-β, IFN-γ, CD154, LT The target mRNA comprises a cytokine selected from IL-12 polypeptide, a fragment or variant thereof, or any combination thereof. In some embodiments, the target mRNA is a single chain IL-12 (scIL-12), a protease sensitive IL-12, a destabilized IL-12, a membrane bound IL-12, an intercalated IL-12. In some embodiments, the target mRNA encodes an IL-12 polypeptide, or a fragment or variant thereof. In some embodiments, the IL-12 is human IL-12. In some embodiments, the target mRNA encodes a p35 subunit of IL-12 and a p40 subunit of IL-12. In some embodiments, the p35 subunit and the p40 subunit are expressed from a single promoter. In some embodiments, the p35 subunit and the p40 subunit are expressed as a single contiguous polypeptide. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more covalent bonds. In some embodiments, the p35 subunit and the p40 subunit are linked by one or more peptide bonds. In some embodiments, the target mRNA encodes a human IL-12 polypeptide comprising a p35 subunit of IL-12 covalently linked to a p40 subunit of IL-12. In some embodiments, the p35 subunit of IL-12 and the p40 subunit of IL-12 are linked by a linker comprising one or more amino acids.

[0129] In some embodiments, the target mRNA comprises a first portion and a second portion, the first portion of the target mRNA encodes the p35 subunit of IL-12 and the second portion of the target mRNA encodes the p40 subunit of IL-12, and the first and second portions are separated by an IRES.

[0130] In some embodiments, the target mRNA encodes a chemokine. Exemplary chemokines include, but are not limited to, CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.

[0131] In some embodiments, the target mRNA encodes an interferon (IFN). Exemplary interferons include, but are not limited to, interferon type I (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω), interferon type II (e.g., IFN-γ) and interferon type III. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[0132] In some embodiments, the target mRNA encodes a growth factor. Exemplary growth factors include, but are not limited to, bone morphogenetic proteins (BMPs), angiopoietins, CNTF, LIF, M-CSF, G-CSF, GM-CSF, epidermal growth factor (EGF), ephrins, erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), growth differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factors insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP; hepatocyte growth factor-like protein, HGFLP). Also known as NF-κB (also known as NF-κB), myostatin (GDF-8), neuregulin (NRG), neurotrophins (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and Wnt signaling pathway members.

[0133] In some embodiments, the target mRNA encodes an enzyme. In some embodiments, the target mRNA encodes cas9. In some embodiments, the target mRNA encodes a zinc finger endonuclease.

[0134] In some embodiments, the target mRNA encodes functional RNA.In some embodiments, the target mRNA encodes miRNA, siRNA, shRNA, dsRNA, antisense oligonucleotide, guide RNA, or any combination thereof.In some embodiments, the target mRNA encodes guide RNA, for example, for use in combination with cas9.

[0135] II.B. Influenza NS1 Any influenza NS1 can be used in the compositions and methods of the present disclosure.In some embodiments, influenza NS1 is influenza A virus NS1, influenza B virus NS1, influenza C virus NS1, or their variants.In some embodiments, influenza NS1 is influenza A virus NS1.In some embodiments, influenza NS1 is influenza B virus NS1.In some embodiments, influenza NS1 is influenza C virus NS1.

[0136] In some embodiments, the influenza NS1 is H1N1 NS1, H1N2 NS1, H2N2 NS1, H3N2 NS1, H5N1 NS1, H7N9 NS1, H7N7 NS1, H9N2 NS1, H7N2 NS1, H7N3 NS1, H5N2 NS1, H10N7 NS1, a mutant thereof, or a combination thereof. In some embodiments, the influenza NS1 is H1N2 NS1. In some embodiments, the influenza NS1 is H2N2 NS1. In some embodiments, the influenza NS1 is H3N2 NS1. In some embodiments, the influenza NS1 is H7N9 NS1. In some embodiments, the influenza NS1 is H7N7 NS1. In some embodiments, the influenza NS1 is H9N2 NS1. In some embodiments, the influenza NS1 is H7N2 NS1. In some embodiments, the influenza NS1 is H7N3 NS1. In some embodiments, the influenza NS1 is H5N2 NS1. In some embodiments, the influenza NS1 is H10N7 NS1.

[0137] In some embodiments, the influenza NS1 is H1N1 NS1. In some embodiments, the influenza is H1N1 TX91 mutant NS1. In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:1 (Table 1). In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the influenza NS1 is H1N1 TX91 mutant NS1 encoded by the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, a first nucleic acid molecule encoding influenza NS1 comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, which nucleotide sequence encodes an influenza NS1 protein. In some embodiments, a first nucleic acid molecule encoding an influenza NS1 protein comprises the nucleotide sequence set forth in SEQ ID NO: 1, which nucleotide sequence encodes an influenza NS1 protein.

[0138] In some embodiments, the influenza NS1 is H5N1 NS1. In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:2 (Table 1). In some embodiments, the influenza NS1 encoded by the first nucleic acid molecule comprises an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:2. In some embodiments, the influenza NS1 is H1N1 TX91 mutant NS1 encoded by the nucleotide sequence shown in SEQ ID NO:2. In some embodiments, a first nucleic acid molecule encoding influenza NS1 comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:2, which nucleotide sequence encodes an influenza NS1 protein. In some embodiments, a first nucleic acid molecule encoding an influenza NS1 protein comprises the nucleotide sequence set forth in SEQ ID NO:2, which nucleotide sequence encodes an influenza NS1 protein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7]

[0139] II.C. Vector In some embodiments, one or more of the first nucleic acid molecule encoding influenza NS1 protein and the second nucleic acid molecule encoding target mRNA are present in a vector. Any vector can be used in the compositions and methods disclosed herein. In some embodiments, the vector comprises a viral vector, a mammalian vector, a bacterial vector, or a combination or variant thereof. In some embodiments, the vector is selected from the group consisting of an adenovirus vector, a lentivirus, a Sendai virus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, a hybrid vector, and an adeno-associated virus (AAV) vector.

[0140] In some embodiments, the vector comprises a replicon. In some embodiments, the replicon derived from an alphavirus comprises a positive-stranded RNA encoding an RNA-dependent RNA polymerase that co-transcribes a therapeutic payload and self-amplifies the replicon upon entry into the cytoplasm. In some embodiments, the vector comprises a replicon, the replicon comprising (1) the UTRs of the parent virus and nonstructural proteins and the subgenomic promoter (SGP) of the parent virus, and (2) a first nucleic acid molecule encoding an influenza NS1 protein, a second nucleic acid molecule encoding a target mRNA, or both a first nucleic acid molecule encoding an influenza NS1 protein and a second nucleic acid molecule encoding a target mRNA. Any replicon can be used in the compositions and methods disclosed herein. In some embodiments, the replicon comprises a Venezuelan equine encephalitis (VEE) replicon or a derivative or portion thereof. In some embodiments, the replicon comprises one or more point mutations relative to the parent virus replicon, for example, one or more point mutations relative to the VEE replicon sequence. Modifications to the sequence of the replicon can be used to increase expression of the target mRNA, increase persistence of the target mRNA (e.g., by reducing immune responses to the target mRNA or polynucleotide encoding the target mRNA and / or by reducing type I interferon activity in the target cell, e.g., the target cancer cell), or both. In some embodiments, the vector comprises a VEE replicon, and the VEE replicon comprises a Q739L mutation relative to the parent VEE replicon. In some embodiments, the first vector, the second vector, or both, comprise one or more additional regulatory elements. In some embodiments, the first vector, the second vector, or both, comprise a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific silencer, or any combination thereof.

[0141] In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA; and (v) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0142] In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; and (vi) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0143] In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA; (v) an E1 sequence; and (vi) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0144] In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; (vi) an E1 sequence; and (vii) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0145] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE.

[0146] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an influenza NS1 protein; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE.

[0147] In some embodiments, the vector comprises (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a second nucleic acid molecule encoding a target mRNA; and (v) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0148] In some embodiments, the vector comprises (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA; (vi) an E1 sequence; and (vii) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE.

[0149] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE.

[0150] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1 or an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE.

[0151] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H1N1 TX91 mutant NS1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE. In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE. In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:1; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0152] In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule encoding an H5N1 NS1; (iv) a P2A linker; (v) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; (vi) an E1 sequence; and (vii) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE. In some embodiments, the vector comprises: (i) a 5'UTR from a parental replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parental replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO:2; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parental replicon, e.g., a 3'UTR from VEE. In some embodiments, the vector comprises: (i) a 5'UTR from a parent replicon, e.g., a 5'UTR from VEE; (ii) one or more nonstructural proteins (nsPs) from a parent replicon, e.g., one or more nsPs from VEE, e.g., nsP2, nsP3, and nsP4 from VEE; (iii) a first nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO:2; (iv) a second nucleic acid molecule encoding a target mRNA, wherein the target mRNA encodes a human IL-12 polypeptide; and (v) a 3'UTR from a parent replicon, e.g., a 3'UTR from VEE.

[0153] II.D. Lipid Nanoparticles Some aspects of the present disclosure relate to lipid nanoparticles that contain, for example encapsulate, polynucleotides or sets of polynucleotides disclosed herein.In some aspects, polynucleotides or sets of polynucleotides disclosed herein are packaged and / or delivered in lipid nanoparticles.Thus, in some aspects, the present disclosure relates to the polynucleotides described herein that are encapsulated by lipid nanoparticles, compositions thereof, and the use of the compositions thereof.

[0154] As used herein, "lipid nanoparticle" (LNP) refers to a vesicle, such as a spherical vesicle, that has a continuous lipid bilayer. Lipid nanoparticles can be used in a method for delivering drug therapy to a target site. Non-limiting examples of LNPs include liposomes, bolaamphiphiles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), and single-layer membrane structures (e.g., archaeal and micelles).

[0155] In some embodiments, the lipid nanoparticles include one or more types of lipids. Lipids, as used herein, refer to a group of organic compounds including, but not limited to, esters of fatty acids, and in some embodiments, are characterized by being insoluble in water, but soluble in many organic solvents. They are generally divided into at least three classes: (1) "simple lipids," including fats and oils, as well as waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derived lipids," such as steroids. Non-limiting examples of lipids include triglycerides (e.g., tristearin), diglycerides (e.g., glycerol behenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate). In some embodiments, one or more types of lipids in the LNPs include cationic lipids. In some embodiments, one or more types of lipids in the LNPs include lipidoids, such as TT3.

[0156] Such lipids useful in the present disclosure include, but are not limited to, N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); Lipofectamine; 1,2-Dilinoleyloxy-N,N-Dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-Dimethylaminopropane (DLenDMA); Dioctadecyldimethylammonium (DODMA), Distearyldimethylammonium (D N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N-N-distearyl-N,N-dimethylammonium bromide (DDAB); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0157] In some embodiments of the present disclosure, the lipid, e.g., lipidoid, is TT3. As used herein, TT3 can form lipid nanoparticles for delivering various bioactive agents to cells.

[0158] In some embodiments of the present disclosure, cationic lipid is DOTAP.The DOTAP used herein can also form lipid nanoparticles.DOTAP can be used to efficiently transfect DNA containing yeast artificial chromosome (YAC) into eukaryotic cells to express genes transiently or stably, and is also suitable for efficiently introducing other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins into mammalian cell research samples.

[0159] In some embodiments of the present disclosure, the cationic lipid is lipofectamine. As used herein, lipofectamine is manufactured and sold by Invitrogen and is a common transfection reagent used in molecular and cell biology. It is used to enhance the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine contains lipid subunits that can form liposomes or lipid nanoparticles in an aqueous environment, which capture the transfection payload, e.g., modRNA. RNA-containing liposomes (positively charged on the surface) can fuse with the negatively charged cell membrane of live cells due to the neutral co-lipids that mediate the fusion of the liposome with the cell membrane, allowing the nucleic acid cargo molecule to translocate into the cytoplasm for replication or expression.

[0160] In some embodiments, the LNPs are composed primarily of cationic lipids along with other lipid components. These typically include, but are not limited to, other lipid molecules belonging to the phosphatidylcholine (PC) class (e.g., 1,s-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sterol (e.g., cholesterol) and polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000 (DSPE-PEG2000) and C14-PEG2000). Table 2 shows the formulation of exemplary LNPs, TT3-LNPs and DOTAP-LNPs. [Table 2]

[0161] In some embodiments, the LNP comprises C14-PEG2000. In some embodiments, the C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG2000), or both. In some embodiments, the C14-PEG2000 (or other lipid components disclosed herein) can be embedded in the LNP prior to encapsulation of the polynucleotide. In some embodiments, the C14-PEG2000 (or other lipid components disclosed herein) can be added to the LNP after encapsulation of the polynucleotide.

[0162] The particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucoadhesion, cellular water uptake and buffer exchange into the nanoparticle interior, and protein diffusion. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm, about 50 to about 400 nm, about 70 to about 300 nm, about 100 to about 200 nm, about 100 to about 175 nm, or about 100 to about 160 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of 100 to 160 nm. In some embodiments of the present disclosure, the diameter of the LNP can be about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm, about 117 nm, about 118 nm, about 119 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, or about 160 nm. In some embodiments, the lipid nanoparticle has a diameter of about 140 nm.

[0163] Zeta potential is a measure of the effective charge on the surface of a lipid nanoparticle. The magnitude of the zeta potential provides information about particle stability. In some embodiments of the present disclosure, the zeta potential of the LNP is in the range of about 3 to about 6 mv. In some embodiments of the present disclosure, the zeta potential of the LNP can be about 3mv, about 3.1mv, about 3.2mv, about 3.3mv, about 3.4mv, about 3.5mv, about 3.6mv, about 3.7mv, about 3.8mv, about 3.9mv, about 4mv, about 4.1mv, about 4.2mv, about 4.3mv, about 4.4mv, about 4.5mv, about 4.6mv, about 4.7mv, about 4.8mv, about 4.9mv, about 5mv, about 5.1mv, about 5.2mv, about 5.3mv, about 5.4mv, about 5.5mv, about 5.6mv, about 5.7mv, about 5.8mv, about 5.9mv, or about 6mv.

[0164] In some embodiments, the present disclosure relates to a polynucleotide or set of polynucleotides encapsulated with lipid nanoparticles (LNPs). In some embodiments of the present disclosure, the mass ratio of lipid to polynucleotide or set of polynucleotides of the LNP ranges from about 1:2 to about 15:1. In some embodiments, the mass ratio of lipid to polynucleotide or set of polynucleotides ranges from about 1:2, about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, about 1:1.2, about 1:1.1, about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about The lipid mass ratio may be about 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, about 10:1, about 10.5:1, about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, or about 15:1. In some embodiments of the present disclosure, the mass ratio of lipid to polynucleotide or set of polynucleotides is about 10:1.

[0165] II.E. Cell In some embodiments, provided herein is a cell that is modified to include a polynucleotide or set of polynucleotides described herein. In some embodiments, the cell includes a vector or set of vectors that includes a polynucleotide or set of polynucleotides described herein. In some embodiments, the cell includes a lipid nanoparticle that includes a polynucleotide or set of polynucleotides described herein. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell includes a T cell (e.g., a CD4+ T cell, a CD8+ T cell, or both), a natural killer cell (NK cell), a tumor-infiltrating lymphocyte, a dendritic cell, a B cell, a bone marrow cell, a monocyte, or a PBMC.

[0166] In some embodiments, the cells described herein (i.e., comprising a polynucleotide or set of polynucleotides of the present disclosure, or a vector or set of vectors comprising the same) can produce (i) human influenza NS1 and (ii) target heterologous mRNA. In some embodiments, the cells described herein (i.e., comprising a polynucleotide or set of polynucleotides of the present disclosure, or a vector or set of vectors comprising the same) can produce (i) human influenza NS1 and (ii) target heterologous mRNA in vivo. For example, in some embodiments, the polynucleotide or set of polynucleotides of the present disclosure, or a vector or set of vectors comprising the same, can be introduced into a cell ex vivo (e.g., via transfection), and the cell can then be administered to a subject (e.g., adoptive cell therapy), whereby (i) human influenza NS1 and (ii) target heterologous mRNA are produced in the subject after administration. In some embodiments, the polynucleotide or set of polynucleotides of the present disclosure, or a vector or set of vectors comprising the same, can be administered to a subject, for example, as part of gene therapy. In some aspects, the cells described herein (i.e., comprising a polynucleotide or set of polynucleotides of the present disclosure, or a vector or set of vectors comprising a polynucleotide or set of polynucleotides of the present disclosure) are capable of producing (i) human influenza NS1 and (ii) a target heterologous mRNA both in vitro and in vivo.

[0167] In some embodiments, the cell is a host cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of a mammalian cell, an insect cell, a yeast cell, a transgenic mammalian cell, a plant cell, and any combination thereof. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0168] In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian host cells suitable for the present disclosure include CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, HBK, NSO, HT1080, HsS78Bst cells, and combinations thereof.

[0169] II.F. Pharmaceutical Compositions As will be apparent from the present disclosure, any of the polynucleotides, vectors, lipid nanoparticles, and cells (also referred to herein as "active") described herein can be incorporated into a pharmaceutical composition suitable for administration. Thus, in some aspects, the pharmaceutical composition comprises an active compound and a pharma- ceutical acceptable excipient.

[0170] As used herein, the term "pharmaceutically acceptable excipient" (also referred to herein as "pharmaceutically acceptable carrier") includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active compounds is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0171] In some aspects, disclosed herein are pharmaceutical compositions comprising (a) a polynucleotide or set of polynucleotides described herein and (b) a pharma- ceutically acceptable excipient. In some aspects, disclosed herein are pharmaceutical compositions comprising (a) a vector or set of vectors described herein and (b) a pharma- ceutically acceptable excipient. In some aspects, disclosed herein are pharmaceutical compositions comprising (a) a lipid nanoparticle described herein and (b) a pharma- ceutically acceptable excipient. In some aspects, disclosed herein are pharmaceutical compositions comprising (a) a cell described herein (e.g., modified to include a polynucleotide or set of polynucleotides of the present disclosure) and (b) a pharma- ceutically acceptable excipient.

[0172] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. In some embodiments, suitable routes of administration that can be used with the present disclosure include intramuscular administration. In some embodiments, suitable routes of administration include intranasal administration. Further examples of suitable routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral, transdermal (topical), and transmucosal, and any combination thereof. Another route of administration includes pulmonary administration. In addition, it may be desirable to administer a therapeutically effective amount of the pharmaceutical composition locally to the area requiring treatment. This can be accomplished, for example, by infusion or perfusion of the area or area during surgery, topical application, injection, catheter, suppository, or implant (e.g., implant formed from porous, non-porous, or gelatinous material, including membranes such as silastic membranes or fibers), and the like. In some embodiments, a therapeutically effective amount of the pharmaceutical composition is delivered in a vesicle, such as a liposome (see, e.g., Langer, Science 249:1527-33, 1990 and Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, NY, pp. 353-65, 1989).

[0173] In some embodiments, the pharmaceutical compositions described herein can be delivered in a controlled release system. For example, in some embodiments, pumps can be used (see, e.g., Langer, Science 249:1527-33, 1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987; Buchwald et al., Surgery 88:507-16, 1980; Saudek et al., N Engl. J Med. 321:574-79, 1989). In some embodiments, polymeric materials can be used (see, e.g., Levy et al., Science 228:190-92, 1985; During et al., Ann. Neural. 25:351-56, 1989; Howard et al., J Neurosurg. 71:105-12, 1989). Other controlled release systems, such as those discussed by Langer (Science 249:1527-33, 1990), can also be used.

[0174] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g. such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0175] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharma- ceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multi-dose containers. These include phenol or cresol, mercury, benzyl alcohol, chlorobutanol, p-hydroxybenzoic acid methyl and propyl esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Metal ion sequestrants or chelators include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water-miscible vehicles; sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0176] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol, methylparabens; antioxidants such as ascorbic acid, sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0177] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELS (BASF; Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0178] Sterile injection can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed components as required, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.For the preparation of sterile powder for sterile injection, the preparation method can be vacuum drying and freeze-drying, which makes the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0179] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means.

[0180] For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, active compound is formulated into ointments, salves, gels or creams generally known in the art.The compound can also be prepared in the form of suppositories (for example, using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0181] In some embodiments, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0182] In some embodiments, the active compound of the present disclosure can be formulated in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage to the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a required pharmaceutical carrier.The specification of the dosage unit form of the present disclosure is determined and directly depends on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such functional compounds for the treatment of individuals.The pharmaceutical composition can be included in a container, pack, or dispenser together with instructions for administration.

[0183] III. Methods of the Disclosure Some aspects of the present disclosure relate to nucleic acid molecules that increase the expression of target heterologous mRNA.Accordingly, the compositions of the present disclosure can be used in a method for increasing the expression of target in cells, including the cells of human subjects that require treatment.

[0184] Some embodiments of the present disclosure relate to a method of expressing a target mRNA in a cell, comprising co-expressing the target mRNA and an influenza NS1 protein in the cell, where the target mRNA is not an influenza mRNA. In some embodiments, the influenza NS1 protein is encoded by a first nucleic acid molecule and the target mRNA is encoded by a second nucleic acid molecule, e.g., as described herein.

[0185] Some aspects of the present disclosure relate to a method of treating a disease or condition in a subject in need of treatment, comprising administering to the subject a composition disclosed herein, such as a polynucleotide or set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a lipid nanoparticle, a cell, or a pharmaceutical composition disclosed herein. For example, the compositions described herein can be administered to cells in culture in vitro or ex vivo, or to a human subject, for example in vivo, to induce selective and sustained expression of the encoded target heterologous mRNA in cells (e.g., tumor cells and / or immune cells), which in some aspects can be useful for treating a disease or disorder.

[0186] Thus, in some aspects, the present disclosure relates to therapeutic methods using the compositions described herein. In some aspects, disclosed herein is a method for expressing a target heterologous mRNA in a subject in need thereof, comprising administering to the subject a composition of the present disclosure.

[0187] In some embodiments, the disease or disorder that can be treated with the present disclosure includes cancer. In some embodiments, the cancer includes squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, gastrointestinal cancer, renal cancer (e.g., renal clear cell carcinoma), ovarian cancer, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), thyroid cancer, pancreatic cancer, cervical cancer, gastric cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer (or carcinoma), stomach cancer, germ cell tumor, pediatric sarcoma, sinonasal cancer, pulmonary natural killer, melanoma (e.g. metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus (e.g. gastroesophageal junction), cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the ureter, carcinoma of the renal pelvis, tumor angiogenesis, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, asbestos-induced Environmentally induced cancers, including cancers, virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or HPV-origin tumors)), and hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lines (producing B cells, T cells, NK cells and plasma cells), such as all types of leukemias, lymphomas and myelomas, e.g., acute leukemia (ALL), acute myeloid leukemia (AML), acute myeloma ... acute, chronic, lymphocytic and / or myeloid leukemias, such as myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (Ml), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma, and chloroma;Lymphomas, e.g. Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g. B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g. Ki1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, enteropathic T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic Lymphoma, T-lymphoblastic, etc.; as well as lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of the lymphatic system, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B lymphoma, Pablastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytoid lymphoma with Waldenström's macroglobulinemia (LPL); myelomas, such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of the myeloid lineage, including fibrosarcoma and rhabdomyosarcoma Tumors of mesenchymal origin; tumors of mesenchymal origin, including seminoma, teratocarcinoma, fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroid follicular carcinoma, and teratocarcinoma; hematopoietic tumors of the lymphoid system, including, but not limited to, T-cell disorders such as T-cell prolymphocytic leukemia (T-PLL), including small cell and encephalo-like cell types; large granular lymphocytic leukemia (LGL) of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head and neck, kidney, rectum, thyroid; acute myeloid lymphoma, or any combination thereof;

[0188] In some embodiments, the compositions described herein are administered to a subject in need thereof in an amount sufficient to reduce tumor burden or cancer cell proliferation in vivo by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more. In some embodiments, the compositions described herein are administered in an amount effective to increase immune activity by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0189] In some embodiments, the disease or disorder that can be treated with the present disclosure includes autoimmune disease. As used herein, the term "autoimmune disease" refers to a disease caused by the host's immune system's inability to distinguish between foreign and self molecules, resulting in the host's immune system attacking and destroying the self molecule. As used herein, "self molecule" (e.g., protein or DNA) refers to a molecule that originates from or is indigenous to the host. As used herein, "foreign molecule" refers to a molecule that originates from another and is of non-natural origin. Non-limiting examples of autoimmune diseases include multiple sclerosis, peripheral neuritis, Sjogren's syndrome, rheumatoid arthritis, alopecia, autoimmune pancreatitis, Behcet's disease, bullous pemphigoid, celiac disease, Devic's disease (neuromyelitis optica), glomerulonephritis, IgA nephropathy, various vasculitides, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, systemic lupus erythematosus, Graves' disease, myasthenia gravis, pemphigus vulgaris, anti-glomerular basement membrane disease (Goodpasture's syndrome), Hashimoto's thyroiditis, autoimmune hepatitis, and combinations thereof.

[0190] In some embodiments, when the disease or disorder to be treated includes an autoimmune disease, administering a composition (e.g., a polynucleotide, vector, lipid nanoparticle, or pharmaceutical composition described herein) to a subject can reduce immune activity, such as T cell activity, in the subject. For example, as is evident from the present disclosure, in some embodiments, immune activity can be reduced using a polynucleotide described herein by encoding a target heterologous mRNA that can reduce immune cell function and / or promote immunosuppressive activity (e.g., promote developmental regulatory T cells). In some embodiments, the immune activity is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to the immune activity of a reference subject (e.g., a subject prior to administration of the composition or a corresponding subject that did not receive the composition). EXAMPLES

[0191] Example 1 The replicon plasmids used in this example included: Strand-EGFP (SEQ ID NO:3; Table 6); Strand-mCherry (SEQ ID NO:4; Table 6); non-cytopathic EGFP (SEQ ID NO:5; Figure 1A; Table 6); Strand-NS1-H5N1 (SEQ ID NO:2; Table 1); Strand-NS1-TX91 (SEQ ID NO:1; Table 1); Strand-NS1-EGFP (SEQ ID NO:6; Table 6); Strand-NS1-mCherry (SEQ ID NO:7; Table 6); and non-cytopathic-NS1-EGFP (SEQ ID NO:8; Figure 1B; Table 6).

[0192] method

[0193] Template preparation

[0194] For replicon RNA, a VEE replicon vector containing an mRNA payload encoding EGFP was prepared by an appropriate plasmid preparation method. The plasmid was further linearized by BspQI treatment. Briefly, 5 μg of replicon plasmid DNA was treated with BspQI in NEB3.1 buffer at 50 °C for 3 h. The enzyme was heat inactivated at 80 °C for 20 min and the sample proceeded to the DNA cleanup step. [Table 3]

[0195] For replicon plasmids containing mCherry (Strand-mCherry and Strand-NS1-mCherry) and plasmids containing only NS1 (Strand-NS1-H5N1 and Strand-NS1-TX91), ISceI was used to digest the replicon template. Reactions contained Cutsmart buffer and were incubated at 37°C for 3 hours.

[0196] ModRNA template generation

[0197] For modified RNA (modRNA) templates, DNA was generated by PCR using a replicon plasmid with a forward primer containing a T7 promoter and a subgenomic promoter and a reverse primer in the 3'-UTR with a stretch of 120 amino acids. [Table 4A] [Table 4B]

[0198] DpnI digestion

[0199] Plasmid DNA (template) in the PCR reaction was digested with DpnI. Add 1 uL of DpnI per 1 ug of starting plasmid to the PCR sample and incubate at 37° C. for 1 hour.

[0200] DNA cleanup

[0201] The PCR amplified DNA samples were cleaned using the NEB PCR clean-up kit according to the manufacturer's protocol, and the DNA was eluted in 20 μL of water.

[0202] PCR (modRNA template) and BspQI-treated replicon DNA (repRNA template) were checked on precast gels to confirm purity (PCR) and integrity (replicon template). 1 μg of purified template was used in each 20 μL IVT reaction below.

[0203] Up to 20 ng of total DNA was loaded onto a 1.2% DNA gel and run at 275 V for 7–10 min.

[0204] In vitro transcription

[0205] The NEBHiScribe High yield T7 kit was used for RNA production. For modified RNA (modRNA) synthesis, the UTP component of the kit was replaced with N1-methylpseudouridine-5'-triphosphate.

[0206] When the IVT reaction was prepared, the necessary kit components were thawed on ice, mixed, and pulse-spun in a microcentrifuge to collect the solution at the bottom of the tube. Samples were kept on ice and the enzyme was not vortexed.

[0207] Co-transcriptional capping method

[0208] For production using the cap analog replicon plasmid and modRNA template, reactions were assembled at room temperature in the following order: [Table 4C]

[0209] The samples were then mixed thoroughly, pulse-spun in a microcentrifuge, and incubated at 400 rpm in a thermomixer for 3 hours at 37° C. A 1 uL aliquot was saved for quality control.

[0210] DNase treatment

[0211] Template DNA was digested using Turbo DNase enzyme. No 10x buffer was added since the enzyme is active in the IVT reaction. Reactions were diluted to 200uL with nuclease-free water.

[0212] 20 μL of enzyme (2 U / μL) was used per 100 μL IVT reaction and incubated for 60 min at 37° C. After the reaction, RNA was purified using the Monarch RNA cleanup kit. A 1 μL aliquot of RNA was saved for quality control.

[0213] RNA quality control

[0214] Sample concentration was checked with Nanodrop and quality control was confirmed on a gel containing RNA from all intermediate steps. Up to 200ng of RNA was run on a 1.2% RNA gel. 1uL of Lonza RNA ladder was used as a size marker. RNA was denatured by adding 50% formaldehyde sample buffer at 65°C for 5 min and samples were immediately placed on ice and kept for at least 1 min.

[0215] Up to 5 uL of total sample was loaded onto the gel and visualized. RNA integrity can also be checked by running it on a fragment analyzer.

[0216] Prepare conventional TT3 LNP formulations with T-junctions

[0217] The lipid materials were each weighed and dissolved in ethanol. The ethanol phase was prepared by mixing all lipid materials according to the following composition ratio of the form: The aqueous phase was prepared by diluting the silica column purified repRNA / modRNA with 20 mM citrate buffer (pH 4.0), 300 mM NaCl and water so that the final salt composition was 10 mM citrate buffer (pH 4.0, 150 mM NaCl). Conventional TT3 LNPs were obtained by mixing the ethanol phase and aqueous phase of LNPs at a flow ratio of 3:1 (aqueous phase:ethanol phase) by T-junction mixing. [Table 5]

[0218] Preparation of PEG-post-micellar TT3 LNP formulations with T-junctions

[0219] The lipid materials were weighed and dissolved in ethanol respectively. The ethanol phase was prepared by mixing all lipid materials except DMG-PEG-2K according to the composition ratio of the forms above. The aqueous phase was prepared by diluting the silica column purified repRNA / modRNA with 20 mM citrate buffer (pH 4.0), 300 mM NaCl and water so that the final salt composition was 10 mM citrate buffer (pH 4.0, 150 mM NaCl). The PEG micelle phase was prepared by adding the corresponding volume of DMG-PEG-2K to TBS buffer and mixing thoroughly by vortexing. Finally, the post-PEG micellar TT3 LNPs were obtained by first mixing the ethanol and aqueous phases of LNPs by T-junction mixing at a flow ratio of 3:1 (aqueous phase:ethanol phase), followed by immediate in-line dilution with the PEG micelle phase by T-junction mixing at a flow ratio of 1:1 (LNP phase:PEG phase). The final lipid composition of the post-PEG micellar TT3 LNPs is listed in Table B. [Table A] [Table B]

[0220] Buffer exchange and freeze / thaw of TT3 LNPs

[0221] The obtained TT3 LNP was transferred to a dialysis cassette and dialyzed in TBS buffer for 2 hours.Then, the TT3 LNP was concentrated by tangential flow filtration.Then, 40% sucrose (W / V) in TBS stock solution was added to all prepared TT3 LNP to make a final solution of TT3 LNP in 10% sucrose.The final RNA concentration of LNP was measured by dissociating LNP with 2% TE+Triton® and further detected by Qubit assay.The TT3 LNP was divided into aliquots of 50 μl / tube and frozen at -80°C.Before cells were treated with LNP, the TT3 LNP was thawed at room temperature.

[0222] Lipid transfection

[0223] For cell lines, 50 ng of TT3:RNA was added to each well. In some experiments, Lipofectamine MessengerMax (ThermoFisher) was used to deliver the payload according to the manufacturer's recommendations.

[0224] 2. Preparation of FACS Samples

[0225] ZombieNIR staining buffer was prepared by diluting 100x dye stock in PBS. Cells were washed with PBS, transferred to deep well 96 well plates and centrifuged at 500g, 10 min for suspension cells. For adherent cells, cells are trypsinized for 5 min followed by centrifugation at 500g for 10 min. Cells are resuspended in PBS, transferred to 96V bottom plates and subsequently live / dead stained for 10 min at RT in the dark with 100uL PBS containing ZombieNIR dye. The reaction is stopped by adding 200uL FACS buffer (containing BSA to quench the dye), centrifuged again and finally resuspended in 200uL FACS buffer. Cells are analyzed on a flow cytometer for reporter (GFP or mCherry) signal and viability.

[0226] result

[0227] Tandem transfection of NS1 with repRNA improved replicon expression in the 4T1 mouse tumor cell line. 4T1 cells were transfected with Strand-mCherry, mCherry modRNA, and NS1 modRNA mRNA using Lipofectamine MessengerMax. In this tandem transfection, NS1 modRNA was transfected 6 hours prior to transfecting repRNA. This allows the cells an opportunity to be provided with NS1 protein before they experience the replicon. 24 hours after transfection, cells were visualized by microscopy as well as processed by flow cytometry to examine quantitative signals (Figure 2A-2D). Tandem transfection of NS1 from both H5N1 and TX91 with repRNA improved replicon expression (Figure 2E).

[0228] Next, 4T1 cells were transfected with Strand-mCherry, mCherry modRNA, and NS1 modRNA mRNA using TT3 lipid nanoparticles. In this co-transfection, NS1 modRNA was transfected along with repRNA. 24 hours after transfection, cells were visualized by microscope and processed by flow cytometry to examine quantitative signals (Figure 3A-3F). Tandem NS1 expression (Figure 3E) and NS1 co-transfection (Figure 3F) mediated higher MFI from repRNA in 4T1 cells.

[0229] Co-transfection of NS1 with repRNA improves replicon expression in various human cancer cell lines. Hcc38 tumor cells were transfected with NS1 modRNA (Figures 4B and 4F), NS1 repRNA (Figures 4C and 4G), or NS1-P2A-mCherry mRNA (Figures 4D and 4H) using Lipofectamine MessengerMax. In this experiment, NS1 was provided as a modRNA (NS1 modRNA; Figures 4B and 4F) or as a separate (NS1 repRNA; Figures 4C and 4G) or bicistronic repRNA vector (NS1-P2A-mCherry; Figures 4D and 4H). 24 hours (Figures 4A-4D) or 48 hours (Figures 4E-4H) after transfection, cells were visualized by microscopy as well as processed by flow cytometry to examine quantitative signals. Similarly, median fluorescence intensity (MFI) was observed to increase with NS1 expression in Scc9 HNSCC (FIG. 4I) and FaDu HNSCC cells (FIGS. 4J-4K).

[0230] When vector engineering was combined with NS1, additive improvements were observed. BT20 cancer cells were transfected using TT3 LNP with a replicon vector encoding EGFP containing either the original (strand) backbone (Figures 5A-5B) or the Q739L mutation (non-cytopathic; Figures 5C-5D), and cells were analyzed using flow cytometry to quantify reporter signal. When combining NS1 in the Q739L vector, additive improvements were readily apparent (Figures 5A-5F).

[0231] Next, B16.F10 (Figures 6A-6C) or 4T1 (Figures 6D-6F) cells were electroporated with replicons generated using unmodified rNTPs (Unmodified Rep; Figures 6A and 6D) or a ratio of UTP:N1-methyl-pseudo-UTP (indicated in psi) of 1:1 (50%; Figures 6B and 6E) or 1:3 (25%; Figures 6C and 6F). A ratio of 1:1 results in a significant decrease in payload expression, while a ratio of 1:3 maintains expression as well as signal intensity (Figure 6G).

[0232] An additive effect was observed in the combination of vector engineering and the use of modified rNTPs in replicon-driven payload expression (Figure 7). A replicon encoding firefly luciferase (Fluc) was engineered using either unmodified rNTPs (unmod) or a specific rNTP at a 1:3 ratio. [ka] ;1:3 C:5me-C (M2), where C refers to cytidine and 5me-C refers to 5-methyl-cytidine. Luminescence was measured 24 and 48 hours after transfection by electroporation in 4T1 cells (Figure 7). These Fluc replicons were then transfected into an interferon-inducible cell line, B16-ISG (Invivogen), and payload expression was measured in luminescence units (Figure 8A) and type I IFN activity was measured using a SEAP reporter assay using a colorimetric method (Figure 8B). Single (M1) or double modified (M2) replicons showed improved payload expression as well as reduced type I IFN activity.

[0233] The use of modified rNTPs in replicon-driven payload expression improved expression and reduced IFN activation (Figures 9A-9C). B16-ISG cells were transfected with Q739L replicons expressing NS1-EGFP (P2A linker) made using unmodified or single (M1) or doubly modified rNTPs as described above. The doubly modified replicons improve payload expression (Figure 9A), signal intensity (Figure 9B), and reduced type I IFN activity (Figure 9C) compared to poly(I:C)-treated cells. Poly(I:C) is a dsRNA analog and a potent type I IFN agonist.

[0234] LNP delivery in T cells activated for 2 days with an anti-CD3 / CD28 / CD2 cocktail containing high-dose IL-2 was improved by the addition of recombinant protein (enhancer) to the medium, resulting in a robust signal in cells transfected with lipid 2-cholesterol (Figure 10E) or lipid 2-b-sitosterol (Figure 10F) compared to cells transfected with lipid 1 and cells that were not exposed to enhancer (Figures 10A-10D).

[0235] The use of modified rNTPs in replicon-driven payload expression improved expression and reduced IFN activation. Primary human T cells were activated with IL-2 and anti-CD3 / CD28 / CD2 for 2 days after thawing and transfected with the Q739L replicon driving NS1-EGFP, as used above, using unmodified or single (M1) or double modified (M2) mRNA. In addition, unmodified vectors were spiked with 1 or 10% poly(I:C), a dsRNA analog, and co-encapsulated in lipid. 24 hours after transfection, cells were analyzed by flow cytometry to quantify GFP signal and cell supernatants were used to analyze pro-inflammatory cytokines, e.g., IFN-γ. The M2 replicon shows the highest level of expression (Figures 11A-11B), while maintaining the lowest amount of IFN-γ secretion (Figure 11C).

[0236] Human PBMCs were isolated from three healthy donors and grown for 2 days with low dose IL-2 (resting) or high dose IL-2 in the presence of anti-CD3 / CD28 / CD2 cocktail (activating), followed by mRNA:lipid delivery with NS1-EGFP or M2-modified Q739L replicon driving EGFP or conventional EGFP mRNA (EGFP-mod). 24 hours after transfection, cells were stained with live / dead viability stain and analyzed for GFP signal (Figure 12A). Supernatants were collected for cytokine profiling and subjected to ELISA for IFN-α detection. NS1 encoding replicon reduced IFN-α activation in both resting and activated states (Figure 12B). array [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Claims

[Claim 1] The invention described in the present specification.

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