Expression constructs and their use

Abstract

Providing expression constructs and their use. [Solution] This disclosure relates to isolated polynucleotides comprising sequences encoding the interleukin (IL)-12 molecule, and delivery systems capable of carrying the polynucleotides. This disclosure also includes methods for producing polynucleotides and methods for treatment using them. This specification provides isolated polynucleotides comprising nucleic acid molecules encoding the beta subunit of the IL-12 protein ("IL-12β"). This specification also provides isolated polynucleotides comprising nucleic acid molecules encoding the alpha subunit of the IL-12 protein ("IL-12α").

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 135,501, filed on 8 January 2021, which is incorporated herein by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (name: 4597_005PC01_SequenceListing_ST25.txt; size: 246,918 bytes; and creation date: January 9, 2022) in the ASCII text file submitted with this application are incorporated herein by reference in their entirety. [Background technology]

[0003] Background of Disclosure Due to its ability to activate both NK cells and cytotoxic T cells, the IL-12 protein has been studied as a promising anti-cancer drug since 1994. See Nastala, CL et al., J Immunol 153: 1697-1706 (1994). However, Despite high expectations, early clinical studies did not yield satisfactory results. Lasek W. et al., Cancer Immunol Immunother 63: 419-435, 424 (2014). Repeated administration of IL-12 resulted in adaptive responses and a progressive reduction in blood IL-12-induced interferon-gamma (IFN-γ) levels in most patients. Ibid. It was also recognized that IL-12-induced anticancer activity is primarily mediated by secondary secretion of IFNγ, but co-induction of IFN-γ by IL-12 in combination with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG) caused severe toxicity. Ibid. In addition to negative feedback and toxicity, the limited efficacy of IL-12 therapy in clinical settings can be caused by a strongly immunosuppressive environment in humans. Ibid. To minimize the toxicity of IFN-γ and improve the efficacy of IL-12, scientists have tried different approaches, such as different dose and timing protocols for IL-12 therapy. Sacco, S. et al., Blood 90: 4473-4479 (1997); Leonard, JP et al., Blood 90: 2541-2548 (1997);Coughlin, CM et al., Cancer Res. 57: See also 2460-2467 (1997); Asselin-Paturel, C. et al., Cancer 91: 113-122 (2001); and Saudemont, A. et al., Leukemia 16: 1637-1644 (2002). Nevertheless, these approaches did not have a significant impact on patient survival. Kang, WK, et al., Human Gene Therapy 12: 671-684 (2001). Therefore, there is a need in the art for improved therapeutic approaches to treat tumors using IL-12. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nastala, CL et al., J Immunol 153: 1697-1706 (1994) [Non-Patent Document 2] Lasek W. et al., Cancer Immunol Immunother 63: 419-435, 424 (2014) [Non-Patent Document 3] Sacco, S. et al., Blood 90: 4473-4479 (1997)

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Summary of the Invention

Means for Solving the Problems

[0005] Brief Summary of the Present Disclosure This specification provides isolated polynucleotides comprising a nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"). In some embodiments, the nucleic acid molecule comprises at least about 75%, at least about 76%, at least about 77%, and at least about 78% of the sequence shown in SEQ ID NOs. 51, SEQ ID NOs. 52, SEQ ID NOs. 53, SEQ ID NOs. 54, SEQ ID NOs. 55, SEQ ID NOs. 56, SEQ ID NOs. 57, SEQ ID NOs. 58, SEQ ID NOs. 59, SEQ ID NOs. 60, SEQ ID NOs. 61, SEQ ID NOs. 62, SEQ ID NOs. 63, SEQ ID NOs. 64, SEQ ID NOs. 65, SEQ ID NOs. 66, SEQ ID NOs. 67, SEQ ID NOs. 68, SEQ ID NOs. 69, SEQ ID NOs. 70, SEQ ID NOs. 71, SEQ ID NOs. 72, SEQ ID NOs. 73, SEQ ID NOs. 74, or SEQ ID NOs. 75. Contains nucleotide sequences that are identical by at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100%.

[0006] In some embodiments, the nucleic acid molecule encoding IL-12β comprises a nucleotide sequence that is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in 51. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 52. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 54.In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 55. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 56. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 57. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 58. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 59. In some embodiments, the nucleic acid molecule encoding IL-12β comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 65, 69, or 74.In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 66, 70, or 75. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 62. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 99% or 100% identical to the sequence shown in SEQ ID NO: 63. In some embodiments, the nucleic acid molecule encoding IL-12β includes a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 64.

[0007] This specification also provides isolated polynucleotides comprising a nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"). In some embodiments, the nucleic acid molecule comprises at least about 77% of the sequence shown in SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 125, at least Contains nucleotide sequences that are approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100% identical.

[0008] In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 101. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 102. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 103. In some embodiments, the nucleic acid molecule encoding IL-12α comprises a nucleotide sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 104.In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 105. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 106. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 107. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 108. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 109. In some embodiments, the nucleic acid molecule encoding IL-12α comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 115, 119, or 124.In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 116, 120, or 125. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 112. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 113. In some embodiments, the nucleic acid molecule encoding IL-12α includes a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 114.

[0009] This disclosure further relates to an isolated polynucleotide comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes the beta subunit of the IL-12 protein ("IL-12β") and the sequence shown in SEQ ID NOs. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. and containing nucleotide sequences that are identical by at least approximately 75%, at least approximately 76%, at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100%;The second nucleic acid molecule encodes the alpha subunit of the IL-12 protein ("IL-12α") and is at least approximately 77% of the sequence shown in SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125. Provides isolated polynucleotides containing nucleotide sequences that are identical by at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100%.

[0010] In some embodiments, the first nucleic acid molecule is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 51. A second nucleic acid molecule containing a nucleotide sequence and / or containing a nucleotide sequence that is at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 101.In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 52. and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 102.In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 53. and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 103. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 54, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 104.In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 55, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 105. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 56, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 106. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 57, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 107.In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 58, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 108. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 59, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 109. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 65, 69, or 74, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 115, 119, or 124.In some embodiments, the first nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 66, 70, or 75, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 116, 120, or 125. In some embodiments, the first nucleic acid molecule contains a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 62, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 112. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 99% or 100% identical to the sequence shown in SEQ ID NO: 63, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 113. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 64, and / or the second nucleic acid molecule comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 114.

[0011] In some embodiments, the isolated polynucleotides disclosed herein further comprise a third nucleic acid molecule encoding a linker that connects a first nucleic acid molecule and a second nucleic acid molecule. In certain embodiments, the linker comprises an amino acid linker having at least about 2, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acids. In some embodiments, the linker comprises a (GS) linker. In some embodiments, the (GS) linker has the formula (Gly3Ser)n or S(Gly3Ser)n, where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the (Gly3Ser)n linker is (Gly3Ser)3 or (Gly3Ser)4. In certain embodiments, the third nucleic acid molecule encoding the linker contains the sequence shown in any one of sequence numbers 168-170.

[0012] In some embodiments, the isolated polynucleotides of the Disclosure further comprise additional nucleic acid molecules encoding half-life extension moieties. In certain embodiments, the half-life extension moieties comprise Fc, albumin or a fragment thereof, an albumin-binding moiety, PAS, HAP, transferrin or a fragment thereof, XTEN, or any combination thereof.

[0013] In some embodiments, the isolated polynucleotides described herein further comprise additional nucleic acid molecules encoding a leader sequence. In certain embodiments, the additional nucleic acid molecules encoding a leader sequence comprise one of the sequences shown in SEQ ID NOs. 26-50.

[0014] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 26; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 51; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 76; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 101; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 126; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 147.

[0015] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 27; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 52; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 77; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 102; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 127; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 148.

[0016] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. Isolated polynucleotides are provided, comprising: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 28; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 53; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 78; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 103; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 128; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 149.

[0017] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. Isolated polynucleotides are provided, comprising: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 29; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 54; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 79; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 104; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 129; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 150.

[0018] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 30; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 55; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 80; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 105; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 130; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 151.

[0019] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 31; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 56; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 81; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 106; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 131; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 152.

[0020] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 32; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 57; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 82; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 107; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 132; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 153.

[0021] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. Isolated polynucleotides are provided, comprising: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 33; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 58; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 83; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 108; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 133; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 154.

[0022] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. Isolated polynucleotides are provided, comprising: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 34; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 59; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 84; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 109; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 134; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 155.

[0023] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 37; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 62; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 87; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 112; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 137; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 158.

[0024] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 38; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 63; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 88; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 113; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 138; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 159.

[0025] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 39; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 64; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 89; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 114; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 139; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 160.

[0026] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 44; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 69; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 94; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 119; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 140; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 161.

[0027] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 45; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 70; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 95; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 120; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 141; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 162.

[0028] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 46; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 71; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 96; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 121; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 142; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 163.

[0029] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 47; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 72; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 97; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 122; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 143; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 164.

[0030] In this specification, (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. The provided isolated polynucleotides include: (a) a first nucleic acid molecule comprising the sequence shown in SEQ ID NO: 36; (b) a second nucleic acid molecule comprising the sequence shown in SEQ ID NO: 61; (c) a third nucleic acid molecule comprising the sequence shown in SEQ ID NO: 86; (d) a fourth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 111; (e) a fifth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 136; and (f) a sixth nucleic acid molecule comprising the sequence shown in SEQ ID NO: 157.

[0031] This specification describes a single nucleic acid molecule comprising (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, (vi) a sixth nucleic acid molecule encoding human serum albumin, (vii) a seventh nucleic acid molecule encoding the third linker, and (viii) an eighth nucleic acid molecule encoding lumican protein. Isolated polynucleotides are provided, wherein (a) a first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 48; (b) a second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 73; (c) a third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 98; (d) a fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 123; (e) a fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 144; (f) a sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 165; (g) a seventh nucleic acid molecule comprises the sequence shown in SEQ ID NO: 168; and (h) an eighth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 171.

[0032] This specification describes a single nucleic acid molecule comprising (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, (vi) a sixth nucleic acid molecule encoding human serum albumin, (vii) a seventh nucleic acid molecule encoding the third linker, and (viii) an eighth nucleic acid molecule encoding lumican protein. Isolated polynucleotides are provided, wherein (a) a first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 49; (b) a second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 74; (c) a third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 99; (d) a fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 124; (e) a fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 145; (f) a sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 166; (g) a seventh nucleic acid molecule comprises the sequence shown in SEQ ID NO: 169; and (h) an eighth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 172.

[0033] This specification describes a single nucleic acid molecule comprising (5' to 3') (i) a first nucleic acid molecule encoding the leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding the first linker, (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding the second linker, (vi) a sixth nucleic acid molecule encoding human serum albumin, (vii) a seventh nucleic acid molecule encoding the third linker, and (viii) an eighth nucleic acid molecule encoding lumican protein. Isolated polynucleotides are provided, wherein (a) a first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 50; (b) a second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 75; (c) a third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 100; (d) a fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 125; (e) a fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 146; (f) a sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 167; (g) a seventh nucleic acid molecule comprises the sequence shown in SEQ ID NO: 170; and (h) an eighth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 173.

[0034] This specification provides an isolated polynucleotide comprising (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 40; (b) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 65; (c) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 90; and (d) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 115.

[0035] This specification provides an isolated polynucleotide comprising (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 41; (b) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 66; (c) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 91; and (d) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 116.

[0036] This specification provides an isolated polynucleotide comprising (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 42; (b) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 67; (c) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 92; and (d) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 117.

[0037] As used herein, an isolated polynucleotide comprising, from 5' to 3', (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding the beta subunit of IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding the alpha subunit of IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 43; (b) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 68; (c) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 93; (d) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 118, is provided.

[0038] In some embodiments, the isolated polynucleotides described herein further comprise a 5'-cap. In certain embodiments, the 5'-cap is m2 7,2’-O Gpp s pGRNA, m 7 GpppG, m 7 Gppppm 7 G, m2 (7,3’-O) GpppG, m2 (7,2’-O) GppspG(D1), m2 (7,2’-O) GppspG(D2), m2 7,3’-O Gppp(m1 2’-O )ApG, (m 7 G-3'mppp-G; which can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G), N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G, N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G, N7-(4-chlorophenoxyethyl)-m 3’-OThe following are selected from the group consisting of G(5')ppp(5')G, 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2 mp, m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N1-methylpsoiduridine, m7G(5')ppp(5')(2'OMeA)pG, and combinations thereof.

[0039] In some embodiments, the isolated polynucleotides of the present disclosure further comprise a regulatory element. In certain embodiments, the regulatory element is selected from the group consisting of at least one translational enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or its seed, a 3' tailing region of a ligated nucleoside, an AU-rich element (ARE), a post-transcriptional regulatory modulator, and combinations thereof.

[0040] In some embodiments, the isolated polynucleotides described herein further comprise a 3' tailing region of a ligated nucleoside. In certain embodiments, the 3' tailing region of the ligated nucleoside comprises a poly-A tail, a poly-AG quartet, or a stem-loop sequence.

[0041] In some embodiments, the isolated polynucleotides of the present disclosure comprise at least one modified nucleoside. In certain embodiments, the at least one modified nucleoside is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-psoidouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-gu The following are selected from the group consisting of anosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolocytidine, 5-methylcytidine, N4-acetylcytidine, 5-methyluridine, 5-iodocytidine, and combinations thereof.

[0042] In some embodiments, the isolated polynucleotides described herein are capable of self-replication. In certain embodiments, the polynucleotide is a self-amplifying replicon RNA. In some embodiments, the self-amplifying replicon RNA is derived from an alphavirus. In certain embodiments, the alphavirus includes Venezuelan encephalitis virus, Semliki forest virus, Sindbis virus, or a combination thereof.

[0043] This disclosure further provides vectors comprising any of the isolated polynucleotides described herein.

[0044] This disclosure also provides (i) any of the isolated polynucleotides described herein, and (ii) lipid nanoparticles (LNPs) comprising one or more lipids. In certain embodiments, one or more lipids comprises cationic lipids. In some embodiments, the lipids are ionizable lipids. In some embodiments, the lipids are lipidoids, e.g., N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3). In some embodiments, the LNPs comprise 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, C14-PEG2000, or any combination thereof.

[0045] In some embodiments, the LNPs described herein have a diameter of about 30 to 500 nm. In certain embodiments, the LNPs have a diameter of about 50 to 400 nm. In some embodiments, the LNPs have a diameter of about 70 to 300 nm. In some embodiments, the LNPs have a diameter of about 100 to 200 nm. In some embodiments, the LNPs have a diameter of about 100 to 175 nm. In some embodiments, the LNPs have a diameter of about 100 to 160 nm.

[0046] In some embodiments, the lipid and the isolated polynucleotide (e.g., modified RNA) have a mass ratio of about 1:2 to about 2:1. In some embodiments, the lipid and the isolated polynucleotide (e.g., modified RNA) have a mass ratio of 1:2, 1:1.5, 1:1.2, 1:1.1, 1:1, 1.1:1, 1.2:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1. In some embodiments, the lipids and isolated polynucleotides (e.g., modified RNA) have a mass ratio of approximately 10:1.

[0047] Pharmaceutical compositions comprising any of the isolated polynucleotides, vectors, or LNPs described herein, and a pharmaceutically acceptable carrier are provided herein. In certain embodiments, the pharmaceutical compositions are formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, lymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, percutaneous, or transmucosal administration.

[0048] Cells comprising any of the isolated polynucleotides, vectors, or LNPs described herein are provided herein. In some embodiments, the cells are in vitro cells, ex vivo cells, or in vivo cells.

[0049] A method for producing polynucleotides is provided herein, comprising enzymatically or chemically synthesizing any of the isolated polynucleotides described herein. A method for producing IL-12 protein is also provided herein, comprising contacting cells with any of the isolated polynucleotides, cells, or LNPs described herein. In certain embodiments, the contact occurs in vivo or ex vivo.

[0050] This disclosure further provides methods for treating a disease or disorder in a subject that requires it, comprising administering one of the isolated polynucleotides, vectors, LNPs, or pharmaceutical compositions described herein to the subject. In certain embodiments, the disease or disorder includes cancer. In some embodiments, cancer includes melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatome, 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, head and neck cancer, or combinations thereof.

[0051] In some embodiments, a method for treating a disease or disorder disclosed herein further includes administering at least one additional therapeutic agent. In certain embodiments, the at least one additional therapeutic agent includes chemotherapeutic agents, targeted anticancer therapies, oncolytic agents, cytotoxic agents, immunotherapy, cytokines, surgical procedures, radiotherapy, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some embodiments, the immune checkpoint inhibitor includes anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CTLA-4 antibodies, anti-GITR antibodies, anti-TIM3 antibodies, or any combination thereof. [Brief explanation of the drawing]

[0052] [Figure 1A-1B] Figures 1A and 1B provide a comparison of tumor volumes in tumor-carrying mice treated with a single intratumor administration of one of the following: (i) PBS (control; white circles); (ii) repRNA capped contemporaneously with a 5' cap analog (black circles in Figure 1A; solid lines in Figure 1B); and (iii) repRNA capped post-transcriptionally by enzymatic addition of a 5' cap (black squares in Figure 1A; dashed lines in Figure 1B). Tumor volume was measured at various time points after administration (x-axis). Figure 1A shows the mean tumor volume. Figure 1B shows the tumor volume of individual animals.

[0053] [Figure 2] Figure 2 provides a comparison of IL-12 protein expression in mouse tumors treated with one of the following: (i) PBS (control; first column from the left); (ii) repRNA prepared using A3G+E1 vector and cap analog co-transcription capping (second column); (iii) modified mRNA (non-self-replicating) prepared using cap analog co-transcription capping (third column); (iv) repRNA prepared using alternative evolutionary vector and cap analog co-transcription capping (fourth column); and (v) repRNA prepared using alternative evolutionary vector and enzymatic post-translational capping (last column).

[0054] [Figure 3] Figure 3 provides a comparison of Kaplan-Meier survival analyses of tumor-carrying mice treated with one of the following single intratumoral administrations: (i) PBS (control; white circle); (ii) repRNA prepared using A3G+E1 vector and cap analog co-transcription capping (white triangle); (iii) modified mRNA (non-self-replicating) prepared using cap analog co-transcription capping (white square); (iv) repRNA prepared using alternative evolutionary vector and cap analog co-transcription capping (black circle); and (v) repRNA prepared using alternative evolutionary vector and enzymatic post-translational capping (black square).

[0055] [Figure 4A]Figure 4A provides a comparison of the in vitro transfection efficiencies of the different RNA constructs described herein, as measured using FACS analysis. Figure 4B provides a comparison of the concentrations of IL-12 protein detected in the supernatant of cells transfected with the different RNA constructs described herein. In both Figures 4A and 4B, the RNA constructs tested included: (i) repRNA prepared using the A3G+E1 vector and simultaneous transcription capping of a cap analog (black circle); (ii) repRNA prepared using the A3G+E1 vector and post-translational capping (black square); (iii) repRNA prepared using the Alternative Evolution+E1 vector and simultaneous transcription capping of a cap analog (black triangle); (iv) repRNA prepared using the Alternative Evolution+E1 vector and post-translational capping (black inverted triangle); (v) repRNA prepared using the Alternative+E1 vector and simultaneous transcription capping of a cap analog (diamond); (vi) repRNA prepared using the A3G+E1 evolution vector and simultaneous transcription capping of a cap analog (white circle); (vii) repRNA prepared using the A3G-E1 vector and simultaneous transcription capping of a cap analog (white square); and (viii) repRNA prepared using the Alternative Evolution-E1 SGP wound-end vector (white triangle). Transfection efficiency is shown as the frequency of IL-12+ cells observed in different groups. The x-axis represents the time after transfection when transfection efficiency was evaluated. [Figure 4B]Figure 4A provides a comparison of the in vitro transfection efficiencies of the different RNA constructs described herein, as measured using FACS analysis. Figure 4B provides a comparison of the concentrations of IL-12 protein detected in the supernatant of cells transfected with the different RNA constructs described herein. In both Figures 4A and 4B, the RNA constructs tested included: (i) repRNA prepared using the A3G+E1 vector and simultaneous transcription capping of a cap analog (black circle); (ii) repRNA prepared using the A3G+E1 vector and post-translational capping (black square); (iii) repRNA prepared using the Alternative Evolution+E1 vector and simultaneous transcription capping of a cap analog (black triangle); (iv) repRNA prepared using the Alternative Evolution+E1 vector and post-translational capping (black inverted triangle); (v) repRNA prepared using the Alternative+E1 vector and simultaneous transcription capping of a cap analog (diamond); (vi) repRNA prepared using the A3G+E1 evolution vector and simultaneous transcription capping of a cap analog (white circle); (vii) repRNA prepared using the A3G-E1 vector and simultaneous transcription capping of a cap analog (white square); and (viii) repRNA prepared using the Alternative Evolution-E1 SGP wound-end vector (white triangle). Transfection efficiency is shown as the frequency of IL-12+ cells observed in different groups. The x-axis represents the time after transfection when transfection efficiency was evaluated.

[0056] [Figure 5]Figure 5 provides a comparison of the in vitro transfection efficiencies of the following RNA constructs, measured using FACS analysis: (i) repRNA prepared using the cap analog co-transcription capping method and an A3G+E1 vector with a restriction enzyme wound at the 3' end (round); (ii) repRNA prepared using the cap analog (alternative origin) method and an A3G+E1 vector with a restriction enzyme wound at the 3' end (square); (iii) repRNA prepared using the cap analog co-transcription capping method and an A3G+E1 vector with an intact poly(A) sequence at the 3' end (triangle); (iv) repRNA prepared using the cap analog (alternative origin) method and an A3G+E1 vector with an intact poly(A) sequence at the 3' end (inverted triangle). Cells treated with PBS were used as a control (diamond). Transfection efficiency is shown as the frequency of IL-12+ cells observed in different groups. The x-axis represents the time after transfection when transfection efficiency was assessed.

[0057] [Figure 6A] Figures 6A and 6B provide a comparison of IL-12 protein concentrations observed in tumors and serum of tumor-carrying mice treated with a single intratumoral administration of an RNA construct encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. Figure 6A shows the concentrations of IL-12 protein in tumors (upper graph) and serum (lower graph) 24 hours after administration. Figure 6B shows the concentrations of IL-12 protein in tumors (upper graph) and serum (lower graph) 96 hours after administration. [Figure 6B]Figures 6A and 6B provide a comparison of IL-12 protein concentrations observed in tumors and serum of tumor-carrying mice treated with a single intratumoral administration of an RNA construct encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. Figure 6A shows the concentrations of IL-12 protein in tumors (upper graph) and serum (lower graph) 24 hours after administration. Figure 6B shows the concentrations of IL-12 protein in tumors (upper graph) and serum (lower graph) 96 hours after administration.

[0058] [Figure 7A] Figures 7A, 7B, and 7C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 24 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. [Figure 7B]Figures 7A, 7B, and 7C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 24 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. [Figure 7C] Figures 7A, 7B, and 7C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 24 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis.

[0059] [Figure 8A] Figures 8A, 8B, and 8C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 96 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. [Figure 8B]Figures 8A, 8B, and 8C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 96 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis. [Figure 8C] Figures 8A, 8B, and 8C provide a comparison of IL-12 protein concentrations observed in the spleen, aspiration lymph nodes, and non-aspiration lymph nodes, respectively, 96 hours after a single intratumoral administration of RNA constructs encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone ("IL-12"); (ii) mIL-12 conjugated to albumin ("IL-12-alb"); and (iii) mIL-12 conjugated to albumin and lumican ("IL-12-alb-lum"). Different RNA constructs were administered to animals at one of two doses shown along the x-axis.

[0060] [Figure 9A-9B] Figures 9A and 9B provide a comparison of IL-12 and IFN-γ protein concentrations, respectively, measured in the serum of tumor-carrying mice treated with a single intratumoral administration of an RNA construct encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone (triangle); (ii) mIL-12 conjugated to albumin (square); and (iii) mIL-12 conjugated to albumin and lumican (circle). The RNA constructs were administered to the animals at the following doses: 0.25 μg (black symbol) or 2.5 μg (white symbol). The x-axis shows the time point (after administration) at which the concentrations of IL-12 and IFN-γ were measured.

[0061] [Figure 10] Figure 10 shows a graphical representation of data regarding optimality (mean codon score) versus minimum folding free energy (kcal / mol) (MFE) for 1137 distinct sequences encoding human light chain leader-hIL12p40-GGS(GGGS)3 linker-hIL12p35-GSGGGS linker-human serum albumin, according to Example 5. L1, L2, L3, M1, M2, M3, H1, H2, and H3 codon-optimized constructs are shown. As described in Example 5, diamond symbols represent sequences with very high codon optimality and MFE. Triangles represent codon-optimized sequences containing the most frequently used triplets at each amino acid position.

[0062] [Figure 11] Figure 11 provides a comparison of IL-12 protein secretion observed in the supernatant of cells transfected with different RNA constructs (x axis) containing codon-optimized IL-12 sequences. As shown, IL-12 in constructs A1–A4 was not conjugated to any other part. IL-12 in constructs B1–B4 was conjugated to albumin. IL-12 in constructs C1–C3 was conjugated to albumin and lumican. Individual triplicate measurements are shown as points on triangles, squares, octagons, or circles, and the horizontal bar shows the average of the triplicate measurements. The x-axis represents the variant used, and the y-axis represents the concentration of IL-12 (ng / ml).

[0063] [Figures 12A-12B]Figures 12A, 12B, and 12C provide a comparison of IL-12 protein concentrations observed in tumors (Figure 12A), serum (Figure 12B), and spleen (Figure 12C) of tumor-carrying mice that received a single intratumoral administration of RNA constructs containing (i) codon-optimized IL-12 sequences conjugated to albumin and lumican (the first set of circles from the left in each of PDX1, PDX2, and PDX3); (ii) codon-optimized IL-12 sequences conjugated to albumin (the second set of circles in each of PDX1, PDX2, and PDX3); or (iii) codon-optimized IL-12 sequences alone (the third set of circles in each of PDX1, PDX2, and PDX3). Untreated animals were used as controls (the last set of circles in each of PDX1, PDX2, and PDX3). "PDX1," "PDX2," and "PDX3" represent xenografts derived from tumor resections from three individual patients with triple-negative breast cancer (TNBC). PDX1 and PDX3 were established from primary resected ER, PR, HER2-negative lesions, while PDX2 was established from metastatic ER, PR, HER2-negative lesions in the lungs of TNBC patients. [Figure 12C]Figures 12A, 12B, and 12C provide a comparison of IL-12 protein concentrations observed in tumors (Figure 12A), serum (Figure 12B), and spleen (Figure 12C) of tumor-carrying mice that received a single intratumoral administration of RNA constructs containing (i) codon-optimized IL-12 sequences conjugated to albumin and lumican (the first set of circles from the left in each of PDX1, PDX2, and PDX3); (ii) codon-optimized IL-12 sequences conjugated to albumin (the second set of circles in each of PDX1, PDX2, and PDX3); or (iii) codon-optimized IL-12 sequences alone (the third set of circles in each of PDX1, PDX2, and PDX3). Untreated animals were used as controls (the last set of circles in each of PDX1, PDX2, and PDX3). "PDX1," "PDX2," and "PDX3" represent xenografts derived from tumor resections from three individual patients with triple-negative breast cancer (TNBC). PDX1 and PDX3 were established from primary resected ER, PR, HER2-negative lesions, while PDX2 was established from metastatic ER, PR, HER2-negative lesions in the lungs of TNBC patients.

[0064] [Figure 13] Figure 13 provides a comparison of tumor volume in TNBC mice treated with vehicle control (white square) or IL-12 encoding repRNA (repRNA) (weekly for a total of four doses). The repRNA was administered to the animals in one of the following doses: (i) 5 μg (black circle), (ii) 0.5 μg (black square), or (iii) 0.05 μg (black triangle).

[0065] [Figure 14]Figure 14 provides a comparison of tumor volumes in TNBC mice treated with rIL-12 coding repRNA modified to contain different amounts of modified nucleoside triphosphate (modNTP): (i) 0% modNTP (i.e., no modified repRNA) (black circle); (ii) 25% modNTP (black square); (iii) 37.5% modNTP (black triangle); or (iv) 50% modNTP (black inverted triangle). Animals treated with vehicle controls were used as controls (white square).

[0066] [Figures 15A-15B] Figures 15A and 15B show the effects of the repRNA constructs described herein on both treated and untreated tumors, respectively, in a B16-F10 mouse syngeneic cancer model. B16-F10 tumor cells were subcutaneously injected into the left and right flanks of animals, as further described in Example 8. Once the optimal tumor size was reached, either a vehicle control or repRNA (2.5 μg) was administered intratumorally into the left flank (i.e., treated tumor). Tumor volume was then assessed in both the left and right flanks (i.e., untreated tumors).

[0067] [Figure 16] Figure 16 provides a comparison of luciferase expression (shown as a bioluminescent signal) in a TNBC mouse model after remedication. As further described in Example 9, mice were injected with an initial dose of one of two firefly luciferase-coding repRNA constructs (mRNA1 and mRNA2) (5 μg) ("Dose 1"), and then, one week later, a second dose of the same repRNA construct (5 μg) was injected ("Dose 2"). Some animals received additional weekly doses (a total of two doses) of anti-IFNAR1 antibody (10 mg / kg).

[0068] [Figure 17]Figure 17 provides a comparison of IFN-γ concentrations in supernatants collected from activated human PBMCs treated with recombinant human IL-12 protein (rhIL12) or the conditional medium described in Example 10 (i.e., supernatant collected from BT20 cells transfected with IL-12-coding repRNA). PBMCs were treated with one of the following concentrations of rhIL12: 100 ng / mL ("3"), 10 ng / mL ("4"), 1 ng / mL ("5"), 0.1 ng / mL ("6"), or 0.01 ng / mL ("7"). The conditional mediums contained one of the following amounts of IL-12: 1 ng / mL ("1") and 10 ng / mL ("2"). Untreated cells were used as a control ("8"). [Modes for carrying out the invention]

[0069] Detailed explanation of disclosure definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which this disclosure pertains. In case of any conflict, the definitions provided in this application shall prevail. Unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0070] Throughout this disclosure, the terms “a” or “an” entity refer to one or more such entities; for example, “a polynucleotide” is understood to represent one or more polynucleotides. Thus, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably as herein.

[0071] Furthermore, when used herein, “and / or” should be taken as a specific disclosure of each of the two identified characteristics or components, with or without the other. Thus, when the term “and / or” is used herein in phrases such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term “and / or” is used in phrases such as “A, B, and / or C,” it is intended to include the following embodiments: 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 (alone); B (alone); and C (alone).

[0072] The term “approximately” is used herein to mean roughly, about, approximately, or within a range. When the term “approximately” is used in conjunction with a numerical range, it modifies that range by extending the upper and lower boundaries of the numerical value being expressed. Generally, unless otherwise indicated herein, the term “approximately” is used to modify a numerical value above and below a stated value by an upper or lower (higher or lower) 10 percent variation.

[0073] The term "at least" preceding a number or a set of numbers is understood to include the number adjacent to the term "at least," as well as all subsequent numbers or integers that may logically be included, where the context makes it clear. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a set of numbers or a range, it is understood that "at least" can modify each of the numbers in the set or range. "At least" is also not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, and 5.18%, without considering significant figures).

[0074] As used herein, “polynucleotide” or “nucleic acid” means a sequence of nucleotides linked by phosphodiester linkages. Polynucleotides exist in the 5'-to-3' orientation as herein. The polynucleotides of this disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0075] As used herein, the term “polypeptide” encompasses both peptides and proteins unless otherwise specified.

[0076] The terms “coding sequence” or “coding” sequence are used herein to mean a DNA or RNA region (transcribed region) that “codes” a particular protein, such as IL-12. When controlled by an appropriate regulatory region, such as a promoter, coding sequences are transcribed (DNA) and translated (RNA) into polypeptides in vitro or in vivo. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Examples of coding sequences, but not limited to, include prokaryotic or eukaryotic cDNA, prokaryotic or eukaryotic genomic DNA, and synthetic DNA sequences. A transcription stop sequence may be located 3' relative to the coding sequence.

[0077] The Kosack consensus sequence, Kosack consensus, or Kosack sequence is a known sequence found in eukaryotic mRNA, having a consensus (gcc)gccRccAUGG (SEQ ID NO: 174) (where R is a purine (adenine or guanine)) three bases upstream of the start codon (AUG), followed by another "G". In some embodiments, the polynucleotide comprises a nucleic acid sequence having at least about 95% or higher, e.g., at least about 99%, sequence identity with the Kosack consensus sequence. In some embodiments, the polynucleotide comprises the Kosack consensus sequence.

[0078] The term “RNA” is used herein to mean a molecule containing at least one ribonucleotide residue. “Ribonucleotide” refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, e.g., modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. The term “mRNA” refers to “messenger RNA” and refers to a “transcript” produced by using a DNA template that encodes a peptide or protein. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro. In the context of this disclosure, mRNA can be produced by in vitro transcription from a DNA template. Methodologies for in vitro transcription are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits. In some aspects of this disclosure, RNA, preferably mRNA, is modified with a 5'-cap structure.

[0079] The term “sequence identity” is used herein to mean the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing sequences. In certain embodiments, sequence identity is calculated based on the total length or portion thereof of two given sequence numbers. That portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or any other specified percentage of both sequence numbers. The term “identity” may also, in some cases, mean the degree of sequence relationship between amino acid or nucleic acid sequences, as determined by the matching of strings of such sequences.

[0080] In certain embodiments, methods for determining identity are designed to give the greatest match between sequences being tested. Methods for determining identity and similarity are systematized in publicly available computer programs.

[0081] "Substantial homology" or "substantial similarity," when referring to nucleic acids or fragments thereof, means that at least approximately 95–99% of the nucleotide sequence identity exists when optimally aligned with appropriate nucleotide insertions or deletions by another nucleic acid (or its complementary strand).

[0082] As used herein, the terms “effective amount,” “therapeutic effective amount,” and “sufficient amount” for compositions comprising polynucleotides disclosed herein refer to an amount sufficient to produce beneficial or desired results, including clinical outcomes, when administered to a subject including humans, and for example, “effective amount” or its synonyms depend on the context in which it is applied.

[0083] The amount of a given therapeutic agent or composition varies depending on various factors, such as the given drug, pharmaceutical formulation, route of administration, type of disease or disorder, the subject being treated (e.g., age, sex, and / or weight), or the host's identity.

[0084] The term “half-life” refers to the time required for half of the activity, quantity, or number of a molecule to be eliminated. In the context of this disclosure, the half-life of RNA represents the stability of said RNA. “Onset” or “progression” of a disease means the initial symptom onset and / or subsequent progression of the disease. The onset of a disease can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression, which may be unpredictable. As used herein, onset or progression refers to the biological course of symptoms. Onset includes appearance, recurrence, and initiation. As used herein, the initiation or appearance of a target disease or disorder includes initial onset and / or recurrence.

[0085] IL-12 expressing nucleotides This disclosure pertains to polynucleotides (e.g., isolated polynucleotides) including nucleic acid molecules encoding the IL-12 protein. Where described herein, the polynucleotides disclosed herein possess one or more properties that result in them being different from naturally occurring reference polynucleotides (e.g., structurally and / or functionally). For example, as further described elsewhere in this disclosure, the nucleic acid molecules encoding the IL-12 protein (e.g., the IL-12α subunit and / or the IL-12β subunit) are codon-optimized (i.e., synthetic).

[0086] In some embodiments, the nucleotide sequence encoding IL-12 includes a translateable region and one, two, or more modifications. In some embodiments, the nucleotide sequence encoding IL-12 exhibits reduced degradation in the cell into which the nucleic acid is introduced compared to the corresponding unmodified nucleic acid.

[0087] In some embodiments, the modification may be located on the sugar portion of the nucleotide. In some embodiments, the modification may be located on the phosphate backbone of the nucleotide.

[0088] In some embodiments, for example, when precise timing of protein production is desired, it is desirable that the modified nucleic acid introduced into the cell be degraded within the cell. Therefore, in some embodiments, the nucleotide sequence encoding IL-12 includes a degradation domain, which can act in a directed manner within the cell.

[0089] In some embodiments, the nucleotide sequence encoding IL-12 includes at least one of a modified 5'-cap, a half-life extension moiety, or a regulatory element.

[0090] In some embodiments, the modified 5'-cap increases RNA stability, increases RNA translation efficiency, prolongs RNA translation, and increases total RNA protein expression compared to the same RNA without the 5'-cap structure.

[0091] In some embodiments, the nucleotide sequence encoding IL-12 is cyclized or concatemerized to generate a translation-ready molecule that facilitates the interaction between the poly(A) binding protein and the 5' end binding protein. The mechanism of cyclization or concatemerization can occur through at least three different pathways: 1) a chemical pathway, 2) an enzymatic pathway, and 3) a ribozyme-catalyzed pathway. The newly formed 5' / 3' linkage can be intramolecular or intermolecular.

[0092] In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5' and 3' ends of the molecule. The 5' end may contain an NHS-ester reactive group, and the 3' end may contain a 3'-amino-terminal nucleotide, so that in an organic solvent, the 3'-amino-terminal nucleotide on the 3' end of the synthetic mRNA molecule undergoes nucleophilic attack at the 5'-NHS-ester moiety to form a new 5' / 3' amide bond.

[0093] In a second pathway, a T4 RNA ligase can be used to enzymatically link a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of the nucleic acid, forming a new phosphorodiester linkage. In an example reaction, 1 μg of nucleic acid molecule is incubated at 37°C for 1 hour with 1–10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. The ligation reaction may occur in the presence of fragmented oligonucleotides that can base-pair with both the juxtaposed 5' and 3' regions to support the enzymatic ligation reaction.

[0094] In the third pathway, either the 5' or 3' end of the cDNA template encodes a ligase ribozyme sequence such that, during in vitro transcription, the resulting nucleic acid molecule may contain an active ribozyme sequence capable of ligating the 5' end of the nucleic acid molecule to the 3' end of the nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, a group I intron, a delta hepatitis virus, a hairpin ribozyme, or it may be selected by SELEX (phylogenetic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at a temperature of 0 to 37°C.

[0095] In some embodiments, multiple distinct nucleic acids, IL-12 encoding nucleotide sequences, or primary constructs may be ligated together through their 3' ends using nucleotides modified at the 3' ends. Chemical conjugation can be used to control the stoichiometry of delivery to cells. For example, isocitrate lyase and malate synthase, enzymes of the glyoxylate cycle, can be supplied to HepG2 cells in a 1:1 ratio to alter the cellular fatty acid metabolism. This ratio can be controlled by chemically ligating the nucleic acids or modified RNAs using a 3'-azide terminal nucleotide on one nucleic acid or modified RNA species, as well as a C5-ethynyl or alkynyl-containing nucleotide on the opposite nucleic acid or IL-12 encoding nucleotide sequence species. The IL-12 encoding nucleotide sequences are post-transcribed using terminal transferase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. Following the addition of a 3'-modified nucleotide, the two nucleic acids or nucleotide sequences encoding IL-12 can be joined in aqueous solution, in or without copper, to form a new covalent linkage via click chemistry mechanisms described in the literature.

[0096] In some embodiments, more than two polynucleotides can be linked together using a functionalization linker molecule. For example, a functionalization saccharide molecule can be chemically modified to contain multiple chemically reactive groups (SH-, NH2-, N3, etc.) that react with the congeneral moiety on a 3'-functionalized mRNA molecule (i.e., 3'-maleimide ester, 3'-NHS- ester, alkynyl). The number of reactive groups on the modified saccharide can be controlled in a stoichiometric manner, allowing for direct control of the stoichiometric ratio of the conjugated nucleic acid or mRNA.

[0097] In some embodiments, to further enhance protein production, the nucleotide sequence, polynucleotide, or primary construct encoding IL-12 of the Disclosure may be combined with other polynucleotides, dyes, inserts (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaffyrin, sapphyrin), or polycyclic compounds. Aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins (e.g., glycoproteins), peptides (e.g., molecules with specific affinity for coligands), antibodies (e.g., antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes), hormones and hormone receptors, non-peptide species (e.g., lipids, lectins, carbohydrates, vitamins, cofactors), or drugs can be designed to be conjugated.

[0098] Conjugation can result in increased stability and / or half-life, and may be particularly useful for targeting IL-12-encoding nucleotide sequences or primary constructs to specific sites in cells, tissues, or organisms.

[0099] In some embodiments, a primary construct is designed to encode one or more polypeptides or fragments thereof of interest. Polypeptides of interest may include, but are not limited to, an entire polypeptide, multiple polypeptides or polypeptide fragments that can be independently encoded by one or more nucleic acids, multiple nucleic acids, or fragments or variants of any of the aforementioned nucleic acids. As used herein, the term “polypeptide of interest” means any polypeptide selected to be encoded in the primary construct of this disclosure. As used herein, “polypeptide” means, in most cases, a polymer of amino acid residues (natural or unnatural) linked together by peptide bonds. As used herein, the terms refer to proteins, polypeptides, and peptides of any size, structure, or function. In some examples, the encoded polypeptide is smaller than about 50 amino acids, and as a result, the polypeptide is referred to as a peptide. If a polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues long. Polypeptides, therefore, include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and the aforementioned analogs. Polypeptides can be single molecules or complexes of multiple molecules, such as dimers, trimers, or tetramers. They can also be single-chain or multi-chain polypeptides, such as antibodies or insulin, and may be associated or linked. The most common disulfide linkages are found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers, in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0100] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from that of the native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least about 50% identity (homology) with the native or reference sequence, and preferably they are at least about 80%, more preferably at least 90% identical (homologous) to the native or reference sequence.

[0101] Accordingly, polynucleotides encoding a polypeptide of interest, including substitutions, insertions and / or additions, deletions, and covalent modifications with respect to a reference sequence, are included within the scope of this disclosure. For example, a sequence tag or amino acids, such as one or more lysines, can be added to the peptide sequence of this disclosure (e.g., at the N-terminus or C-terminus). Sequence tags can be used for the purification or localization of the peptide. Lysines can be used to increase the solubility of the peptide or to enable biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein can be deleted as needed to provide a truncated sequence. Alternatively, certain amino acids (e.g., C-terminal or N-terminal residues) can be deleted depending on the use of the sequence, for example, whether soluble or as part of a larger sequence linked to a solid support.

[0102] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides for the purposes of this disclosure. For example, any protein fragment of a reference protein longer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 100 amino acid lengths (meaning a polypeptide sequence that is otherwise identical but at least one amino acid residue shorter than the reference polypeptide sequence) is provided herein. In another example, any protein containing a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids that is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized pursuant to this disclosure. In certain embodiments, the polypeptides utilized pursuant to this disclosure contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, as shown in any of the sequences provided or referenced herein.

[0103] In some embodiments, the nucleotide sequence encoding IL-12 includes a modified 5'-cap, a half-life extension moiety, a regulatory element, or a combination thereof.

[0104] In some embodiments, the modified 5'-cap is m2 7,2’-O Gpp s pGRNA, m 7 GpppG, m 7 Gppppm 7 G, m2 (7,3’-O) GpppG, m2 (7,2’-O) GppspG(D1), m2 (7,2’-O) GppspG(D2), m2 7,3’-O Gppp(m1 2’-O )ApG, (m 7 G-3'mppp-G; this can be specified as equivalent to 3'O-Me-m7G(5')ppp(5')G), N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G, N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G, N7-(4-chlorophenoxyethyl)-m3’-O The following are selected from the group consisting of G(5')ppp(5')G, 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2 mp, m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N1-methylpsoiduridine, m7G(5')ppp(5')(2'OMeA)pG, and combinations thereof.

[0105] The disclosure also includes polynucleotides comprising both a 5' cap and a nucleotide sequence encoding IL-12 of the disclosure (e.g., polynucleotides comprising nucleotide sequences encoding IL12B polypeptide, IL12A polypeptide, and / or IL12B and IL12A fusion polypeptide).

[0106] The 5' cap structure of natural mRNA is involved in nuclear export, increases mRNA stability, and binds mRNA cap-binding proteins (CBPs), which, through association with poly(A)-binding proteins of CBPs, contribute to mRNA stability and translational readiness in cells, forming mature circular mRNA species. The cap further assists in the removal of 5' proximal introns during mRNA splicing.

[0107] Endogenous mRNA molecules can be 5'-capped, creating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcription sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcription nucleotides at the 5' end of the mRNA can also be 2'-O-methylated as needed. 5'-cap removal (decapping) by hydrolysis and cleavage of the guanylate cap structure targets nucleic acid molecules such as mRNA molecules for degradation. It is possible.

[0108] According to this disclosure, the 5'-terminated cap may include an endogenous cap or a cap analog. According to this disclosure, the 5'-terminated cap may include a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0109] In some embodiments, the 5'-terminal cap structure is CapO, Capl, ARC A, inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5'methyl G cap, or an analog thereof.

[0110] An example of an unrestricted additional cap is the 5' cap disclosed in WO / 2017 / 201350, published November 23, 2017, which is incorporated herein by reference.

[0111] In some embodiments, the half-life extension portion includes Fc, albumin or a fragment thereof, an albumin-binding portion, a PAS sequence, a HAP sequence, transferrin or a fragment thereof, XTEN, or any combination thereof.

[0112] In some embodiments, the half-life extension portion includes Fc. In some embodiments, the half-life extension portion includes albumin or a fragment thereof.

[0113] In some embodiments, the regulatory element is selected from the group consisting of at least one translation enhancer element (TEE), a translation initiation sequence, at least one microRNA binding site or its seed, the 3' tailing region of a ligated nucleoside, an AU-rich element (ARE), a post-transcriptional regulatory modulator, and combinations thereof.

[0114] In some embodiments, the regulatory element further comprises a poly-A region. In some embodiments, the nucleotide sequence encoding IL-12 of the Disclosure (e.g., a polynucleotide comprising a nucleotide sequence encoding an IL12B polypeptide, an IL12A polypeptide, and / or an IL12B and IL12A fusion polypeptide) further comprises a poly-A tail. In some embodiments, terminal groups on the poly-A tail can be incorporated for stabilization. In other embodiments, the poly-A tail comprises a des-3' hydroxyl tail. During RNA processing, a long chain of adenine nucleotides (poly-A tail) can be added to a polynucleotide, such as an mRNA molecule, to increase stability.

[0115] Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group. Then, poly(A) polymerase adds a chain of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly(A) tail that can be between approximately 80 and 250 residues long, for example, containing approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues.

[0116] Poly-A tails can also be added after the structure has been transported from the nucleus.

[0117] According to this disclosure, terminal groups on the poly(A) tail can be incorporated for stabilization. The polynucleotides of this disclosure may contain a des-3' hydroxyl tail. These are described in Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, August 23, This content may also include structural parts or 2'-O-methyl modifications taught by (2005, which is incorporated herein by reference in whole). For further poly-A tails, see also WO / 2017 / 201350, published November 23, 2017, which is incorporated herein by reference.

[0118] In some embodiments, the nucleotide sequence encoding IL-12 includes any modification or combination of modifications described herein.

[0119] Terminal structure modification: Untranslated region (UTR) The untranslated region (UTR) of a gene is transcribed but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not contain the start codon, while the 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is growing evidence for the regulatory role that UTRs play in terms of nucleic acid molecule and translational stability. The regulatory properties of UTRs can be incorporated into the RNA of this disclosure (e.g., modified RNA) to enhance molecular stability. Certain properties can also be incorporated to ensure controlled downregulation of transcripts in case they are misdirected to undesirable organ sites.

[0120] 5'UTR and translation start Natural 5'UTRs possess properties that play a role in translation initiation. They possess signatures similar to Kozak sequences, which are generally known to be involved in the process by which ribosomes initiate translation of many genes. Kozak sequences have a consensus CCR(A / G)CCAUGG (where R is a purine (adenine or guanine)) three bases upstream of the start codon (AUG), followed by another "G". 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0121] The 5'UTR secondary structure involved in elongation factor binding can interact with other RNA-binding molecules in the 5'UTR or 3'UTR to regulate gene expression. For example, the elongation factor EIF4A2, which binds to the secondary structural element in the 5'UTR, is required for microRNA-mediated repression (the entire Meijer HA is incorporated herein by reference). et al., Science, 2013, 340, 82-85). Different secondary structures in the 5'UTR can be incorporated into adjacent regions to stabilize or selectively destabilize (destalize) mRNA in specific tissues or cells.

[0122] The stability and protein production of nucleic acids or mRNAs of this disclosure can be enhanced by manipulating properties typically found in abundantly expressed genes in specific target organs. For example, the expression of nucleic acid molecules such as mRNA in the hepatocyte line or liver can be enhanced by introducing the 5'UTR of hepatically expressed mRNAs such as albumin, serum amyloid A, apolipoproteins A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII. Similarly, the use of 5'UTRs derived from other tissue-specific mRNAs to improve expression in those tissues is possible for muscle (MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (Tie-1, CD36), myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0123] Other non-UTR sequences can be incorporated into the 5'UTR (or 3'UTR). For example, an intron or a portion of an intron sequence can be incorporated into an adjacent region of the nucleic acid or mRNA of this disclosure. Incorporation of an intron sequence may increase protein production and mRNA levels.

[0124] In certain aspects of this disclosure, at least one fragment of an IRES sequence derived from a GTX gene may be included in the 5'UTR. In a non-limiting example, the fragment may be an 18-nucleotide sequence derived from the IRES of a GTX gene. In another non-limiting example, the 18-nucleotide sequence fragment derived from the IRES sequence of a GTX gene may be repeated in tandem in the 5'UTR of the polynucleotide described herein. The 18-nucleotide sequence may be repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or more than ten times in the 5'UTR.

[0125] Nucleotides can be mutated, replaced, and / or removed from the 5' (or 3') UTR. For example, one or more nucleotides upstream of the start codon can be replaced with another nucleotide. The number of nucleotides replaced can be more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, or 60 nucleotides upstream of the start codon. As another example, one or more nucleotides upstream of the start codon can be removed from the UTR.

[0126] 5'UTR, 3'UTR, and Translation Enhancer Element (TEE) In some embodiments, the 5'UTR of a nucleotide sequence encoding IL-12 includes at least one translational enhancer polynucleotide, a translational enhancer element, and a translational enhancer element (collectively referred to as "TEE"). In some embodiments, the TEE is located between the transcription promoter and the start codon. In some embodiments, RNA having at least one TEE in the 5'UTR (e.g., modified RNA) includes a cap in the 5'UTR. In some embodiments, at least one TEE can be located in the 5'UTR of a nucleotide sequence encoding IL-12 that undergoes cap-dependent or cap-independent translation.

[0127] The term “translational enhancer element” or “translation enhancer element” (collectively referred to as “TEE” in this specification) refers to a sequence that increases the amount of polypeptide or protein produced from mRNA.

[0128] In one embodiment, TEEs are conserved elements in the UTR that can promote the translational activity of nucleic acids, such as cap-dependent or cap-independent translation, though not limited to these. The conservation of these sequences has been studied across 14 species, including human, by Panek et al. (Nucleic Acids Research, 2013, 1-10; the entire work is incorporated herein by reference). This has been previously shown.

[0129] In some embodiments, the nucleotide sequence encoding IL-12 has at least one TEE having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with the one disclosed in whole in U.S. Application No. 2014 / 0147454, which is incorporated herein by reference. In some embodiments, RNA (e.g., modified RNA) is used, each of which is incorporated herein by reference in whole, as described in U.S. Patent Publications US20090226470, US20070048776, US20130177581 and US20110124100, International Patent Publications WO1999024595, WO2012009644, WO2009075886 and WO2007025008, and European Patent Publications EP2610341A1 and EP26 The invention includes at least one TEE having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with the TEEs described in U.S. Patent No. 10340A1, U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, and U.S. Patent No. 7,183,395.

[0130] In some embodiments, the 5'UTR of a nucleotide sequence encoding IL-12 may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or 60 TEE sequences. In some embodiments, the TEE sequences in the 5'UTR of RNA (e.g., modified RNA) may be the same or different TEE sequences. In some embodiments, the TEE sequences may be repeated more than once, twice, or three times in patterns such as ABABAB, or AABBAABBAABB, or ABCABCABC, or variants thereof. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0131] In some embodiments, the spacers used to separate the two TEE sequences include, but are not limited to, other sequences known in the art that regulate the translation of RNA (e.g., modified RNA), such as miR sequences (e.g., miR binding sites and miR seeds) described herein. In some embodiments, each spacer used to separate the two TEE sequences includes a different miR sequence or a component of a miR sequence (e.g., a miR seed sequence).

[0132] In some embodiments, the TEEs used in the 5'UTR of the nucleotide sequence encoding IL-12 in this disclosure are, but are not limited to, each an IRES sequence, such as those described in U.S. Patent No. 7,468,275 and International Patent Publication No. WO2001055369, which are incorporated herein by reference in whole.

[0133] In some embodiments, the TEEs described herein are located at the 5'UTR and / or 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, the TEE located at 3'UTR is the same as and / or different from the TEE located at 5'UTR and / or described for incorporation therein.

[0134] In some embodiments, the 3'UTR of a nucleotide sequence encoding IL-12 may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or 60 TEE sequences. In some embodiments, the TEE sequences in the 3'UTR of a nucleotide sequence encoding IL-12 of the present disclosure are the same or different TEE sequences. The TEE sequences are repeated one, two or three times or more in patterns such as ABABAB, or AABBAABBAABB, or ABCABCABC, or variants thereof. In these patterns, each letter A, B, or C represents a different TEE sequence at the nucleotide level.

[0135] In some embodiments, the 3'UTR includes a spacer that separates two TEE sequences. In some embodiments, the spacer is a 15-nucleotide spacer and / or other spacers known in the art. In some embodiments, the 3'UTR may include a TEE sequence-spacer module that is repeated at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times and at least nine times, or more than nine times.

[0136] In some embodiments, the spacers separating the two TEE sequences include, but are not limited to, other sequences known in the art that modulate the translation of IL-12 encoding nucleotide sequences, such as miR sequences (e.g., miR binding sites and miR seeds) described herein. In some embodiments, each spacer used to separate the two TEE sequences includes a different miR sequence or a component of a miR sequence (e.g., a miR seed sequence).

[0137] Integration of microRNA binding sites In some embodiments, the nucleotide sequence encoding IL-12 further includes a sensor sequence. Examples of sensor sequences include microRNA binding sites, transcription factor binding sites, structured mRNA sequences and / or motifs, and artificial binding sites engineered to act as pseudo-receptors for endogenous nucleic acid-binding molecules. A non-limiting example of a polynucleotide containing a sensor sequence is described in U.S. Patent Application No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0138] In some embodiments, microRNA (miRNA) profiling of target cells or tissues is performed to determine the presence or absence of miRNAs in the cells or tissues.

[0139] MicroRNAs (or miRNAs) are 19-25 nucleotide-long non-coding RNAs that bind to the 3'UTR of nucleic acid molecules and downregulate gene expression by either reducing the stability of the nucleic acid molecule or inhibiting translation. In some embodiments, RNA (e.g., modified RNA) comprises one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences may correspond to any known microRNAs, such as those taught in U.S. Publication US2005 / 0261218 and U.S. Publication US2005 / 0059005, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, known microRNAs in the human genome, their sequences and seed sequences, are described in U.S. Patent Application 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0140] The microRNA sequence includes a "seed" region, i.e., the sequence of the region from positions 2 to 8 of the mature microRNA, which has complete Watson-Crick complementarity with the miRNA target sequence. The microRNA seed includes positions 2 to 8 or 2 to 7 of the mature microRNA. In some embodiments, the microRNA seed includes 7 nucleotides (e.g., nucleotides 2 to 8 of the mature microRNA), where the seed complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite position 1 of the microRNA. In some embodiments, the microRNA seed includes 6 nucleotides (e.g., nucleotides 2 to 7 of the mature microRNA), where the seed complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite position 1 of the microRNA. For example, Grimson A, Farh K, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol See Cell. 2007 Jul. 6; 27(1):91-105. MicroRNA seed bases The microRNA has complete complementarity with the target sequence. Provided the microRNA of the present disclosure is available, the molecule can be targeted for degradation or reduced translation by manipulating the microRNA target sequence to the 3'UTR of the nucleic acid or mRNA of this disclosure. This process reduces the risk of off-target effects during nucleic acid molecule delivery. MicroRNAs, microRNA target regions, and their expression patterns have been identified, and their biological roles have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi: 10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 2012 80:393-403 and all of them). References; each of these is incorporated herein by reference in its entirety.

[0141] For example, if mRNA is not intended to be delivered to the liver but ends up there, the liver-abundant microRNA miR-122 can inhibit the expression of a target gene when one or more target sites of miR-122 are manipulated on the 3'UTR of a modified nucleic acid, enhanced modified RNA, or ribonucleic acid. Manipulating the introduction of one or more binding sites to different microRNAs can further reduce the longevity, stability, and protein translation of modified nucleic acids, enhanced modified RNA, or ribonucleic acid. As used herein, the term “microRNA site” refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It should be understood that “binding” may follow the rules of conventional Watson-Crick hybridization or may reflect any stable association of the microRNA with a microRNA site or an adjacent target sequence.

[0142] Conversely, for the purposes of the nucleotide sequences encoding IL-12 of this disclosure, naturally occurring microRNA binding sites can be manipulated to remove them from the sequence (i.e., eliminated) in order to increase protein expression in specific tissues. For example, the miR-122 binding site can be removed to improve protein expression in the liver.

[0143] In some embodiments, the nucleotide sequence encoding IL-12 includes, but is not limited to, at least one miRNA binding site in the 3'UTR to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells, such as normal and / or cancerous cells (e.g., HEP3B or SNU449).

[0144] Tissues in which microRNAs are known to regulate mRNA and thereby protein expression include, but are not limited to, the liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126).

[0145] Specifically, microRNAs are known to be differentially expressed in immune cells (also called hematopoietic cells), such as antigen-presenting cells (APCs) (e.g., dendritic cells and macrophages), macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes, and natural killer cells. Immune cell-specific microRNAs are involved in immunogenicity, autoimmunity, immune responses to infection, inflammation, and unwanted immune responses after gene therapy and tissue / organ transplantation. Immune cell-specific microRNAs also regulate many aspects of hematopoietic cell (immune cell) development, proliferation, differentiation, and apoptosis. For example, miR-142 and miR-146 are exclusively expressed in immune cells and are particularly abundant in bone marrow dendritic cells. It has been demonstrated in the art that immune responses to exogenous nucleic acid molecules are blocked by the addition of a miR-142 binding site to the 3'UTR of delivered gene constructs, enabling more stable gene transfer in tissues and cells. miR-142 efficiently degrades exogenous mRNA in antigen-presenting cells and suppresses cytotoxic elimination of transduced cells (Annoni A et al., blood, 2009, 114, 5152-5161; Brown BD, et al., Nat med. 2006, 12(5), 585-591; Brown BD, et al., blood, 2007, 110(13): 4144-4152, each of which is incorporated herein by reference in whole).

[0146] Numerous microRNA expression studies have been conducted in the art to profile differential microRNA expression in various cancer cells / tissues and other diseases. Some microRNAs are abnormally overexpressed in certain cancer cells, while others are underexpressed. For example, microRNAs are expressed in cancer cells (WO2008 / 154098, US2013 / 0059015, US2013 / 0042333, WO2011 / 157294); cancer stem cells (US2012 / 0053224); pancreatic cancer and disease (US2009 / 0131348, US2011 / 0171646, US2010 / 0286232, US Patent No. 8,389,210); asthma and inflammation (US Patent No. 8,415,096); prostate cancer (US2013 / 0053 264); Hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 054828, US Patent No. 8,252,538); Lung cancer cells (WO2011 / 076143, WO2013 / 033640, WO2009 / 070653, US2010 / 0323357); Cutaneous T-cell lymphoma (WO2013 / 011378); Colorectal cancer cells (WO2011 / 0281756, WO2011 / 076142); Cancer-positive lymph nodes (lympho node) (WO2009 / 100430, US2009 / 0263803); nasopharyngeal cancer (EP2112235); chronic obstructive pulmonary disease (US2012 / 0264626, US2013 / 0053263); thyroid cancer (WO2013 / 066678); ovarian cancer cells (US2012 / 0309645, WO2011 / 095623); breast cancer cells (WO2008 / 1540) These are expressed differentially in 98, WO2007 / 081740, US2012 / 0214699, leukemia and lymphoma (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563, the contents of each of these are incorporated herein by reference in their entirety).

[0147] At least one microRNA site can be manipulated into the 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more microRNA sites can be manipulated into the 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 are the same or different microRNA sites. In some embodiments, the microRNA sites incorporated into the nucleotide sequence encoding IL-12 target the same or different tissues of the body. As a non-limiting example, the introduction of tissue, cell type, or disease-specific microRNA binding sites in the 3'UTR of modified nucleic acid mRNA can reduce the level of expression in specific cell types (e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.).

[0148] In some embodiments, the microRNA site is manipulated near the 5' end of the 3'UTR, approximately midway between the 5' and 3' ends of the 3'UTR, and / or near the 3' end of the 3'UTR. In some embodiments, the microRNA site is manipulated near the 5' end of the 3'UTR, and approximately midway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is manipulated near the 3' end of the 3'UTR, and approximately midway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is manipulated near the 5' end of the 3'UTR, and near the 3' end of the 3'UTR.

[0149] In some embodiments, the modified messenger RNA includes a microRNA binding region that is 100% identical to a known seed sequence or less than 100% identical to the seed sequence. The seed sequence can be partially mutated to reduce its microRNA binding affinity, thus resulting in reduced downmodulation of its mRNA transcript. Essentially, the degree of match or mismatch between the target mRNA and the microRNA seed can act as a rheostat, finer-tuning the microRNA's ability to modulate protein expression. In addition, mutations in the non-seed region of the microRNA binding site can also affect the microRNA's ability to modulate protein expression.

[0150] RNA motifs for RNA-binding proteins (RBPs) RNA-binding proteins (RBPs) can regulate many aspects of co-transcription and post-transcriptional gene expression, including, but are not limited to, RNA splicing, localization, translation, turnover, polyadenylation, capping, modification, efflux, and localization. RNA-binding domains (RBDs), such as RNA recognition motifs (RRs) and hnRNP K homology (KH) domains, typically regulate the association of sequences between RBPs and their RNA targets (Ray et al. Nature 2013. 499:172-177; the whole is incorporated herein by reference). In some aspects, canonical RBDs bind short RNA sequences. In some aspects, canonical RBDs recognize RNA structures.

[0151] Non-limiting examples of RNA-binding proteins and related nucleic acids and protein sequences are described in U.S. Patent Application No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0152] In some embodiments, to increase the stability of the target mRNA, mRNA encoding HuR is co-transfected or co-injected into cells or tissues together with the target mRNA. These proteins can also be tethered to the target mRNA in vitro and then administered together to cells. The poly(A) tail-binding protein PABP interacts with the eukaryotic translation initiation factor eIF4G to stimulate translation initiation. Co-administration of mRNA encoding these RBPs together with mRNA drugs, and / or tethering these proteins to mRNA drugs in vitro, as well as administration of protein-binding mRNA to cells, can increase the translation efficiency of the mRNA. The same concept can be extended to co-administration of mRNA with mRNAs encoding various transcription factors and promoters, as well as with their own proteins, to influence RNA stability and / or translation efficiency.

[0153] In some embodiments, the nucleotide sequence encoding IL-12 includes, but is not limited to, at least one RNA-binding motif, such as an RNA-binding domain (RBD).

[0154] In some embodiments, a first region of the linked nucleoside and / or at least one adjacent region includes at least one RBD. In some embodiments, the first region of the linked nucleoside includes an RBD related to a splicing factor, and at least one adjacent region includes an RBD for stability and / or a translation factor.

[0155] Other modulatory elements in 3'UTR In addition to microRNA binding sites, other regulatory sequences in the 3'-UTR of native mRNA that regulate mRNA stability and translation in different tissues and cells can be removed or introduced into modified messenger RNA. Such cis-regulatory elements include, but are not limited to, cis-RNP (ribonucleoprotein) / RBP (RNA-binding protein) regulatory elements, AU-rich elements (AUEs), structured stem-loops, constitutive decay elements (CDEs), GC abundance, and other structured mRNA motifs (Parker BJ et al., Genome Research, 2011, 21, 1929-1943, which is incorporated herein by reference in its entirety). For example, CDEs are a class of regulatory motifs that mediate mRNA degradation through their interaction with Roquin proteins. In particular, CDEs are found in many mRNAs encoding developmental and inflammatory regulators that limit cytokine production in macrophages (Leppek K et al., 2013, Cell, 153, 869-881, which is incorporated herein by reference in its entirety).

[0156] In some embodiments, RNA (e.g., modified mRNA) is nutrient-requiring. As used herein, “nutrient-requiring” means mRNA having at least one property that induces, promotes, or induces the degradation or inactivation of mRNA in response to a spatial or temporal cue, such that protein expression is substantially prevented or reduced. Such spatial or temporal cues include the location of the mRNA being translated, such as a particular tissue or organ or cellular environment. Cues may also include temperature, pH, ionic strength, water content, etc.

[0157] 3'UTR and AU rich elements 3'UTRs are known to have stretches into which adenosine and uridine are embedded. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on the characteristics and functional properties of their sequences, AU-rich elements (AREs) can be classified into three classes (Chen et al.). (al, 1995). Class I AREs contain several AUUUA motifs within the U-rich region. It contains dispersed copies. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) notamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are not very well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, most notably HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. Manipulating the HuR-specific binding site to the 3'UTR of nucleic acid molecules results in HuR binding, and therefore in vivo stabilization of the message.

[0158] The stability of the nucleic acids or mRNAs of this disclosure can be modulated by introducing, removing, or modifying the AU-rich element (ARE) in the 3'UTR. When manipulating a particular nucleic acid or mRNA, one or more copies of the ARE can be introduced to make the nucleic acid or mRNA of this disclosure more unstable, thereby suppressing the translation of the resulting protein and reducing its production. Similarly, the ARE can be identified and removed or mutated to increase intracellular stability and, therefore, increase the translation and production of the resulting protein. Transfection experiments can be performed using the nucleic acids or mRNAs of this disclosure in relevant cell systems, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different ARE-manipulating molecules, and the produced proteins can be assayed approximately 6 hours, 12 hours, 24 hours, 48 ​​hours, and / or 7 days after transfection by using an ELISA kit for the relevant protein.

[0159] 3'UTR and Triple Helix In some embodiments, the nucleotide sequence encoding IL-12 includes a triple helix at the 3' end of a modified nucleic acid, an enhanced IL-12 encoding nucleotide sequence, or ribonucleic acid. In some embodiments, the 3' end of the IL-12 encoding nucleotide sequence includes a triple helix alone or in combination with a poly-A tail.

[0160] In some embodiments, the nucleotide sequence encoding IL-12 includes at least first and second U-rich regions, a conserved stem-loop region between the first and second regions, and an A-rich region. In some embodiments, the first and second U-rich regions and the A-rich region associate to form a triple helix at the 3' end of the nucleic acid. This triple helix can stabilize the nucleic acid, enhance the translation efficiency of the nucleic acid, and / or protect the 3' end from degradation. Exemplary triple helices include, but are not limited to, the triple helix sequences of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), MEN-β, and polyadenylated nucleus (PAN)RNA (see Wilusz et al., Genes & Development 2012 26:2392-2407; the whole is incorporated herein by reference).

[0161] stem loop In some embodiments, the nucleotide sequence encoding IL-12 includes a stem-loop, for example, a histone stem-loop, though not limited to this. In some embodiments, the stem-loop is a nucleotide sequence, though not limited to this, that is approximately 25 or approximately 26 nucleotides long, such as sequence numbers 7-17 described in International Patent Publication WO2013103659, which is incorporated herein by reference. The histone stem-loop may be located at 3' relative to the coding region (e.g., at the 3' end of the coding region). In non-limiting examples, the stem-loop may be located at the 3' end of nucleic acids described herein.

[0162] In some embodiments, a nucleotide sequence encoding IL-12, including a histone stem loop, can be stabilized by the addition of at least one strand-terminating nucleoside. While we do not wish to be constrained by theory, the addition of at least one strand-terminating nucleoside can slow down the degradation of the nucleic acid and, therefore, increase its half-life.

[0163] In some embodiments, the chain-terminating nucleoside is described in International Patent Publication WO2013103659, which is incorporated herein by reference in its entirety. In some embodiments, the chain-terminating nucleoside is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleoside, for example, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleoside, or -O-methylnucleoside.

[0164] In some embodiments, the nucleotide sequence encoding IL-12 includes a histone stem-loop, a poly(A) tail sequence, and / or a 5' cap structure. In some embodiments, the histone stem-loop is before and / or after the poly(A) tail sequence. Nucleic acids comprising the histone stem-loop and poly(A) tail sequence may include the strand termination nucleosides described herein.

[0165] In some embodiments, the nucleotide sequence encoding IL-12 includes a histone stem-loop and a 5' cap structure. Examples of 5' cap structures include, but are not limited to, those described herein and / or known in the art.

[0166] 5' capping The 5' cap structure of mRNA is involved in nuclear export, increases mRNA stability, and binds mRNA cap-binding proteins (CBPs), which, through association with poly(A)-binding proteins of CBPs, contribute to mRNA stability and translational readiness in cells, forming mature circular mRNA species. The cap further assists in the removal of 5' proximal introns during mRNA splicing.

[0167] Endogenous mRNA molecules can be 5'-capped, creating a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcription sense nucleotide of the mRNA. This 5'-guanylate cap can then be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or pre-terminal transcription nucleotides at the 5' end of the mRNA can also be 2'-O-methylated as needed. Removal of the 5'-cap by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules such as mRNA molecules for degradation.

[0168] The RNA modifications described herein generate a non-hydrolyzable cap structure that prevents cap removal and thus can increase the half-life of the mRNA. Since hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage, the modified nucleotides can be used during the capping reaction. For example, the vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) can be used with α-thio-guanosine nucleotides, according to the manufacturer's instructions, to create the phosphorothioate linkage at the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can be used.

[0169] Additional modifications, though not limited to them, include 2'-O-methylation of the ribose sugar at the 5'-terminal and / or pre-5'-terminal nucleotides of mRNA at the 2'-hydroxyl group of the sugar ring (as mentioned above). Multiple distinct 5'-cap structures can be used to generate 5'-caps for nucleic acid molecules such as mRNA molecules.

[0170] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5'-caps, but retain cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically, and / or linked to nucleic acid molecules.

[0171] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, where one guanine contains an N7 methyl group and a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; this can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is linked to the 5' terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). The N7- and 3'-O-methylated guanines provide the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0172] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-β-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0173] In some embodiments, the cap is a dinucleotide cap analog. In some embodiments, the dinucleotide cap analog is modified with a boranophosphate group or a phosphoroselenoate group at different phosphate positions, for example, the content of which is incorporated herein by reference in whole, of the dinucleotide cap analog described in U.S. Patent No. 8,519,110.

[0174] In some embodiments, the cap analog is an N7-(4-chlorophenoxyethyl)-substituted dinucleotide (dicucleotide) form of a cap analog known in the art and / or described herein. Non-limiting examples of the N7-(4-chlorophenoxyethyl)-substituted dinucleotide form of a cap analog include the N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, for example, Kore et al. Bioorganic & Medicinal Chemistry 2013 21:4570-4574, which describes various cap analogs and methods for synthesizing cap analogs; this is incorporated herein by reference in its entirety). In some embodiments, the cap analog of this disclosure is a 4-chloro / bromophenoxyethyl analog.

[0175] Capping analogs enable the simultaneous capping of nucleic acid molecules in in vitro transcription reactions, but up to approximately 20% of the transcript remains uncapped. This structural difference between the capping analog and the endogenous 5'-cap structure of nucleic acids produced by the endogenous cellular transcription mechanism can result in reduced translational eligibility and reduced cellular stability. Therefore, in some embodiments, the methods provided herein (see, for example, Examples 1-3) can increase the capping efficiency of IL-12-expressing nucleotides produced as described herein. In some embodiments, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and about 100% of the polynucleotide are capped by the methods provided herein. In some embodiments, at least about 50% of the polynucleotide are capped by the methods provided herein. In some embodiments, at least about 60% of the polynucleotide are capped. In some embodiments, at least about 70% of the polynucleotide are capped. In some embodiments, at least about 80% of the polynucleotide are capped. In some embodiments, at least about 85% of the polynucleotide are capped. In some embodiments, at least about 90% of the polynucleotide are capped. In some embodiments, at least about 95% of the polynucleotide is capped. In some embodiments, about 100% of the polynucleotide is capped. In some embodiments, at least about 80% to about 100% of the polynucleotide is capped.

[0176] In some embodiments, providing RNA having a 5'-cap or a 5'-cap analog is achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap is co-transcriptionally incorporated into the resulting RNA strand.

[0177] In some embodiments, the RNA can be generated, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcription using a capping enzyme, for example, a vaccinia virus capping enzyme. In some embodiments, the nucleotide sequence encoding IL-12 is capped post-transcriptionally using an enzyme to produce a more authentic 5'-cap structure. As used herein, the term “more authentic” refers to a property that, either structurally or functionally, more closely reflects or mimics an endogenous or wild-type property. That is, a “more authentic” property is a better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure compared to a synthetic property or analog of the prior art, or is superior in one or more respects to the corresponding endogenous, wild-type, natural or physiological property. Non-limiting examples of more authentic 5' cap structures of this disclosure include, among many, those having enhanced binding of cap-binding proteins, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' cap removal compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can create a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of mRNA and the guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5' terminal nucleotide of mRNA contains a 2'-O-methylation. This cap results in higher translational qualification and cellular stability, as well as reduced activation of pro-inflammatory cytokines, compared to, for example, other 5' cap analog structures known in the art. Cap structures include 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2 mp, and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up.

[0178] In some embodiments, the 5'-terminated cap may be an endogenous cap or a cap analog. In some embodiments, the 5'-terminated cap may include a guanine analog. Useful guanine analogs include inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0179] In some embodiments, the 5' cap contains a 5'-5' triphosphate linkage. In some embodiments, the 5' cap contains a 5'-5' triphosphate linkage with a triphosphate modification. In some embodiments, the 5' cap contains a 2'-O or 3'-O-ribose methylated nucleotide. In some embodiments, the 5' cap contains a modified guanosine nucleotide or a modified adenosine nucleotide. In some embodiments, the 5' cap contains 7-methylguanylate. Exemplary cap structures include m7G(5')ppp(5')G, m7,2'O-mG(5')ppSp(5')G, m7G(5')ppp(5')2'O-mG, and m7,3'O-mG(5')ppp(5')2'O-mA.

[0180] In some embodiments, the nucleotide sequence encoding IL-12 includes a modified 5' cap. Modifications at the 5' cap can increase mRNA stability, increase mRNA half-life, and increase mRNA translation efficiency. In some embodiments, the modified 5' cap includes one or more of the following modifications: modification of the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), replacement of the oxygen of the sugar ring (which gives rise to a carbon ring) with a methylene moiety (CH2), modification of the triphosphate crosslinking portion of the cap structure, or modification of the nucleoside (G) portion.

[0181] Examples of 5' cap structures that can be modified include, but are not limited to, the caps described in U.S. Patent Application No. 2014 / 0147454 and WO2018 / 160540, which are incorporated herein by reference in their entirety.

[0182] IRES array In some embodiments, nucleotide sequences encoding IL-12 include intra-sequence ribosome entry sites (IRESs). Characteristic picornavirus RNAs and initially identified IRESs play a crucial role in initiating protein synthesis in the absence of a 5' cap structure. IRESs can act as a single ribosome binding site or as one of multiple ribosome binding sites in mRNA. Nucleic acids or mRNAs containing more than one functional ribosome binding site can encode several peptides or polypeptides that are independently translated by ribosomes ("polycistronic nucleic acid molecules"). If nucleic acids or mRNAs are provided with IRESs, a second translatable region is further provided as needed. Examples of IRES sequences that can be used pursuant to this disclosure include, but are not limited to, picornaviruses (e.g., FMDV), pest virus (CFFV), poliovirus (PV), and encephalomyocarditis virus. Examples include those derived from ecclesiastical virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0183] End structure modification: Poly A tail During RNA processing, a long chain of adenine nucleotides (poly-A tail) is typically attached to the messenger RNA (mRNA) molecule to increase its stability. Immediately after transcription, the 3' end of the transcript is cleaved, releasing the 3' hydroxyl group. Poly-A polymerase then attaches the chain of adenine nucleotides to the RNA. This process, called polyadenylation, involves attaching a poly-A tail that is between 100 and 250 residues long.

[0184] In some embodiments, the 3' tail is longer than about 30 nucleotides. In some embodiments, the poly-A tail is longer than about 35 nucleotides. In some embodiments, the length is at least about 40 nucleotides. In some embodiments, the length is at least about 45 nucleotides. In some embodiments, the length is at least about 55 nucleotides. In some embodiments, the length is at least about 60 nucleotides. In some embodiments, the length is at least about 70 nucleotides. In some embodiments, the length is at least about 80 nucleotides. In some embodiments, the length is at least about 90 nucleotides. In some embodiments, the length is at least about 100 nucleotides. In some embodiments, the length is at least about 120 nucleotides. In some embodiments, the length is at least about 140 nucleotides. In some embodiments, the length is at least about 160 nucleotides. In some embodiments, the length is at least about 180 nucleotides. In some embodiments, the length is at least about 200 nucleotides. In some embodiments, the length is at least about 250 nucleotides. In some embodiments, the length is at least about 300 nucleotides. In some embodiments, the length is at least about 350 nucleotides. In some embodiments, the length is at least about 400 nucleotides. In some embodiments, the length is at least about 450 nucleotides. In some embodiments, the length is at least about 500 nucleotides. In some embodiments, the length is at least about 600 nucleotides. In some embodiments, the length is at least about 700 nucleotides. In some embodiments, the length is at least about 800 nucleotides. In some embodiments, the length is at least about 900 nucleotides. In some embodiments, the length is at least about 1000 nucleotides. In some embodiments, the length is at least about 1100 nucleotides. In some embodiments, the length is at least about 1200 nucleotides. In some embodiments, the length is at least about 1300 nucleotides. In some embodiments, the length is at least about 1400 nucleotides. In some embodiments, the length is at least about 1500 nucleotides. In some embodiments, the length is at least about 1600 nucleotides.In some embodiments, the length is at least about 1700 nucleotides. In some embodiments, the length is at least about 1800 nucleotides. In some embodiments, the length is at least about 1900 nucleotides. In some embodiments, the length is at least about 2000 nucleotides. In some embodiments, the length is at least about 2500 nucleotides. In some embodiments, the length is at least about 3000 nucleotides.

[0185] In some embodiments, the nucleotide sequence encoding IL-12 is designed to include a polyAG quartet. A G quartet is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G quartet is incorporated at the end of a polyA tail. The resulting nucleic acid or mRNA can be assayed for other parameters, including stability, protein production, and half-life at various time points. It has been found that the polyAG quartet yields protein production equivalent to at least 75% of the protein production seen when using a 120-nucleotide polyA tail alone.

[0186] In some embodiments, the nucleotide sequence encoding IL-12 includes a poly-A tail, which is stabilized by the addition of a chain termination nucleoside. In some embodiments, the nucleotide sequence encoding IL-12 having a poly-A tail further includes a 5' cap structure.

[0187] In some embodiments, the nucleotide sequence encoding IL-12 includes a poly-AG quartet. In some embodiments, the nucleotide sequence encoding IL-12 having a poly-AG quartet further includes a 5' cap structure.

[0188] In some embodiments, a nucleotide sequence encoding IL-12, including a poly-A tail or poly-AG quartet, is stabilized by the addition of oligonucleotides ending with a 3'-deoxynucleoside, 2',3'-dideoxynucleoside, 3'-O-methylnucleoside, 3'-O-ethylnucleoside, 3'-arabinoside, and other modified nucleosides known in the art and / or described herein.

[0189] Modified Nucleoside In some embodiments, the nucleotide sequence encoding IL-12 comprises one or more modified nucleosides. In some embodiments, one or more modified nucleosides are 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-psoidouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-o This includes xo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloropurine, N6-methyl-2-aminopurine, 6-chloropurine, N6-methyladenosine, α-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methylcytidine, N4-acetylcytidine, 5-methyluridine, 5-iodocytidine, and combinations thereof.

[0190] In some embodiments, one or more uridine molecules in the nucleotide sequence encoding IL-12 are replaced by a modified nucleoside. In some embodiments, the modified nucleoside replacing the uridine is pseudouridine (ψ), N1-methylpsoiduridine (m1ψ), or 5-methyluridine (m5U).

[0191] In some embodiments, the nucleotide sequences encoding IL-12 include nucleotide sequences encoding IL-12 described in U.S. Patent Application No. 2014 / 0147454, International Application WO2018160540, International Application WO2015 / 196118, or International Application WO2015 / 089511, which are incorporated herein by reference in their entirety.

[0192] Cytotoxic nucleosides In some embodiments, the nucleotide sequence encoding IL-12 comprises one or more cytotoxic nucleosides. For example, the cytotoxic nucleosides may be incorporated into a bifunctional nucleotide sequence encoding IL-12 or into a polynucleotide such as mRNA. Examples of cytotoxic nucleoside anticancer agents include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[0193] Several cytotoxic nucleoside analogs are in clinical use or are being studied in clinical trials as anticancer agents. Examples of such analogs include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacitidine, 4'-thio-aracitidine, cyclopentenylcytosine, and 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Another example of such a compound is fludarabine phosphate. These compounds can be administered systemically and may have side effects typical of cytotoxic agents, such as little to no specificity to tumor cells compared to proliferating normal cells.

[0194] Several prodrugs of cytotoxic nucleoside analogs have also been reported in the art. Examples include, but are not limited to, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidic acid). Generally, these prodrugs can be converted to active drugs primarily in the liver and systemic circulation, and may exhibit little to no selective release of the active drug in tumor tissue. For example, capecitabine, a prodrug of 5'-deoxy-5-fluorocytidine (ultimately 5-fluorouracil), is metabolized in both the liver and tumor tissue. A series of capecitabine analogs containing "radicals readily hydrolyzable under physiological conditions" are claimed by Fujiu et al. (U.S. Patent No. 4,966,891) and incorporated herein by reference. The series described by Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine, and the implication that these compounds are activated by hydrolysis under normal physiological conditions to produce 5'-deoxy-5-fluorocytidine.

[0195] A series of cytarabine N4-carbamates have been reported by Fadl et al (Pharmazie. 1995, 50, 382-7, the whole of which is incorporated herein by reference), and these The compounds were designed to be converted to cytarabine in the liver and plasma. WO2004 / 041203, incorporated herein in its entirety by reference, discloses a prodrug of gemcitabine, a portion of which is an N4-carbamate. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine and were intended to provide gemcitabine by hydrolysis and release in the liver and plasma after absorption of the intact prodrug from the gastrointestinal tract. Nomura et al (Bioorg Med. Chem. 2003, 11, 2453-61, incorporated herein in its entirety by reference) describe an acetal derivative of 1-(3-C-ethynyl-β-D-ribo-pentofaranosyl)cytosine that, in vivo reduction, yields an intermediate requiring further hydrolysis under acidic conditions to produce a cytotoxic nucleoside compound.

[0196] Cytotoxic nucleotides that can be used as chemotherapeutic agents include, but are not limited to, pyrazolo[3,4-D]-pyrimidine, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloropurine, and inosine, or combinations thereof.

[0197] Code array In some aspects of this disclosure, the nucleotide sequence encoding IL-12 includes a sequence encoding the interleukin (IL)-12 molecule. In some aspects, the IL-12 molecule includes IL-12, an IL-12 subunit (e.g., an IL-12 beta subunit or an IL-12 alpha subunit), or a mutant IL-12 molecule that retains immunomodulatory function.

[0198] IL-12 is a heterodimer cytokine with multiple biological effects on the immune system. It is composed of two subunits, p35 (also known as the alpha subunit) and p40 (also known as the beta subunit), which interact to produce an active heterodimer. The IL-12 p35 subunit is also known in the art as IL-12α; IL-12A; natural killer cell-stimulating factor 1; cytotoxic lymphocyte maturation factor 1, p35; CLMF P35; NKSF1; CLMF; or NFSK. The IL-12 p40 subunit is also known in the art as IL-12β; IL-12B; natural killer cell-stimulating factor 2; cytotoxic lymphocyte maturation factor 2, p40; CLMF P40; NKSF2; CLMF2; IMD28; or IMD29. Unless otherwise indicated, the term "IL-12" (or any grammatical variant thereof) may refer to the IL-12 p35 subunit, the IL-12 p40 subunit, or the heterodimer IL-12 p70.

[0199] The wild-type human IL-12 p35 protein has a length of 219 amino acids. The wild-type human IL-12 p40 protein has a length of 328 amino acids. The amino acids of the wild-type human IL-12 protein are further provided below in Table 1.

[0200] In some embodiments, the IL-12 protein (encoded by, for example, the nucleic acid molecule described herein) comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 182.

[0201] In some embodiments, the IL-12 molecule comprises an IL-12α subunit and / or an IL-12β subunit. In some embodiments, the IL-12α subunit comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 183.

[0202] In some embodiments, the IL-12β subunit comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 184.

[0203] When described herein, the nucleic acid molecules of this disclosure are codon-optimized. Therefore, in some embodiments, the IL-12 protein disclosed herein (e.g., IL-12 The nucleotide sequences encoding the p35 subunit, the IL-12 p40 subunit, or the heterodimer IL-12 p70 are different from those of the wild-type nucleotide sequence (e.g., SEQ ID NO: 185 or SEQ ID NO: 186).

[0204] In some embodiments, the nucleic acid molecules described herein encode an IL-12β subunit and comprise at least about 75%, at least about 76%, of the sequence shown in any one of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75. Contains nucleotide sequences that are identical by at least approximately 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100%. In certain embodiments, the nucleotide sequence includes a nucleotide sequence that is at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any IL-12β subunit sequence of any of the constructs provided in Table 1.

[0205] In some embodiments, IL-12 The nucleic acid molecule encoding the p40 subunit is (i) at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 51; (ii) at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the sequence shown in SEQ ID NO: 52 (iii) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 53; (iii) at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least (iv) identical to the sequence shown in sequence number 54 by at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;(v) at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 55; (vi) at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or less identical to the sequence shown in sequence number 56. (vii) at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 57; (viii) at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 58; (ix) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 59; (x) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, less than 98% identical to the sequence shown in sequence number 65, 69, or 74. (xi) identical to (xii) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (xii) identical to (xii) at least 97%, at least 98%, at least 99%, or 100% identical to (xiii) identical to (xiii) at least 99% or 100% identical to (xii) the sequence shown in (xii) 63;Alternatively, it may include a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in (xiv) Sequence ID No. 64.

[0206] In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 51. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 52. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 53. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 54. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 55. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 56. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 57. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 58. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 59. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 65. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 66. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 67. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 68. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 69. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 70. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 71. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 72. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 73. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 74. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 75.In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 62. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 63. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 64. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 60. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 61.

[0207] In some embodiments, the nucleic acid molecules described herein encode the IL-12 p35 subunit and are at least about 77% of the sequence shown in any one of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125. Contains nucleotide sequences that are at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100% identical. In certain embodiments, the nucleotide sequence includes a nucleotide sequence that is at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any IL-12α subunit sequence of any of the constructs provided in Table 1.

[0208] In some embodiments, IL-12 The nucleic acid molecule encoding the p35 subunit is (i) at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 102; (ii) at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, less (iii) at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% (iv) identical to the sequence shown in sequence number 104 by at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;(v) at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 105; (vi) at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% identical to the sequence shown in sequence number 106. (vii) at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 107; (viii) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 108 (ix) at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in sequence number 109; (x) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, less identical to the sequence shown in sequence numbers 115, 119, or 124. (xi) at least 99% or 100% identical to the sequence shown in sequence number 116, 120, or 125; (xii) at least 98% or 99% identical to the sequence shown in sequence number 112; (xiii) at least 98% or 99% identical to the sequence shown in sequence number 113;Alternatively, it includes a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in (xiv) Sequence ID No. 114.

[0209] In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 101. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 102. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 103. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 104. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 105. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 106. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 107. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 108. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 109. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 115. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 116. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 117. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 118. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 119. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 120. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 121. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 122. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 123. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 124. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 125.In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 112. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 113. In some embodiments, the nucleic acid molecule encoding the IL-12α subunit includes the sequence shown in SEQ ID NO: 114. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 110. In some embodiments, the nucleic acid molecule encoding the IL-12β subunit includes the sequence shown in SEQ ID NO: 111.

[0210] In some embodiments, nucleic acid molecules encoding the IL-12 p40 subunit and nucleic acid molecules encoding the IL-12 p35 subunit can be conjugated to each other. For example, in some embodiments, the Disclosure provides an isolated polynucleotide comprising a first nucleic acid and a second nucleic acid, wherein the first nucleic acid encodes the IL-12 p40 subunit and the second nucleic acid encodes the IL-12 p35 subunit. In some embodiments, the IL-12α subunit and the IL-12β subunit are linked by a linker. In some embodiments, the linker comprises an amino acid linker of at least about 2, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, or at least about 20 amino acids. In some embodiments, the linker comprises a (GS) linker. In some embodiments, the GS linker has the formula (Gly3Ser)n or S(Gly3Ser)n, where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 100. In some embodiments, the (Gly3Ser)n linker is (Gly3Ser)3 or (Gly3Ser)4.

[0211] In some embodiments, the first nucleic acid molecule encoding the IL-12β subunit is at least about 75%, at least about 76%, at least about Contains nucleotide sequences that are 77%, at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% identical. The second nucleic acid molecule encoding the IL-12α subunit is at least about 77 sequences, including the sequence shown in SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 125. Contains nucleotide sequences that are identical by at least approximately 78%, at least approximately 79%, at least approximately 80%, at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least 100%.

[0212] In some embodiments, the first nucleic acid molecule encoding the IL-12β subunit is at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least 100% identical to any of the IL-12β subunit sequences of the constructs provided in Table 1. A second nucleic acid molecule comprising a nucleotide sequence; which encodes an IL-12α subunit, comprises a nucleotide sequence that is at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to any of the IL-12α subunit sequences of the constructs provided in Table 1.

[0213] In some embodiments, (i) the first nucleic acid molecule is at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 51. The first nucleic acid molecule contains a nucleotide sequence that is identical to the sequence shown in Sequence ID No. 101 by at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%;(ii) The first nucleic acid molecule contains a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 52. The and / or second nucleic acid molecule contains a nucleotide sequence that is at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 102;(iii) The first nucleic acid molecule contains a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 103, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, or at least 9 (iv) The first nucleic acid molecule contains a nucleotide sequence that is identical to at least 2%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; (iv) The first nucleic acid molecule contains a nucleotide sequence that is identical to at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence shown in SEQ ID NO: 54, and / or the second nucleic acid molecule contains a nucleotide sequence that is identical to at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence shown in SEQ ID NO: 104;(v) The first nucleic acid molecule contains a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 55, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, or at least identical to the sequence shown in SEQ ID NO: 105. (vi) The first nucleic acid molecule contains a nucleotide sequence that is 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 56; (vi) the first nucleic acid molecule contains a nucleotide sequence that is at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least (vii) The first nucleic acid molecule contains a nucleotide sequence that is 99% or 100% identical to the sequence shown in SEQ ID NO: 106, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 57 The first nucleic acid molecule contains a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in Sequence ID No. 107;(viii) The first nucleic acid molecule contains a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 58, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 108; (ix) The first nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 59, and / or the second nucleic acid (x) The acid molecule contains a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 109; (x) The first nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 65, 69, or 74, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 115, 119, or 124;(xi) The first nucleic acid molecule contains a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 66, 70, or 75, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 116, 120, or 125; (xii) The first nucleic acid molecule contains a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 62 (xiii) The first nucleic acid molecule contains a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 112, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 99%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 63, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 113; or (xiv) The first nucleic acid molecule contains a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 65, and / or the second nucleic acid molecule contains a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence shown in SEQ ID NO: 115.

[0214] In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 51, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 101. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 52, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 102. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 53, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 103. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 54, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 104. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 55, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 105. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 56, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 106. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 57, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 107. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 58, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 118. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 59, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 119. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 65, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 115. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 66, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 116. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 67, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 117. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 68, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 118. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 69, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 119. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 70, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 120. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 71, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 121.In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 72, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 122. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 73, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 123. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 74, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 124. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 75, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 125. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 62, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 112. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 63, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 113. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 64, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 114. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 60, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 110. In some embodiments, the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 61, and the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 111. In some embodiments, the first nucleic acid molecule includes one of the IL-12β subunit sequences of the constructs provided in Table 1, and the second nucleic acid molecule includes one of the IL-12α subunit sequences of the constructs provided in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23

[0215] In some embodiments, the isolated polynucleotides described herein, i.e., the isolated polynucleotides containing nucleic acid molecules encoding IL-12 (e.g., IL-12α subunit and / or IL-12β subunit), contain one or more heterologous moieties (e.g., genes for experimental and / or therapeutic purposes). As used herein, the term "heterologous moiety" refers to any molecule (chemical or biological) that is different from the IL-12 protein (e.g., IL-12α subunit and / or IL-12β subunit) encoded by the nucleic acid molecules disclosed herein. Such heterologous moieties can be genetically fused, conjugated, and / or otherwise associated with the IL-12 protein. For example, in some embodiments, the heterologous moiety can be fused to the 3' end of the IL-12α subunit. In some embodiments, the heterologous moiety can be conjugated to the 3' end of the IL-12α subunit via a linker (e.g., GS linker). In some embodiments, the heterologous moiety can be fused to the 3' end of the IL-12β subunit. In some embodiments, the heterologous moiety can be conjugated to the 3' end of the IL-12β subunit via a linker (e.g., GS linker).

[0216] In some embodiments, the heterogeneous portion includes a half-life extension portion. The term “half-life extension portion” refers to a pharmaceutically acceptable portion, domain, or molecule covalently linked (“conjugated” or “fused”) to the IL-12 protein encoded by the nucleic acid molecule of this disclosure (e.g., the IL-12α subunit and / or IL-12β subunit) to prevent or mitigate chemical modifications that reduce the in vivo proteolysis or other activity of the IL-12 protein, increase its half-life, and / or improve or modify other pharmacokinetic or biophysical properties, including, but not limited to, increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing the bioactivity and / or target selectivity of the IL-12 protein, increasing manufacturability, and / or reducing the immunogenicity of the IL-12 protein, either directly or via a linker, and optionally via an amino acid not naturally encoded, to the IL-12 protein encoded by the nucleic acid molecule of this disclosure (e.g., the IL-12α subunit and / or IL-12β subunit).

[0217] In the context of this disclosure, the terms “fused” or “conjugated” indicate that at least two polypeptide chains (e.g., encoded by nucleic acid molecules described herein) are operably linked and recombinantly expressed. In some embodiments, two polypeptide chains may be “fused” as a result of chemical synthesis. In the context of this disclosure, the terms “conjugate” or “conjugation” indicate that two molecular entities (e.g., two polypeptides, or a polypeptide and a polymer such as PEG) are chemically linked.

[0218] In some embodiments, the half-life extension portion includes an Fc region, albumin, albumin-binding polypeptide, fatty acid, Pro / Ala / Ser (PAS), glycine-rich homoamino acid polymer (HAP), the β-subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES), a long, structurally indeterminate hydrophilic sequence of amino acids (XTEN), an albumin-binding small molecule, or a combination thereof. See, for example, WO2013 / 041730A1, which is incorporated herein by reference in its entirety. In certain embodiments, the half-life extension portion is albumin (e.g., human serum albumin).

[0219] In some embodiments, the heterologous portion includes lumican. Lumican binds to collagen, which is abundantly and ubiquitously expressed in tumors. In certain embodiments, conjugating lumican to the IL-12 protein (e.g., the IL-12α subunit and / or the IL-12β subunit) can improve the targeting of the IL-12 protein to tumors (e.g., by preventing and / or reducing the entry of the IL-12 protein into the systemic circulation), thereby reducing the toxicity of the IL-12 protein. Additional disclosures (e.g., sequences) of lumicans that may be used are provided elsewhere in this disclosure.

[0220] In some embodiments, the heterogeneous portion encodes a cytokine, chemokine, or growth factor other than IL-12. Cytokines are known in the art, and the term itself refers to a generalized group of small proteins secreted by certain cells within the immune system that have an effect on other cells. Cytokines are known to enhance cellular immune responses, and, as used herein, may include, but are not limited to, TNFα, IFN-γ, IFN-α, TGFβ, IL-1, IL-2, IL-4, IL-10, IL-13, IL-17, IL-18, and chemokines. Chemokines are useful for studies investigating responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction, among other applications. Chemokines are known in the art and are a type of cytokine that induces chemotaxis of neighboring responsive cells, typically leukocytes, to the site of infection. Non-exclusive examples of chemokines include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10. Growth factors are well known in the art, and the term itself may be interchangeable with the term cytokine. As used herein, the term “growth factor” refers to naturally occurring substances that can signal between cells and stimulate cell growth. Cytokines can be growth factors, but certain types of cytokines can also have inhibitory effects on cell growth, and therefore distinguish the two terms.Non-specific examples of growth factors include adremedullin (AM), angiopoietin (Ang), autocrine cell motility stimulants, bone morphogenetic proteins (BMPs), ciliary neurotrophic factor (CNTF), leukemia suppressor factor (LIF), interleukin-6 (IL-6), macrophage colony-stimulating factor (m-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and epithelial growth factors. Growth factors (EFG), ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin (EPO), fibroblast growth factor-1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 3 (FGF3), fibroblast growth factor 4 (FGF4), fibroblast growth factor 5 (FGF5), fibroblast growth factor 6 (FGF6), fibroblast growth factor 7 (FGF7), Fibroblast Growth Factor 8 (FGF8), Fibroblast Growth Factor 9 (FGF9), Fibroblast Growth Factor 10 (FGF10), Fibroblast Growth Factor 11 (FGF11), Fibroblast Growth Factor 12 (FGF12), Fibroblast Growth Factor 13 (FGF13), Fibroblast Growth Factor 14 (FGF14), Fibroblast Growth Factor 15 (FGF15), Fibroblast Growth Factor 16 (FGF16), Fibroblast Growth Factor 17 (FGF 17) Fibroblast growth factor 18 (FGF18), Fibroblast growth factor 19 (FGF19), Fibroblast growth factor 20 (FGF20), Fibroblast growth factor 21 (FGF21), Fibroblast growth factor 22 (FGF22), Fibroblast growth factor 23 (FGF23), Fetal bovine growth hormone (FBS), Glial cell lineage-derived neurotrophic factor (GDNF), Neurturin, Peresphin, Artemin, Proliferation components. Growth factor-9 (GDF9), hepatocyte growth factor (HGF), hepatome-derived growth factor (HDGF), insulin, insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2), interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), chemoproliferative factor (MSF), macrophage-stimulating protein (MSP), myostatin (GDF-8), neuregulin-1 (NRG1), neuregulin-2 Examples include (NRG2), neuregulin 3 (NRG3), neuregulin 4 (NRG4), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), placental growth factor (TCGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF).

[0221] In some embodiments, the polynucleotides of this disclosure (e.g., isolated polynucleotides) further include nucleic acid molecules encoding a leader sequence. As used herein, the term “leader sequence” refers to a sequence located at the amino terminus of the precursor form of a protein. The leader sequence is cleaved during maturation. In certain embodiments, the leader sequence includes a signal peptide. The term “signal peptide” refers to a leader sequence that ensures entry into the secretory pathway of the protein. An additional description of leader sequences is provided, for example, in US2007 / 0141666A1, which is incorporated herein by reference in its entirety. In some embodiments, a leader sequence that may be used herein includes the amino acid sequence MRVPAQLLGLLLLWLPGARCA (SEQ ID NO: 180). In some embodiments, a nucleic acid molecule encoding such a leader sequence includes the sequence shown in any one of SEQ ID NOs: 26-50. In some embodiments, a nucleic acid molecule encoding a leader sequence includes the sequence encoding one of the leader sequences of the constructs provided in Table 1.

[0222] As will be apparent from the above disclosure, in some embodiments, the polynucleotides of this disclosure (e.g., isolated polynucleotides) comprise multiple nucleic acid molecules. For example, in certain embodiments, the polynucleotides (e.g., isolated polynucleotides) comprise (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence; (ii) a second nucleic acid molecule encoding an IL-12β subunit; (iii) a third nucleic acid molecule encoding a linker (e.g., a GS linker); and (iv) a fourth nucleic acid molecule encoding an IL-12α subunit. In some embodiments, as described herein, such polynucleotides further comprise one or more additional properties described herein. For example, in some embodiments, the polynucleotide described herein comprises (1) a 5'-cap (5' to 3'), (2) a first nucleotide sequence encoding a leader sequence, (3) a second nucleotide sequence encoding an IL-12β subunit, (4) a third nucleotide sequence encoding a linker (e.g., a GS linker), (5) a fourth nucleotide sequence encoding an IL-12α subunit, and (6) a poly(A) tail. In some embodiments, the polynucleotide described herein comprises (1) a 5'-cap (5' to 3'), (2) a 5'-UTR, (3) a promoter, (4) a first nucleotide sequence encoding a leader sequence, (5) a second nucleotide sequence encoding an IL-12β subunit, (6) a third nucleotide sequence encoding a linker (e.g., a GS linker), (7) a fourth nucleotide sequence encoding an IL-12α subunit, (8) a 3'-UTR, and (9) a poly(A) tail. Additional descriptions of exemplary constructs are provided below.

[0223] In some embodiments, (i) the first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence shown in SEQ ID NO: 40; (ii) the second nucleic acid molecule (i.e., encoding the IL-12β subunit) comprises the sequence shown in SEQ ID NO: 65; (iii) the third nucleic acid molecule (i.e., encoding the linker) comprises the sequence shown in SEQ ID NO: 90; and (iv) the fourth nucleic acid molecule (i.e., encoding the IL-12α subunit) comprises the sequence shown in SEQ ID NO: 115. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 15. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 40 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 65 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 90 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 115 (i.e., an IL-12α subunit), and (6) a poly(A) tail. Examples of such polynucleotides are described herein as “A1 constructs”.

[0224] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 41; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 66; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 91; and (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 116. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 16. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 41 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 66 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 91 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 116 (i.e., an IL-12α subunit), and (6) a poly(A) tail. Examples of such polynucleotides are described herein as “A2 constructs”.

[0225] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 42; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 67; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 92; and (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 117. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 17. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 42 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 67 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 92 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 117 (i.e., an IL-12α subunit), and (6) a poly(A) tail. Examples of such polynucleotides are described herein as “A3 constructs”.

[0226] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 43; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 68; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 93; and (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 118. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 18. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 43 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 68 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 93 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 118 (i.e., an IL-12α subunit), and (6) a poly(A) tail. Examples of such polynucleotides are described herein as “A4 constructs”.

[0227] In some embodiments, the polynucleotides described herein (e.g., isolated polynucleotides) include (i) a first nucleic acid molecule encoding a leader sequence (5' to 3'); (ii) a second nucleic acid molecule encoding an IL-12β subunit; (iii) a third nucleic acid molecule encoding a first linker (e.g., a first GS linker); (iv) a fourth nucleic acid molecule encoding an IL-12α subunit; (v) a fifth nucleic acid molecule encoding a second linker (e.g., a second GS linker); and (vi) a sixth nucleic acid molecule encoding a half-life extension portion (e.g., human serum albumin). In some embodiments, as described herein, such polynucleotides further include one or more additional properties described herein. For example, in some embodiments, the polynucleotides described herein include (1) a 5'-cap (from 5' to 3'), (2) a first nucleotide sequence encoding a leader sequence, (3) a second nucleotide sequence encoding an IL-12β subunit, (4) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (5) a fourth nucleotide sequence encoding an IL-12α subunit, (6) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (7) a sixth nucleotide sequence encoding a half-life extension portion (e.g., human serum albumin), and (8) a poly(A)tail. In some embodiments, the polynucleotides described herein include (1) a 5'-cap (5' to 3'), (2) a 5'-UTR, (3) a promoter, (4) a first nucleotide sequence encoding a leader sequence, (5) a second nucleotide sequence encoding an IL-12β subunit, (6) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (7) a fourth nucleotide sequence encoding an IL-12α subunit, (8) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (9) a sixth nucleotide sequence encoding a heterogeneous moiety (e.g., albumin), (10) a 3'-UTR, and (11) a poly(A)tail. Further descriptions of such exemplary constructs are provided below.

[0228] In some embodiments, (i) the first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence shown in SEQ ID NO: 26; (ii) the second nucleic acid molecule (i.e., encoding the IL-12β subunit) comprises the sequence shown in SEQ ID NO: 51; (iii) the third nucleic acid molecule (i.e., encoding the first linker) comprises the sequence shown in SEQ ID NO: 76; (iv) the fourth nucleic acid molecule (i.e., encoding the IL-12α subunit) comprises the sequence shown in SEQ ID NO: 101; (v) the fifth nucleic acid molecule (i.e., encoding the second linker) comprises the sequence shown in SEQ ID NO: 126; and (vi) the sixth nucleic acid molecule (i.e., encoding the half-life extension portion) comprises the sequence shown in SEQ ID NO: 147. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 26 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 51 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 76 (i.e., a first GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 101 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 126 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 147 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "L1 constructs".

[0229] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 27; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 52; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 77; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 102; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 127; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 148. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 27 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 52 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 77 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 102 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 127 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 148 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "L2 constructs".

[0230] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 28; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 53; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 78; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 103; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 128; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 149. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 3. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 28 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 53 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 78 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 103 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 128 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 149 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "L3 constructs".

[0231] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 29; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 54; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 79; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 104; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 129; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 150. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 4. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 29 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 54 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 79 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 104 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 129 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 150 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “M1 constructs”.

[0232] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 30; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 55; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 80; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 105; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 130; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 151. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 5. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analog) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 30 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 55 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 80 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 105 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 130 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 151 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “M2 constructs”.

[0233] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 31; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 56; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 81; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 106; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 131; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 152. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 6. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 31 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 56 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 81 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 106 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 131 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 152 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “M3 constructs”.

[0234] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 32; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 57; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 82; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 107; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 132; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 153. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 7. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 32 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 57 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 82 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 107 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 132 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 153 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "H1 constructs".

[0235] In some embodiments, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 33; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 58; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 83; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 108; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 133; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 154. Thus, in certain embodiments, the polynucleotides described herein (e.g., isolated polynucleotides) comprise the sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide comprises one or more additional features described herein. For example, in some embodiments, the polynucleotides provided herein comprise, from 5' to 3', (1) a 5'-cap (or cap analog), (2) a first nucleotide sequence comprising the sequence set forth in SEQ ID NO: 33 (i.e., the leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO: 58 (i.e., the IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO: 83 (i.e., the GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO: 108 (i.e., the IL-12α subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO: 133 (i.e., the second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth in SEQ ID NO: 154 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as the "H2 construct".

[0236] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 34; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 59; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 84; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 109; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 134; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 155. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 9. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 34 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 59 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 84 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 109 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 134 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 155 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "H3 constructs".

[0237] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 37; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 62; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 87; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 112; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 137; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 158. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 12. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 37 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 62 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 87 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 112 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 137 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 158 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "vH1 constructs".

[0238] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 38; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 63; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 88; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 113; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 138; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 159. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 13. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analog) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 38 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 63 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 88 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 113 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 138 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 159 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "vH2 constructs".

[0239] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 39; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 64; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 89; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 114; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 139; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 160. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 14. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 39 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 64 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 89 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 114 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 139 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 160 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "vH3 constructs".

[0240] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 44; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 69; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 94; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 119; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 140; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 161. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 19. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 44 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 69 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 94 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 119 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 140 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 161 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “B1 constructs”.

[0241] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 45; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 70; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 95; (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 120; (v) the fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 141; and (vi) the sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 162. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 20. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 45 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 70 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 95 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 120 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 141 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 162 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “B2 constructs”.

[0242] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 46; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 71; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 96; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 121; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 142; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 163. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 21. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 46 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 71 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 96 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 121 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 142 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 163 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “B3 constructs”.

[0243] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 47; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 72; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 97; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 122; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 143; and (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 164. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 22. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 47 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 72 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 97 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 122 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 143 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 164 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “B4 constructs”.

[0244] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 35; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 60; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 85; (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 110; (v) the fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 135; and (vi) the sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 156. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 10. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 35 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 60 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 85 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 110 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 135 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 156 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as "CO constructs".

[0245] In some embodiments, (i) the first nucleic acid molecule comprises the sequence shown in SEQ ID NO: 36; (ii) the second nucleic acid molecule comprises the sequence shown in SEQ ID NO: 61; (iii) the third nucleic acid molecule comprises the sequence shown in SEQ ID NO: 86; (iv) the fourth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 111; (v) the fifth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 136; and (vi) the sixth nucleic acid molecule comprises the sequence shown in SEQ ID NO: 157. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 11. In some embodiments, the polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include (1) a 5'-cap (or cap analogue) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 36 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 61 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 86 (i.e., a GS linker), (5) a fourth nucleotide sequence including the sequence shown in SEQ ID NO: 111 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 136 (i.e., a second GS linker), (7) a sixth nucleotide sequence including the sequence shown in SEQ ID NO: 157 (i.e., human serum albumin), and (8) a poly(A) tail. Examples of such polynucleotides are described herein as “CP constructs”.

[0246] In some embodiments, the polynucleotides described herein (e.g., isolated polynucleotides) include (i) a first nucleic acid molecule encoding a leader sequence (5' to 3'); (ii) a second nucleic acid molecule encoding an IL-12β subunit; (iii) a third nucleic acid molecule encoding a first linker (e.g., a first GS linker); (iv) a fourth nucleic acid molecule encoding an IL-12α subunit; (v) a fifth nucleic acid molecule encoding a second linker (e.g., a second GS linker); (vi) a sixth nucleic acid molecule encoding a half-life extension portion (e.g., human serum albumin); (vii) a seventh nucleic acid molecule encoding a third linker (e.g., a third GS linker); and (viii) an eighth nucleic acid molecule encoding a lumican. In some embodiments, as described herein, such polynucleotides further include one or more additional properties described herein. In some embodiments, the polynucleotides described herein include (1) a 5'-cap (from 5' to 3'), (2) a first nucleotide sequence encoding a leader sequence, (3) a second nucleotide sequence encoding an IL-12β subunit, (4) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (5) a fourth nucleotide sequence encoding an IL-12α subunit, (6) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (7) a sixth nucleotide sequence encoding a heterogeneous portion (e.g., albumin), (8) a seventh nucleotide sequence encoding a third linker (e.g., a GS linker), (9) an eighth nucleotide sequence encoding an additional portion (e.g., lumican), and (12) a poly(A)tail.In some embodiments, the polynucleotides described herein include (1) a 5'-cap (5' to 3'), (2) a 5'-UTR, (3) a promoter, (4) a first nucleotide sequence encoding a leader sequence, (5) a second nucleotide sequence encoding an IL-12β subunit, (6) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (7) a fourth nucleotide sequence encoding an IL-12α subunit, (8) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (9) a sixth nucleotide sequence encoding a heterogeneous moiety (e.g., albumin), (10) a seventh nucleotide sequence encoding a third linker (e.g., a GS linker), (11) an eighth nucleotide sequence encoding an additional moiety (e.g., lumican), (12) a 3'-UTR, and (13) a poly(A)tail. Further descriptions of such exemplary constructs are provided below.

[0247] In some embodiments, (i) a first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence shown in SEQ ID NO: 48; (ii) a second nucleic acid molecule (i.e., encoding the IL-12β subunit) comprises the sequence shown in SEQ ID NO: 73; (iii) a third nucleic acid molecule (i.e., encoding the first linker) comprises the sequence shown in SEQ ID NO: 98; (iv) a fourth nucleic acid molecule (i.e., encoding the IL-12α subunit) comprises the sequence shown in SEQ ID NO: 123; (v) a fifth nucleic acid molecule (i.e., encoding the second linker) comprises the sequence shown in SEQ ID NO: 144; (vi) a sixth nucleic acid molecule (i.e., encoding the half-life extension portion) comprises the nucleic acid sequence shown in SEQ ID NO: 165; (vii) a seventh nucleic acid molecule (i.e., encoding the third linker) comprises the sequence shown in SEQ ID NO: 168; and (viii) an eighth nucleic acid molecule (i.e., encoding the lumican) comprises the sequence shown in SEQ ID NO: 171. Therefore, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) comprises the sequence shown in SEQ ID NO: 23. In some embodiments, the polynucleotide comprises one or more additional properties described herein.For example, in some embodiments, the polynucleotide provided herein comprises: (1) a 5'-cap (or cap analog) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 48 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 73 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 98 (i.e., a first GS linker), and (5) the sequence shown in SEQ ID NO: 123 (i.e., an IL-12β subunit). (6) a fourth nucleotide sequence including the -12α subunit; (7) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 144 (i.e., the second GS linker); (8) a seventh nucleotide sequence including the sequence shown in SEQ ID NO: 168 (i.e., the third GS linker); (9) an eighth nucleotide sequence including the sequence shown in SEQ ID NO: 171 (i.e., lumican); and (10) a poly(A) tail. Examples of such polynucleotides are described herein as "C1 constructs".

[0248] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 49; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 74; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 99; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 124; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 145; (vi) the sixth nucleic acid molecule includes the sequence shown in SEQ ID NO: 166; (vii) the seventh nucleic acid molecule includes the sequence shown in SEQ ID NO: 169; and (viii) the eighth nucleic acid molecule includes the sequence shown in SEQ ID NO: 172. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 24. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotides provided herein include: (1) a 5'-cap (or cap analog) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 49 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 74 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 99 (i.e., a first GS linker), and (5) the sequence shown in SEQ ID NO: 124 (i.e., an IL-12β subunit). (6) a fourth nucleotide sequence including the -12α subunit; (7) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 145 (i.e., the second GS linker); (8) a seventh nucleotide sequence including the sequence shown in SEQ ID NO: 169 (i.e., the third GS linker); (9) an eighth nucleotide sequence including the sequence shown in SEQ ID NO: 172 (i.e., lumican); and (10) a poly(A) tail. Examples of such polynucleotides are described herein as "C2 constructs".

[0249] In some embodiments, (i) the first nucleic acid molecule includes the sequence shown in SEQ ID NO: 50; (ii) the second nucleic acid molecule includes the sequence shown in SEQ ID NO: 75; (iii) the third nucleic acid molecule includes the sequence shown in SEQ ID NO: 100; (iv) the fourth nucleic acid molecule includes the sequence shown in SEQ ID NO: 125; (v) the fifth nucleic acid molecule includes the sequence shown in SEQ ID NO: 146; (vi) the sixth nucleic acid molecule includes the nucleic acid sequence shown in SEQ ID NO: 167; (vii) the seventh nucleic acid molecule includes the sequence shown in SEQ ID NO: 170; (viii) the eighth nucleic acid molecule includes the sequence shown in SEQ ID NO: 173. Thus, in certain embodiments, the polynucleotide described herein (e.g., isolated polynucleotide) includes the sequence shown in SEQ ID NO: 25. In some embodiments, the polynucleotide includes one or more additional properties described herein. For example, in some embodiments, the polynucleotide provided herein comprises: (1) a 5'-cap (or cap analog) (5' to 3'), (2) a first nucleotide sequence including the sequence shown in SEQ ID NO: 50 (i.e., a leader sequence), (3) a second nucleotide sequence including the sequence shown in SEQ ID NO: 75 (i.e., an IL-12β subunit), (4) a third nucleotide sequence including the sequence shown in SEQ ID NO: 100 (i.e., a first GS linker), and (5) the sequence shown in SEQ ID NO: 125 (i.e., an IL-12β subunit). (6) a fourth nucleotide sequence including the -12α subunit; (7) a fifth nucleotide sequence including the sequence shown in SEQ ID NO: 146 (i.e., the second GS linker); (8) a seventh nucleotide sequence including the sequence shown in SEQ ID NO: 170 (i.e., the third GS linker); (9) an eighth nucleotide sequence including the sequence shown in SEQ ID NO: 173 (i.e., lumican); and (10) a poly(A) tail. Examples of such polynucleotides are described herein as "C3 constructs".

[0250] Lipid nanoparticles and delivery systems In some embodiments, this disclosure relates to the delivery of biologically active molecules (e.g., IL-12 protein) to cells. In certain embodiments, delivery may occur in vivo (e.g., by administering the polynucleotides described herein to a target) or ex vivo (e.g., by culturing the polynucleotides described herein with cells in vitro). In some embodiments, delivery of the polynucleotides described herein (e.g., isolated polynucleotides) may be carried out using any suitable delivery system known in the art. In certain embodiments, the delivery system is a vector. Thus, in some embodiments, this disclosure provides a vector containing the polynucleotides of this disclosure. Suitable vectors that may be used are known in the art. See, for example, Sung et al., Biomater Res 23(8) (2019).

[0251] In some embodiments, the polynucleotides described herein (e.g., isolated polynucleotides containing nucleic acid molecules encoding the IL-12 protein) are delivered using lipid nanoparticles. Accordingly, in some embodiments, this disclosure relates to polynucleotides expressing IL-12 (e.g., RNA) encapsulated by lipid nanoparticles, compositions thereof, and the use of such compositions for treating subjects having or suspected of having cancer.

[0252] As used herein, "lipid nanoparticles" (LNPs) refer to vesicles, for example, spherical vesicles having a continuous lipid bilayer. Lipid nanoparticles can be used in methods for delivering pharmaceutical therapies to targeted sites. Non-limiting examples of LNPs include liposomes, double-headed amphiphilic compounds (bolaamphiles), solid lipid nanoparticles (SLNs), Examples include nanostructured lipid carriers (NLCs) and monolayer membrane structures (e.g., archaeosomes and micelles).

[0253] In some embodiments, lipid nanoparticles contain one or more lipids. Lipids, as used herein, refer to a group of organic compounds, including esters of fatty acids, characterized in some embodiments by being insoluble in water but soluble in many organic solvents. They are typically 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) “derivative 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 lipids in the LNPs contain cationic lipids. In some embodiments, one or more lipids in the LNP include a lipidoid, e.g., TT3. Thus, in some embodiments, any of the polynucleotides described herein (e.g., including the sequence shown in any one of SEQ ID NOs: 1 to 25, a 5'-cap (or cap analog), and a poly(A) tail) can be encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide includes the sequence shown in SEQ ID NO: 1, a 5'-cap (or cap analog), and a poly(A) tail, where the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide includes the sequence shown in SEQ ID NO: 2, a 5'-cap (or cap analog), and a poly(A) tail, where the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 3, a 5'-cap (or cap analogue), and a poly(A) tail, where the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3).In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 4, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 5, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 6, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 7, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 8, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 9, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 10, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 11, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 12, a 5'-cap (or cap analogue), and a poly(A) tail, where the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3).In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 13, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 14, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 15, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 16, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 17, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 18, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 19, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 20, a 5'-cap (or cap analogue), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 21, a 5'-cap (or cap analogue), and a poly(A) tail, where the polynucleotide is encapsulated in lipidoid nanoparticles (e.g., TT3).In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 22, a 5'-cap (or cap analog), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 23, a 5'-cap (or cap analog), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 24, a 5'-cap (or cap analog), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3). In some embodiments, the polynucleotide comprises the sequence shown in SEQ ID NO: 25, a 5'-cap (or cap analog), and a poly(A) tail, wherein the polynucleotide is encapsulated in a lipidoid nanoparticle (e.g., TT3).

[0254] Such lipids useful in this 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), and distearyldimethylammonium. Examples include (DSDMA), N,N-dioleyl-N,N,-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); NN-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).

[0255] In certain aspects of this disclosure, the lipid, e.g., lipidoid, is TT3. When used herein, TT3 can form lipid nanoparticles for the delivery of various biologically active drugs to cells. In addition, this disclosure also demonstrates that unloaded TT3-LNPs can induce immunogenic cell death (ICD) in cancer cells in vivo and in vitro. Immunogenic cell death, as described herein, refers to a form of cell death that can induce an effective immune response through the activation of dendritic cells (DCs) and the resulting activation of a specific T cell response. In certain aspects of this disclosure, the cells undergoing immunogenic cell death are tumor cells. Immunogenic tumor cell death can induce an effective anti-tumor immune response. In certain aspects of this disclosure, the lipid nanoparticles include TT3-LNPs (TT3-LNP-modRNA) that encapsulate a nucleotide sequence (modRNA) encoding IL-12, which encodes only a reporter gene. The nucleotide sequence encoding IL-12 works synergistically with TT3-LNP to induce higher levels of ICD in tumor cells compared to TT3-LNP alone. In certain aspects of this disclosure, the lipid nanoparticles include TT3-LNP encapsulating modRNA encoding the IL-12 molecule. IL-12, an immunomodulatory cytokine, elicits a potent immune response against local tumors. The combination of TT3-LNP, modRNA, and IL-12 expression is effective not only for synergistic inhibition of tumor cells at the site but also for eliciting a systemic anti-tumor immune response, killing distal tumor cells and preventing tumor recurrence.

[0256] In certain aspects of this disclosure, the cationic lipid is DOTAP. DOTAP can also form lipid nanoparticles when used herein. DOTAP can be used for highly efficient transfection of DNA, including yeast artificial chromosomes (YACs), into eukaryotic cells for transient or stable gene expression, and is also suitable for the efficient transfer of other negatively charged molecules, such as RNA, oligonucleotides, nucleotides, ribonucleoprotein (RNP) complexes, and proteins, into mammalian cell research samples.

[0257] In certain aspects of this disclosure, the cationic lipid is lipofectamine. Lipofectamine, as used herein, is a common transfection reagent manufactured and marketed by Invitrogen and used in molecular cell biology. It is used to increase 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 encapsulate the transfection payload, e.g., modRNA. The RNA-containing liposomes (positively charged on their surface) are fused to a neutral copolymer that mediates the fusion of the liposomes with the cell membrane. Consequently, it can fuse with the negatively charged plasma membrane of living cells, allowing nucleic acid cargo molecules to traverse the cytoplasm for replication or expression.

[0258] In some aspects of this disclosure, LNPs are primarily composed of cationic lipids along with other lipid components. These typically include, but are not limited to, phosphatidylcholine (PC) class (e.g., 1,s-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), sterols (e.g., cholesterol), and other lipid molecules belonging to 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 exemplary formulations of LNPs, TT3-LNPs, and DOTAP-LNPs. [Table 2]

[0259] In some embodiments, the LNP contains C14-PEG2000. In certain embodiments, C14-PEG2000 includes 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), or both. As described herein (see, for example, Example 3), in some embodiments, C14-PEG2000 (or other lipid components disclosed herein) may be embedded in the LNP before polynucleotide encapsulation. In some embodiments, C14-PEG2000 (or other lipid components disclosed herein) may be added to the LNP after polynucleotide encapsulation. For example, in a particular embodiment, a polynucleotide (e.g., isolated polynucleotide) containing a nucleic acid molecule encoding the IL-12 protein (e.g., IL-12α and / or IL-12β subunits) is encapsulated in an LNP, and then C14-PEG2000 (or other lipid components disclosed herein) is attached to the LNP, for example, using micelles.

[0260] The particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucosal adhesion, cellular water uptake and buffer exchange into the nanoparticles, and protein diffusion. In some aspects of this disclosure, the diameter of the LNPs is in the range of about 30 to about 500 nm. In some aspects of this disclosure, the diameter of the LNPs 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 aspects of this disclosure, the diameter of the LNPs is in the range of 100 to 160 nm. In certain aspects of this disclosure, the diameter of the LNP may 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 certain aspects, the lipid nanoparticles have a diameter of about 140 nm.

[0261] Zeta potential is a measure of the effective charge on the surface of lipid nanoparticles. The magnitude of the zeta potential provides information about the stability of the particles. In some aspects of this disclosure, the zeta potential of LNPs is in the range of about 3 to about 6 mV. In some aspects of this disclosure, the zeta potential of the LNP may be approximately 3mv, approximately 3.1mv, approximately 3.2mv, approximately 3.3mv, approximately 3.4mv, approximately 3.5mv, approximately 3.6mv, approximately 3.7mv, approximately 3.8mv, approximately 3.9mv, approximately 4mv, approximately 4.1mv, approximately 4.2mv, approximately 4.3mv, approximately 4.4mv, approximately 4.5mv, approximately 4.6mv, approximately 4.7mv, approximately 4.8mv, approximately 4.9mv, approximately 5mv, approximately 5.1mv, approximately 5.2mv, approximately 5.3mv, approximately 5.4mv, approximately 5.5mv, approximately 5.6mv, approximately 5.7mv, approximately 5.8mv, approximately 5.9mv, or approximately 6mv.

[0262] In some embodiments, this disclosure relates to polynucleotides (e.g., mRNA) encapsulated by lipid nanoparticles (LNPs). In some embodiments of this disclosure, the mass ratio of lipid to polynucleotide (e.g., mRNA) in the LNP is in the range of about 1:2 to about 15:1. In some embodiments, the mass ratio of lipid to polynucleotide (e.g., mRNA) is 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 2 The ratios may be approximately 1:1, 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 aspects of this disclosure, the mass ratio between lipids and polynucleotides (e.g., mRNA) is about 10:1.

[0263] Pharmaceutical composition In some embodiments, this disclosure relates to pharmaceutical compositions comprising polynucleotides, vectors, and / or lipid nanoparticles as described herein. In some embodiments of this disclosure, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier (excipient). "Acceptable," as used herein, means that the carrier must be compatible with the active ingredient of the composition and must not be harmful to the subject being treated. In some embodiments, the carrier can stabilize the active ingredient. Examples of pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkoins, Ed. KE Hoover.

[0264] Pharmaceutical compositions used for in vivo administration must be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Lipid nanoparticles can be placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous needle.

[0265] In some aspects of this disclosure, pharmaceutical compositions may be formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, percutaneous, sublingual, submucosal, percutaneous, or transmucosal administration. In some aspects of this disclosure, pharmaceutical compositions may be formulated for intratumoral injection. Intratumoral injection, as used herein, refers to direct injection into the tumor. High concentrations of a composition can be achieved in situ while using small amounts of drug. Local delivery of immunotherapy enables multiple combination therapies while preventing significant systemic exposure and off-target toxicity.

[0266] In some aspects of this disclosure, the pharmaceutical composition may be formulated for intramuscular, intravenous, or subcutaneous injection.

[0267] In certain aspects of this disclosure, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, buffer, excipient, salt, or stabilizer in the form of a lyophilized formulation or aqueous solution. For example, Remington: The Science and Practice of Pharmacy 20 thSee Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover. Acceptable carriers and excipients or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin The following are examples of the following: proteins such as 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 dextran; 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 nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0268] In some embodiments, the pharmaceutical compositions described herein are, for example, Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent No. 4,485,045 This includes lipid nanoparticles that can be prepared by methods known in the art, such as those described in U.S. Patent No. 4,544,545, which are incorporated herein by reference in their entirety. Liposomes having an enhanced circulation time are disclosed in U.S. Patent No. 5,013,556, which are incorporated herein by reference in their entirety. In some embodiments, liposomes can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to obtain liposomes having a desired diameter.

[0269] In some aspects of this disclosure, pharmaceutical compositions are formulated in a sustained-release format. A preferred example of a sustained-release preparation comprises a semipermeable matrix of a solid hydrophobic polymer containing lipid nanoparticles, the matrix of which is in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include, but are not limited to, polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPROM DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyrate.

[0270] In some embodiments, suitable surfactants include, but are not limited to, nonionic agents, such as polyoxyethylene sorbitan (e.g., TWEEN® 20, 40, 60, 80, or 85) and other sorbitans (e.g., SPAN® 20, 30, 60, 80, or 85). In some embodiments, the surfactant-containing composition contains between 0.05% and 5% of the surfactant. In some embodiments, the composition contains 0.1% and 2.5%. It will be recognized that other components, such as mannitol or other pharmaceutically acceptable vehicles, may be added if necessary.

[0271] In some embodiments, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, granules, liquids or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or inhalation.

[0272] To prepare solid compositions such as tablets, the main active ingredient can be mixed with a pharmaceutically acceptable carrier, such as conventional tableting components like corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gum, and other pharmaceutically acceptable diluents, such as water, to form a solid preliminary formulation composition containing a homogeneous mixture of the compound of the Disclosure or a non-toxic, pharmaceutically acceptable salt thereof. When these preliminary formulation compositions are referred to as homogeneous, it means that the active ingredient is uniformly dispersed throughout the composition, and as a result, the composition can be easily further divided into equally effective unit dosage forms such as tablets, pills, and capsules. The solid preliminary formulation composition is then further divided into unit dosage forms of the types described above, containing about 0.1 to about 500 mg of the active ingredient of the Disclosure. Tablets or pills of the novel compositions can be coated or otherwise formulated to provide dosage forms that provide the benefit of extended action. For example, a tablet or pill may contain an inner and outer dosing component, the latter in the form of an envelope covering the former. The two components serve to withstand disintegration in the stomach, and the inner component may be separated by an enteric coating that allows it to pass through the duodenum intact or allows for delayed release. Various materials can be used for such an enteric coating or coating, including several polymer acids, as well as mixtures of polymer acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0273] Suitable emulsions can be prepared using commercially available fat emulsions such as INTRALIPID®, LIPOSYN®, INFONUTROL®, LIPOFUNDIN®, and LIPIPHYSAN®. The active ingredient may be dissolved in a pre-mixed emulsion composition, or dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) to form an emulsion when mixed with phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water. It will be recognized that other components, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain oil up to about 20%, for example, between about 5% and about 20%. The fat emulsion may contain fat droplets of a suitable size and may have a pH in the range of about 5.5 to about 8.0.

[0274] Pharmaceutical compositions for inhalation or inhalation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain the preferred pharmaceutically acceptable excipients listed above. In some embodiments, the compositions are administered orally or via nasal respiration for topical or systemic effects.

[0275] Compositions in pharmaceutically acceptable solvents can be atomized by gas. The atomized solution can be inhaled directly from the atomizing device, or the atomizing device can be attached to a face mask, tent, or intermittent positive pressure respirator. The solution, suspension, or powder composition can be administered from a device that delivers the formulation in an appropriate manner.

[0276] therapeutic application In certain aspects of this disclosure, the polynucleotides, vectors, lipid nanoparticles, and / or pharmaceutical compositions described herein (collectively referred to herein as “Compositions”) are used to treat a disease or disorder. In certain aspects, the disease or disorder includes cancer. Non-limiting examples of cancers that may be treated are provided elsewhere in this disclosure.

[0277] In some embodiments, an effective amount of any of the compositions described herein is administered via a preferred route, e.g., intratumoral administration, intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebospinal, subcutaneous, and recurrent. It is administered to subjects requiring it via intranodal, intrasynovial, intrathecal, oral, inhalation, or local routes. Commercial nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be sprayed, and lyophilized powders can be sprayed after reconstitution. In some embodiments, the pharmaceutical compositions described herein are aerosolized or inhaled as lyophilized and crushed powders using fluorocarbon formulations and metered-dose inhalers. In some embodiments, the pharmaceutical compositions described herein are formulated for intratumoral injection. In some embodiments, the pharmaceutical compositions described herein are administered to subjects via local routes, for example, by injection into a local site such as a tumor site or an infection site. In some embodiments, the subjects are humans.

[0278] As will be apparent from this disclosure, in some embodiments, the compositions described herein are administered to a subject in an effective amount to impart a therapeutic effect, either alone or in combination with one or more other active agents. In some embodiments, the compositions are administered to a subject with cancer, and the therapeutic effect includes reduced tumor load, reduction of cancer cells, increased immune activity, or a combination thereof. Whether the administered composition (e.g., lipid nanoparticles) has achieved a therapeutic effect can be determined using any suitable method known in the art (e.g., measuring tumor volume and / or T cell activity). The effective amount will vary depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, health status, size, sex and weight, the duration of treatment, the nature of any combination therapy, the specific route of administration, and similar factors within the expertise of the healthcare professional, as will be recognized by those skilled in the art.

[0279] Empirical considerations such as half-life generally contribute to the determination of dosage. The frequency of administration may, but not necessarily, be determined and adjusted throughout treatment, generally based on the treatment and / or suppression and / or recovery and / or delay of the target disease / impairment. Alternatively, a sustained continuous-release formulation of the compositions described herein (e.g., lipid nanoparticles) may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0280] In some embodiments of this disclosure, the treatment is a single injection of the composition disclosed herein. In some embodiments, the single injection is administered intratumorally to a subject requiring it.

[0281] In some embodiments of this disclosure, the dosage for the compositions described herein may be determined empirically in an individual given one or more doses of the composition (e.g., lipid nanoparticles described herein). In some embodiments, the individual is given progressively increasing doses of the compositions described herein. To evaluate the efficacy of the compositions described herein, indicators of disease / disorder may be tracked. For repeated administrations over several days or longer, depending on the condition, in some embodiments, the treatment is continued until the desired symptom suppression occurs or until a sufficient therapeutic level is achieved and the target disease or disorder or its symptoms are reduced.

[0282] In some aspects of this disclosure, the dosing frequency is approximately once every week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, approximately once every five weeks, approximately once every six weeks, approximately once every seven weeks, approximately once every eight weeks, approximately once every nine weeks, or approximately once every ten weeks; or approximately once every month, approximately every two months, or approximately every three months, or at longer intervals. The dosing regimen (e.g., dosage and / or dosing frequency) of the composition described herein used (e.g., lipid nanoparticles) can be varied over time.

[0283] In some aspects of this disclosure, the method includes administering one dose or multiple doses of the compositions described herein to a subject requiring it.

[0284] The appropriate dosage of a composition (e.g., lipid nanoparticles described herein) depends on the specific composition (e.g., lipid nanoparticles), the type and severity of the disease / disorder (e.g., cancer), whether the composition (e.g., lipid nanoparticles) is administered for prophylactic or therapeutic purposes, previous treatments, the subject's medical history and response to the composition (e.g., lipid nanoparticles), and the discretion of the attending physician. In some embodiments, a clinician may administer the compositions disclosed herein until a dosage is reached that achieves the desired outcome. In some embodiments, the desired outcome is a reduction in tumor load, a decrease in cancer cells, or increased immune activity. Administration of one or more compositions described herein may be continuous or intermittent, depending, for example, the recipient's physiological state, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the compositions described herein may be essentially continuous over a pre-selected period, or it may be a series of intervald doses, for example, before, during, or after the onset of the target disease or disorder.

[0285] As used herein, mitigating a target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Mitigating a disease does not necessarily require a curative outcome. As used herein, “delaying” the onset of a target disease or disorder means extending, hindering, slowing, delaying, stabilizing, and / or delaying the progression of the disease. This delay can be of varying lengths depending on the disease being treated and / or the patient’s medical history. Methods for delaying or mitigating the onset of a disease, or delaying the onset of a disease, are methods that reduce the probability of developing one or more symptoms of the disease within a given time frame, and / or reduce the severity of symptoms within a given time frame, compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to give statistically significant results.

[0286] In some embodiments, the compositions described herein are administered to a subject in need in an amount sufficient to reduce tumor load or cancer cell growth in vivo, 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 higher. In some embodiments, the compositions described herein are administered in an amount effective to increase immune activity, 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 higher.

[0287] In some embodiments, administration of a composition (e.g., a polynucleotide, vector, lipid nanoparticle, or pharmaceutical composition as described herein) to a subject enhances immune activity, such as T cell activity, in the subject. In certain embodiments, the immune activity is enhanced or increased by at least about 0.5 times, at least about 1 time, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 50 times, or more, compared to the immune activity of a reference subject (e.g., the subject before administration of the composition, or a corresponding subject that was not responsive to administration of the composition).

[0288] In some embodiments, subjects are individuals who have cancer, are suspected of having cancer, or are at risk of developing cancer. In some embodiments, cancer is selected from a group of head and neck cancers, including melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatome, 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 squamous cell head and neck cancer. In some embodiments, cancer may be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, or Hodgkin lymphoma.

[0289] Subjects with a target disease or disorder can be identified through routine health checkups, such as clinical examinations, organ function tests, CT scans, or ultrasounds. Subjects suspected of having a target disease or disorder may exhibit one or more symptoms of the disease or disorder. Subjects at risk for a disease or disorder may have one or more risk factors associated with that disease or disorder. Subjects at risk for a disease or disorder can also be identified through routine medical procedures.

[0290] In some embodiments, the compositions described herein are co-administered with at least one additional preferred therapeutic agent. In some embodiments, the at least one additional preferred therapeutic agent includes an anticancer agent, an antiviral agent, an antibacterial agent, or other agents that enhance and / or complement the immunostimulatory effect of the compositions described herein (e.g., lipid nanoparticles). Further examples of additional therapeutic agents that may be used in combination with the compositions described herein include chemotherapeutic agents, targeted anticancer therapies, oncolytic agents, cytotoxic agents, immunotherapy, cytokines, surgical procedures, radiotherapy, activators of costimulatory molecules, immune checkpoint inhibitors, vaccines, cellular immunotherapy, or any combination thereof. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered to the subject sequentially, i.e., each therapeutic agent is administered at a different time. In some embodiments, the compositions described herein and at least one additional therapeutic agent are administered to the subject substantially simultaneously.

[0291] Those skilled in the art will recognize that any combination of the compositions described herein and other anticancer agents (e.g., chemotherapeutic agents) can be used in any order to treat cancer. The combinations described herein may be selected based on several factors, including, but are not limited to, effectiveness in reducing tumor formation or tumor growth, reducing cancer cells, increasing immune activity, and / or alleviating at least one symptom associated with cancer, or effectiveness in mitigating the side effects of other agents in the combination. For example, the combination therapies described herein may reduce any of the side effects associated with each individual member of the combination, for example, the side effects associated with anticancer agents.

[0292] In some embodiments, other anticancer agents include chemotherapy, radiotherapy, surgery, immunotherapy, or a combination thereof. In some embodiments, chemotherapeutic agents include carboplatin, cisplatin, docetaxel, gemcitabine, nab-paclitaxel (nab-paclitexal), pemetrexed, vinorelbine, or a combination thereof. In some embodiments, radiotherapy includes ionizing radiation, gamma rays, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, close-range radiation therapy, whole-body radioisotopes, radiosensitizers, or a combination thereof. In some embodiments, surgical therapy includes curative surgery (e.g., tumor removal surgery), prophylactic surgery, laparoscopic surgery, laser surgery, or a combination thereof. In some embodiments, immunotherapy includes adoptive cell transfer, therapeutic cancer vaccines, or a combination thereof.

[0293] In some embodiments, the chemotherapeutic agent is a platinum-based agent, e.g., carboplatin, oxaliplatin, cisplatin, nedaplatin, satraplatin, lobaplatin, triplatin, tetranitrate, picoplatin, prolyndac, alloplatin, and other derivatives; a topoisomerase I inhibitor, e.g., camptothecin, topotecan, irinotecan / SN38, rubitecan, berotecan, and other derivatives; a topoisomerase II inhibitor, e.g., etoposide (VP-16), daunorubicin, or doxorubicin (e.g., doxorubicin, doxorubicin HCl, doxorubicin). Doxorubicin analogs, or doxorubicin and its salts or analogs in liposomes), mitoxantrone, acralubicin, epirubicin, idarubicin, amrubicin, amsacrin, pirarubicin, barurubicin, zorubicin, teniposide, and other derivatives; antimetabolites, e.g., folate family (methotrexate, pemetrexed, larcitrexed, aminopterin, and related products); purine antagonists (thioguanine, fludarabine, cladribine, 6-mercaptopurine, pentostatin, clofarabine, and Related substances) and pyrimidine antagonists (cytarabine, phloxuridine, azacitidine, tegafur, carmofur, capecitabine, gemcitabine, hydroxyurea, 5-fluorouracil (5FU), and related substances); alkylating agents, e.g., nitrogen mustards (e.g., cyclophosphamide, melphalan, chlorambucil, mechloretamine, ifosfamide, trophosfamide, prednimustine, bendamustine, uramustine, estramustine, and related substances); nitrosulfame Rare compounds (e.g., carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozosin, and related compounds); triazenes (e.g., dacarbazine, altoretamine, temozolomide, and related compounds); alkyl sulfonates (e.g., busulfan, mannosulfan, treosulfan, and related compounds); procarbazines; mitobronitol, and aziridines (e.g., carbocone, triadiquan, thiotepa, triethylenemalamine, and related compounds);Antibiotics, e.g., hydroxyureas, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and other derivatives); anthracendions (e.g., mitoxantrone and related products); Streptomyces family (e.g., bleomycin, mitomycin C, actinomycin, plicamycin); ultraviolet light; and combinations thereof.

[0294] In some embodiments, other anticancer agents are antibodies. Antibodies (preferably monoclonal antibodies) achieve their therapeutic effects against cancer cells through various mechanisms. They can have a direct effect in inducing apoptosis or programmed cell death. They can efficiently inhibit tumor cell proliferation by blocking components of signaling pathways, such as growth factor receptors. In cells expressing monoclonal antibodies, they can induce anti-idiotype antibody formation. Indirect effects include mobilizing cytotoxic cells such as monocytes and macrophages. This type of antibody-mediated cell death is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind to complement, resulting in direct cytotoxicity known as complement-dependent cell-mediated cytotoxicity (CDC). Combining surgical procedures with immunotherapy drugs or methods is a successful approach, as demonstrated, for example, in Gadri et al. 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 32(4): 333-40. The following list provides some non-limiting examples of anticancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present invention: avagovomab (CA-125), absiximab (CD41), adecatumumab (EpCAM), aftuzumab (CD20), aracizumab pegol (VEGFR2), artumomab penteate (CEA), amatsuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), alsitumomab (CEA), bavituximab (phosphatidylserine), vectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), vibatuzumab meltansine (CD44) v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab meltansine (mucin CanAg), cantuzumab lavtansine (MUC1), capromab pendetide (prostate cancer cells), carrumab (CNT0888), catumakisomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab vogatox (EpCAM), cizutumumab (IGF-1 receptor), clodiximab (claudin), criba Tuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), darotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), droditumab (DR5), eclomeximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enabatuzumab (PDL1) 92), encituximab (NPC-1C), epratuzumab (CD22), erzmakisomab (HER2 / neu, CD3), etalacizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficratuzumab (SCH900105), figtumumab (IGF-1 receptor), frambotumab (glycoprotein 75), fresolimmab (TG) F-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-1β), girentuximab (carbonic anhydrase 9 (CA-IX)), glenbatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), iclucumab (VEGFR-1), igovoma (CA-125), indatur Mavlav tansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), rabetuzumab (CEA), lexatumumab (TRAIL-R2), rivivirumab (hepatitis B surface antigen), lintuzumab (CD33), rorbotuzumab meltansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matsuzuma BB (EGFR), mepolizumab (IL-5), milatuzumab (CD74), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab pasdotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), narutuzumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumumab (PDGF-R α), onarutuzumab (human scatter factor receptor kinase)kinase), oportuzumab monatox (EpCAM), olegobomab (CA-125), oxerumab (OX-40), panitumumab (EGFR), patritumumab (HER3), pemtumoma (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), lacosumomab (N-glycolylneuraminic acid), radretumumab (fibronectin extradomain-B), rafibirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samarizumab (CD200), sibrotuzumab (FAP), siltz Ximab (IL-6), Tabalmab (BAFF), Takatuzumab Tetraxetan (Alpha-fetoprotein), Tapritumomab Paptox (CD19), Tenatumomab (Tenascin C), Teprotumumab (CD221), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL-13), Tositumomab (CD20), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Tucothumab cermoloykin (EpCAM), Ublituximab (MS4A1), Urelumab (4-1BB), Boroxiximab (integrin α5β1), Botumumab (tumor antigen CTAA16.88), Saltumumab (EGFR), Zanolimumab (CD4).

[0295] In some embodiments, other anticancer agents include cytokines, chemokines, costimulatory molecules, fusion proteins, or combinations thereof. Examples of chemokines, but not limited to, include CCR7 and its ligands CCL19 and CCL21, as well as CCL2, CCL3, CCL5, and CCL16. Other examples include CXCR4, CXCR7, and CXCL12. Furthermore, costimulatory or regulatory molecules, such as B7 ligands (B7.1 and B7.2), may also be useful. For example, interleukins, especially (e.g., IL-1 to IL-17), interferons (e.g., IFN-alpha 1 to IFN-alpha 8, IFN-alpha 10, IFN-alpha 13, IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, IFN-alpha 21, IFN-beta 1, IFN-W, IFNE-1 and IFNK), hematopoietic factors, TGF (e.g., TGF-α, TGF-β, and other members of the TGF family), but not limited to, 41BB, 41BB-L, CD137, CD137L, CTLA-4GITR, GITRL, Fas, Fas-L, TNFR1, TRAIL-R1, TRAIL-R2 The final members of the receptor tumor necrosis factor family and their ligands, including p75NGF-R, DR6, LT.beta.R, RANK, EDAR1, XEDAR, Fn114, Troy / Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT, and CD95 (Fas / APO-1), as well as other co-stimulatory molecules, glucocorticoid-induced TNFR-related proteins, TNF receptor-associated apoptosis-mediated proteins (TRAMP), and other cytokines such as cell death receptor 6 (DR6), are also useful. In particular, CD40 / CD40L and OX40 / OX40L are important targets for combination immunotherapy due to their direct effects on T cell survival and proliferation.For a review article, please refer to Lechner et al. 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3 (11), 1317-1340.

[0296] In some forms, other anticancer drugs are bacterial treatments. Researchers are using anaerobic bacteria, such as Clostridium novyi, to destroy the oxygen-poor interior of tumors. These should then die upon contact with the oxygenated side of the tumor, meaning they will be harmless to the rest of the body. Another strategy is to use anaerobic bacteria transformed with an enzyme that can convert non-toxic prodrugs into toxic drugs. Due to tumor necrosis and bacterial growth in hypoxic areas, the enzyme is expressed only in the tumor. Thus, a prodrug applied systemically is metabolized into a toxic drug only in the tumor. This has been demonstrated to be effective with the non-pathogenic anaerobic organism Clostridium sporogenes.

[0297] In some embodiments, other anticancer agents are kinase inhibitors. Cancer cell growth and survival are closely linked to the deregulation of kinase activity. A wide range of inhibitors are used to restore normal kinase activity and thus reduce tumor growth. The group of targeted kinases includes receptor tyrosine kinases, e.g., BCR-ABL, B-Raf, EGFR, HER-2 / ErbB2, IGF-IR, PDGFR-α, PDGFR-β, c-Kit, Flt-4, Flt3, FGFR1, FGFR3, FGFR4, CSF1R, c-Met, RON, c-Ret, ALK; cytoplasmic tyrosine kinases, e.g., c-SRC, c-YES, Abl, JAK-2; serine / threonine kinases, e.g., ATM, Aurora A and B, CDK, mTOR, PKCi, PLK, b-Raf, S6K, STK11 / LKB1; and lipid kinases, e.g., PI3K, SK1. Small molecule kinase inhibitors include, for example, PHA-739358, nilotinib, dasatinib, and PD166326, NSC 743411, lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), batalanib (PTK787 / ZK222584), sutent (SU11248), sorafenib (BAY 43-9006), and leflunomide (SU101). For more information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

[0298] In some embodiments, other anticancer agents are Toll-like receptors. Members of the Toll-like receptor (TLR) family are important links between innate and adaptive immunity, and the effects of many adjuvants rely on TLR activation. Numerous established cancer vaccines incorporate ligands for TLRs to boost the vaccine response. In addition to TLR2, TLR3, TLR4, and especially TLR7 and TLR8 have been investigated for cancer treatment in passive immunotherapy approaches. Closely related TLR7 and TLR8 contribute to the antitumor response by influencing immune cells, tumor cells, and the tumor microenvironment and can be activated by nucleoside analog structures. All TLRs have been used as standalone immunotherapeutic agents or cancer vaccine adjuvants and can be synergistically combined with the formulations and methods of this disclosure. For more information, see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

[0299] In some embodiments, other anticancer agents are angiogenesis inhibitors. Angiogenesis inhibitors prevent the extensive growth of blood vessels (angiogenesis) necessary for tumor survival. Angiogenesis, which is promoted by tumor cells in response to their increased demand for nutrients and oxygen, can be blocked, for example, by targeting different molecules. Non-limiting examples of angiogenesis-mediated molecules or angiogenesis inhibitors that can be combined with this disclosure include soluble VEGF (VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1 and VEGFR2, and coreceptors neuropilin-1 and neuropilin-2)1 and NRP...

Claims

[Claim 1] The invention described in the specification.

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