Expression constructs and uses thereof

Abstract

The present disclosure relates to isolated polynucleotides comprising sequences encoding interleukin (IL)-12 molecules, and delivery systems capable of carrying the polynucleotides. The present disclosure also includes methods of making the polynucleotides and methods of treatment using the same. Provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the beta subunit of the IL-12 protein ("IL-12β"). Also provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α").

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 135,501, filed January 8, 2021, which is incorporated by reference herein in its entirety.

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

[0003] Background of the disclosure Due to its ability to activate both NK cells and cytotoxic T cells, IL-12 protein has been investigated as a promising anti-cancer therapeutic since 1994. See Nastala, CL et al., J Immunol 153: 1697-1706 (1994). However, despite high expectations, early clinical studies did not provide satisfactory results. Lasek W. et al., Cancer Immunol Immunother 63: 419-435, 424 (2014). Repeated administration of IL12 resulted in adaptive responses and a progressive decrease in IL-12-induced interferon gamma (IFN-γ) levels in the blood in most patients. Ibid. It was also recognized that IL-12-induced anticancer activity was primarily mediated by secondary secretion of IFNγ, but co-induction of IFN-γ by IL-12 in conjunction with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG) caused severe toxicity. Id. In addition to negative feedback and toxicity, the limited efficacy of IL-12 therapy in clinical settings may be caused by the strong 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 time protocols for IL12 therapy. See 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: 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 have not significantly affected patient survival. Kang, WK, et al., Human Gene Therapy 12: 671-684 (2001). Thus, 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) [Non-Patent Document 4] Leonard, JP et al., Blood 90: 2541-2548 (1997) [Non-Patent Document 5] Coughlin, CM et al., Cancer Res. 57: 2460-2467 (1997) [Non-Patent Document 6] Asselin-Paturel, C. et al., Cancer 91: 113-122 (2001) [Non-Patent Document 7] Saudemont, A. et al., Leukemia 16: 1637-1644 (2002) [Non-Patent Document 8] Kang, WK, et al., Human Gene Therapy 12: 671-684 (2001) Summary of the Invention [Means for solving the problem]

[0005] BRIEF SUMMARY OF THE DISCLOSURE Provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"). In some embodiments, the nucleic acid molecule has a sequence identity at least about 75%, at least about 76%, at least about 77%, at least about 78%, or similar to the sequence set forth in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. %, 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 the nucleotide sequence of the present invention.

[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 set forth in 51. In some embodiments, a nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO:52. In some embodiments, a nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO: 53. In some embodiments, a nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO:54.In some embodiments, the nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO: 55. In some embodiments, the nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO:56. 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 set forth in SEQ ID NO: 57. In some embodiments, the nucleic acid molecule encoding IL-12β 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 set forth in SEQ ID NO: 58. 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 set forth 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 set forth in SEQ ID NO:65, 69, or 74.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 set forth in SEQ ID NO:66, 70, or 75. In some embodiments, the nucleic acid molecule encoding IL-12β comprises a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:62. In some embodiments, the nucleic acid molecule encoding IL-12β comprises a nucleotide sequence that is at least 99% or 100% identical to the sequence set forth in SEQ ID NO:63. In some embodiments, the nucleic acid molecule encoding IL-12β comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:64.

[0007] Also provided herein is an isolated polynucleotide comprising a nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"). In some embodiments, the nucleic acid molecule has a sequence identity of at least about 77%, at least about 80%, or at least about 90% identical to the sequence set forth 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. The nucleic acid sequence may be 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.

[0008] In some embodiments, a nucleic acid molecule encoding IL-12α comprises 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 set forth in SEQ ID NO:101. In some embodiments, a nucleic acid molecule encoding IL-12α 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 set forth in SEQ ID NO:102. In some embodiments, a nucleic acid molecule encoding IL-12α 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 set forth in SEQ 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 set forth in SEQ ID NO:104.In some embodiments, a nucleic acid molecule encoding IL-12α 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 set forth in SEQ ID NO: 105. In some embodiments, a nucleic acid molecule encoding IL-12α 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 set forth in SEQ ID NO: 106. 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 set forth in SEQ ID NO: 107. 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 set forth in SEQ ID NO: 108. In some embodiments, the nucleic acid molecule encoding IL-12α 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 set forth 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 set forth in SEQ ID NO:115, 119, or 124.In some embodiments, the nucleic acid molecule encoding IL-12α 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 set forth in SEQ ID NO:116, 120, or 125. In some embodiments, the nucleic acid molecule encoding IL-12α comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:112. In some embodiments, the nucleic acid molecule encoding IL-12α comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:113. In some embodiments, the nucleic acid molecule encoding IL-12α comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:114.

[0009] The 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 a beta subunit of IL-12 protein ("IL-12β") and has a sequence as set forth in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. comprising 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;a second nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α") and having a sequence identity at least about 77% similar to the sequence set forth 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 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 the nucleotide sequence of the present invention;

[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 set forth in SEQ ID NO:51. The second nucleic acid molecule comprises a nucleotide sequence and / or 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 set forth in SEQ 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 set forth in SEQ ID NO:52; and / or the second 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 set forth in SEQ 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 set forth in SEQ ID NO:53, and / or the second 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 set forth in SEQ 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO:115, 119, or 124.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 set forth in SEQ ID NO:66, 70, or 75, 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 set forth in SEQ ID NO:116, 120, or 125. In some embodiments, the first nucleic acid molecule comprises a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:62, 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 set forth 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 set forth 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 set forth 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 set forth 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 set forth in SEQ ID NO:114.

[0011] In some embodiments, the isolated polynucleotide disclosed herein further comprises a third nucleic acid molecule encoding a linker connecting the first nucleic acid molecule and the second nucleic acid molecule. In certain 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. In certain embodiments, the third nucleic acid molecule encoding the linker comprises a sequence set forth in any one of SEQ ID NOs: 168-170.

[0012] In some embodiments, the isolated polynucleotide of the present disclosure further comprises an additional nucleic acid molecule encoding a half-life extending moiety. In certain embodiments, the half-life extending moiety comprises 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 an additional nucleic acid molecule encoding a leader sequence. In certain embodiments, the additional nucleic acid molecule encoding a leader sequence comprises any one of the sequences set forth in SEQ ID NOs: 26-50.

[0014] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:26; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:51; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:76; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:101; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:126; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:147.

[0015] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:27; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:52; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:77; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:102; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:127; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:148.

[0016] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:28; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:53; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:78; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:103; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:128; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:149.

[0017] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:29; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:54; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:79; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:104; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:129; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:150.

[0018] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:30; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:55; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:80; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:105; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:130; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:151.

[0019] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:31; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:56; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:81; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:106; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:131; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:152.

[0020] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:32; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:57; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:82; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:107; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:132; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:153.

[0021] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:33; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:58; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:83; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:108; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:133; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:154.

[0022] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:34; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:59; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:84; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:109; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:134; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:155.

[0023] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:37; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:62; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:87; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:112; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:137; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:158.

[0024] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:38; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:63; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:88; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:113; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:138; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:159.

[0025] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:39; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:64; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:89; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:114; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:139; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:160.

[0026] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:44; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:69; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:94; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:119; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:140; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:161.

[0027] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:45; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:70; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:95; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:120; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:141; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:162.

[0028] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:46; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:71; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:96; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:121; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:142; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:163.

[0029] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:47; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:72; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:97; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:122; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:143; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:164.

[0030] As used herein, a nucleic acid molecule includes (from 5' to 3') (i) a first nucleic acid molecule encoding a leader sequence, (ii) a second nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, (iv) a fourth nucleic acid molecule encoding an alpha subunit of the IL-12 protein ("IL-12α"), (v) a fifth nucleic acid molecule encoding a second linker, and (vi) a sixth nucleic acid molecule encoding human serum albumin. An isolated polynucleotide is provided, comprising: (a) a first nucleic acid molecule comprising the sequence set forth in SEQ ID NO:36; (b) a second nucleic acid molecule comprising the sequence set forth in SEQ ID NO:61; (c) a third nucleic acid molecule comprising the sequence set forth in SEQ ID NO:86; (d) a fourth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:111; (e) a fifth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:136; and (f) a sixth nucleic acid molecule comprising the sequence set forth in SEQ ID NO:157.

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

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

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

[0034] Provided herein is 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 a beta subunit of an IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding an alpha subunit of an IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:40; (b) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:65; (c) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:90; and (d) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:115.

[0035] Provided herein is 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 a beta subunit of an IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding an alpha subunit of an IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:41; (b) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:66; (c) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:91; and (d) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:116.

[0036] Provided herein is 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 a beta subunit of an IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding an alpha subunit of an IL-12 protein ("IL-12α"), wherein (a) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:42; (b) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:67; (c) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:92; and (d) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:117.

[0037] Provided herein is 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 a beta subunit of an IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding an alpha subunit of an 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; and (d) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:118.

[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’-OG(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-methylpseudouridine, m7G(5')ppp(5')(2'OMeA)pG, and combinations thereof.

[0039] In some embodiments, the isolated polynucleotide of the present disclosure further comprises a regulatory element.In certain 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 linked nucleosides, 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 the linked nucleosides. In certain embodiments, the 3' tailing region of the linked nucleosides comprises a polyA tail, a polyAG quartet, or a stem-loop sequence.

[0041] In some embodiments, the isolated polynucleotides of the present disclosure include 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-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-guanosine, 5-amino ... Selected from the group consisting of anosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

[0042] In some embodiments, the isolated polynucleotide described herein can replicate autonomously. 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 comprises Venezuelan equine encephalitis virus, Semliki Forest virus, Sindbis virus, or a combination thereof.

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

[0044] The present disclosure also provides lipid nanoparticles (LNPs) comprising (i) any of the isolated polynucleotides described herein, and (ii) one or more lipids. In certain embodiments, the one or more lipids comprise cationic lipids. In some embodiments, the lipid is an ionizable lipid. In some embodiments, the lipid is a lipidoid, such as N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3). In some embodiments, the LNP comprises 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 30-500 nm. In certain embodiments, the LNPs have a diameter of about 50-400 nm. In some embodiments, the LNPs have a diameter of about 70-300 nm. In some embodiments, the LNPs have a diameter of about 100-200 nm. In some embodiments, the LNPs have a diameter of about 100-175 nm. In some embodiments, the LNPs have a diameter of about 100-160 nm.

[0046] In some embodiments, the lipids and the isolated polynucleotide (e.g., modified RNA) have a mass ratio of about 1:2 to about 2:1. In some embodiments, the lipids 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 lipid and the isolated polynucleotide (e.g., modified RNA) have a mass ratio of about 10:1.

[0047] Provided herein is a pharmaceutical composition comprising any of the isolated polynucleotides, vectors or LNPs described herein and pharma- ceutical acceptable carriers.In certain embodiments, the pharmaceutical composition is formulated for intratumoral, intrathecal, intramuscular, intravenous, subcutaneous, inhalation, intradermal, intralymphatic, intraocular, intraperitoneal, intrapleural, intraspinal, intravascular, nasal, transdermal, sublingual, submucosal, transdermal or transmucosal administration.

[0048] Provided herein is a cell comprising any of the isolated polynucleotides, vectors, or LNPs described herein. In some embodiments, the cell is an in vitro cell, an ex vivo cell, or an in vivo cell.

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

[0050] The present disclosure further provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any of the isolated polynucleotides, vectors, LNPs, or pharmaceutical compositions described herein.In certain embodiments, the disease or disorder comprises cancer.In some embodiments, the cancer comprises 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, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, head and neck cancer, or combinations thereof.

[0051] In some embodiments, the method of treating a disease or disorder disclosed herein further comprises administering at least one additional therapeutic agent to the subject.In certain embodiments, the at least one additional therapeutic agent comprises a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic agent, a cytotoxic agent, an immune-based therapy, a cytokine, a surgical procedure, a radiation treatment, an activator of costimulatory molecules, an immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.In some embodiments, the immune checkpoint inhibitor comprises an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, an anti-TIM3 antibody, or any combination thereof. [Brief description of the drawings]

[0052] [Figure 1A-1B] Figures 1A and 1B provide a comparison of tumor volumes in tumor-bearing mice treated with a single intratumoral administration of one of the following: (i) PBS (control; open circles); (ii) repRNA cotranscriptionally capped with a 5' cap analog (filled circles in Figure 1A; solid lines in Figure 1B); and (iii) repRNA posttranscriptionally capped by enzymatic addition of a 5' cap (filled squares in Figure 1A; dashed lines in Figure 1B). Tumor volumes were measured at various time points after administration (x-axis). Figure 1A shows the average tumor volume. Figure 1B shows the tumor volumes of individual animals.

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

[0054] [Diagram 3] FIG. 3 provides a comparison of Kaplan-Meier survival analyses of tumor-bearing mice treated with a single intratumoral administration of one of the following: (i) PBS (control; open circles); (ii) repRNA generated using A3G+E1 vector and cap analog cotranscriptional capping (open triangles); (iii) modified mRNA (non-self-replicating) generated using cap analog cotranscriptional capping (open squares); (iv) repRNA generated using alternative evolution vector and cap analog cotranscriptional capping (closed circles); and (v) repRNA generated using alternative evolution vector and enzymatic posttranslational capping (closed squares).

[0055] [Figure 4A]Figure 4A provides a comparison of the in vitro transfection efficiency of different RNA constructs described herein, as measured using FACS analysis, and Figure 4B provides a comparison of the concentration of IL-12 protein detected in the supernatant of cells transfected with different RNA constructs described herein. In both Figures 4A and 4B, the RNA constructs tested included: (i) repRNAs generated using A3G+E1 vector and cap analog co-transcriptional capping (closed circles); (ii) repRNAs generated using A3G+E1 vector and enzymatic post-translational capping (closed squares); (iii) repRNAs generated using alternative evolution+E1 vector and cap analog co-transcriptional capping (closed triangles); (iv) repRNAs generated using alternative evolution+E1 vector and enzymatic post-translational capping (closed inverted triangles); (v) repRNAs generated using alternative+E1 vector and cap analog co-transcriptional capping (diamonds); (vi) repRNAs generated using A3G+E1 evolved vector and cap analog co-transcriptional capping (open circles); (vii) repRNAs generated using A3G-E1 vector and cap analog co-transcriptional capping (open squares); and (viii) repRNAs generated using alternative evolution-E1 SGP scarred end vector (open triangles). Transfection efficiency is shown as the frequency of IL-12+ cells observed in the different groups. The x-axis represents the time post-transfection when transfection efficiency was assessed. [Figure 4B]Figure 4A provides a comparison of the in vitro transfection efficiency of different RNA constructs described herein, as measured using FACS analysis, and Figure 4B provides a comparison of the concentration of IL-12 protein detected in the supernatant of cells transfected with different RNA constructs described herein. In both Figures 4A and 4B, the RNA constructs tested included: (i) repRNAs generated using A3G+E1 vector and cap analog co-transcriptional capping (closed circles); (ii) repRNAs generated using A3G+E1 vector and enzymatic post-translational capping (closed squares); (iii) repRNAs generated using alternative evolution+E1 vector and cap analog co-transcriptional capping (closed triangles); (iv) repRNAs generated using alternative evolution+E1 vector and enzymatic post-translational capping (closed inverted triangles); (v) repRNAs generated using alternative+E1 vector and cap analog co-transcriptional capping (diamonds); (vi) repRNAs generated using A3G+E1 evolved vector and cap analog co-transcriptional capping (open circles); (vii) repRNAs generated using A3G-E1 vector and cap analog co-transcriptional capping (open squares); and (viii) repRNAs generated using alternative evolution-E1 SGP scarred end vector (open triangles). Transfection efficiency is shown as the frequency of IL-12+ cells observed in the different groups. The x-axis represents the time post-transfection when transfection efficiency was assessed.

[0056] [Diagram 5]FIG. 5 provides a comparison of the in vitro transfection efficiency of the following RNA constructs, measured using FACS analysis: (i) repRNAs made using the cap analog co-transcriptional capping method and A3G+E1 vector with a 3' end terminating in a restriction enzyme scar (circle); (ii) repRNAs made using the cap analog (alternate origin) method and A3G+E1 vector with a 3' end terminating in a restriction enzyme scar (square); (iii) repRNAs made using the cap analog co-transcriptional capping method and A3G+E1 vector with a 3' end terminating in an intact polyA sequence (triangle); (iv) repRNAs made using the cap analog (alternate origin) method and A3G+E1 vector with a 3' end terminating in an intact polyA sequence (inverted triangle). PBS-treated cells were used as control (diamond). Transfection efficiency is shown as the frequency of IL-12+ cells observed in the different groups. The x-axis represents the time after transfection when transfection efficiency was evaluated.

[0057] [Figure 6A] Figures 6A and 6B provide a comparison of the concentrations of IL-12 protein observed in the tumors and serum of tumor-bearing mice treated with a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses indicated along the x-axis. Figure 6A shows the concentration of IL-12 protein in the tumor (top graph) and serum (bottom graph) 24 hours after administration. Figure 6B shows the concentration of IL-12 protein in the tumor (top graph) and serum (bottom graph) 96 hours after administration. [Figure 6B]Figures 6A and 6B provide a comparison of the concentrations of IL-12 protein observed in the tumors and serum of tumor-bearing mice treated with a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses indicated along the x-axis. Figure 6A shows the concentration of IL-12 protein in the tumor (top graph) and serum (bottom graph) 24 hours after administration. Figure 6B shows the concentration of IL-12 protein in the tumor (top graph) and serum (bottom graph) 96 hours after administration.

[0058] [Figure 7A] Figures 7A, 7B, and 7C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 24 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated along the x-axis. [Figure 7B]Figures 7A, 7B, and 7C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 24 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated along the x-axis. [Figure 7C] Figures 7A, 7B, and 7C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 24 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated along the x-axis.

[0059] [Figure 8A] Figures 8A, 8B, and 8C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 96 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated along the x-axis. [Figure 8B]Figures 8A, 8B, and 8C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 96 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated along the x-axis. [Figure 8C] Figures 8A, 8B, and 8C provide a comparison of the concentrations of IL-12 protein observed in the spleen, draining lymph nodes, and non-draining lymph nodes, respectively, of tumor-bearing mice 96 hours after a single intratumoral administration of an RNA construct encoding one of the following murine 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"). The different RNA constructs were administered to the animals at one of two doses, as indicated 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-bearing mice treated with a single intratumoral administration of an RNA construct encoding one of the following mouse IL-12 proteins: (i) mIL-12 alone (triangles); (ii) mIL-12 conjugated to albumin (squares); and (iii) mIL-12 conjugated to albumin and lumican (circles). The RNA constructs were administered to animals at the following doses: 0.25 μg (filled symbols) or 2.5 μg (open symbols). The x-axis provides the time points (post-administration) at which the concentrations of IL-12 and IFN-γ were measured.

[0061] [Figure 10] Figure 10 shows a graphical representation of data on optimality (mean_codon_score) versus minimum folding free energy (kcal / mol) (MFE) for 1137 distinct sequences encoding the fusion protein 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. Diamond symbols represent sequences with very high codon optimality and MFE, as described in Example 5. Triangles represent codon-optimized sequences containing the most frequently used triplet at each amino acid position.

[0062] [Figure 11] FIG. 11 provides a comparison of IL-12 protein secretion observed in the supernatants of cells transfected with different RNA constructs (x-axis) containing codon-optimized IL-12 sequences. As indicated, IL-12 in constructs A1-A4 was not conjugated to any other moiety. 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 triangle, square, octagon, or circle dots, and horizontal bars indicate the average of triplicate measurements. The x-axis represents the variant used and the y-axis represents the concentration of IL-12 (ng / ml).

[0063] [Figure 12A-12B]Figures 12A, 12B, and 12C provide a comparison of the concentrations of IL-12 protein observed in the tumor (Figure 12A), serum (Figure 12B), and spleen (Figure 12C) of tumor-bearing mice that received a single intratumoral administration of RNA constructs containing (i) codon-optimized IL-12 sequences conjugated to albumin and lumican (first circle set from the left in PDX1, PDX2, and PDX3, respectively); (ii) codon-optimized IL-12 sequences conjugated to albumin (second circle set in PDX1, PDX2, and PDX3, respectively); or (iii) codon-optimized IL-12 sequences alone (third circle set in PDX1, PDX2, and PDX3, respectively). Untreated animals were used as controls (last circle set in PDX1, PDX2, and PDX3, respectively). "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, and PDX2 was established from a metastatic ER, PR, HER2 negative lesion in the lung of a TNBC patient. [Figure 12C]Figures 12A, 12B, and 12C provide a comparison of the concentrations of IL-12 protein observed in the tumor (Figure 12A), serum (Figure 12B), and spleen (Figure 12C) of tumor-bearing mice that received a single intratumoral administration of RNA constructs containing (i) codon-optimized IL-12 sequences conjugated to albumin and lumican (first circle set from the left in PDX1, PDX2, and PDX3, respectively); (ii) codon-optimized IL-12 sequences conjugated to albumin (second circle set in PDX1, PDX2, and PDX3, respectively); or (iii) codon-optimized IL-12 sequences alone (third circle set in PDX1, PDX2, and PDX3, respectively). Untreated animals were used as controls (last circle set in PDX1, PDX2, and PDX3, respectively). "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, and PDX2 was established from a metastatic ER, PR, HER2 negative lesion in the lung of a TNBC patient.

[0064] [Figure 13] Figure 13 provides a comparison of tumor volumes in TNBC mice treated with vehicle control (open squares) or repRNA encoding IL-12 (repRNA) (weekly for a total of four doses). The repRNA was administered to animals at one of the following doses: (i) 5 μg (closed circles), (ii) 0.5 μg (closed squares), or (iii) 0.05 μg (closed triangles).

[0065] [Figure 14]Figure 14 provides a comparison of tumor volumes in TNBC mice treated with rIL-12-encoding repRNA modified to contain different amounts of modified nucleoside triphosphates (modNTPs): (i) 0% modNTPs (i.e., no modified repRNA) (closed circles); (ii) 25% modNTPs (closed squares); (iii) 37.5% modNTPs (closed triangles); or (iv) 50% modNTPs (closed inverted triangles). Animals treated with vehicle control were used as controls (open squares).

[0066] [Figure 15A-15B] Figures 15A and 15B show the effect of the repRNA constructs described herein on both treated and untreated tumors, respectively, in a B16-F10 mouse syngeneic cancer model. Animals were subcutaneously injected with B16-F10 tumor cells in the left and right flanks, as further described in Example 8. Once optimal tumor size was reached, vehicle control or repRNA (2.5 μg) was administered intratumorally in the left flank (i.e., treated tumors). Tumor volumes were 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 bioluminescence signal) in TNBC mouse models after remedying. As further described in Example 9, mice were injected with an initial dose of one of two firefly luciferase-encoding repRNA constructs (mRNA1 and mRNA2) (5 μg) ("Dose 1"), and then one week later, injected with a second dose of the same repRNA construct (5 μg) ("Dose 2"). In some animals, they additionally received weekly (total of two doses) of anti-IFNAR1 antibody (10 mg / kg).

[0068] [Figure 17]Figure 17 provides a comparison of the concentration of IFN-γ in supernatants collected from activated human PBMCs treated with recombinant human IL-12 protein (rhIL12) or conditioned medium as described in Example 10 (i.e., supernatants collected from BT20 cells transfected with IL-12-encoding repRNA). PBMCs were treated with rhIL12 at one of the following concentrations: 100 ng / mL ("3"), 10 ng / mL ("4"), 1 ng / mL ("5"), 0.1 ng / mL ("6"), or 0.01 ng / mL ("7"). Conditioned medium 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"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description of the Disclosure definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0070] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide" is understood to refer to one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0071] Furthermore, "and / or" as used herein should be taken as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" when used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0072] The term "about" is used herein to mean approximately, roughly, approximately, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 10 percent upward or downward (higher or lower), unless otherwise indicated.

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

[0074] "Polynucleotide" or "nucleic acid" as used herein means a sequence of nucleotides joined by phosphodiester linkages. Polynucleotides, as used herein, are oriented in a 5' to 3' direction. Polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Nucleotide bases, as used herein, are designated by the single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0075] As used herein, the term "polypeptide", unless otherwise indicated, encompasses both peptides and proteins.

[0076] The term "coding sequence" or "encoding" sequence is used herein to mean a DNA or RNA region (transcribed region) that "encodes" a particular protein, such as IL-12. A coding sequence is transcribed (DNA) and translated (RNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory regions, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from prokaryotic or eukaryotic sources, genomic DNA from prokaryotic or eukaryotic sources, and synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0077] The Kozak consensus sequence, Kozak consensus or Kozak sequence is known as a sequence present in eukaryotic mRNA, which has the 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 to the Kozak consensus sequence. In some embodiments, the polynucleotide comprises a Kozak consensus sequence.

[0078] The term "RNA" is used herein to mean a molecule that contains at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide that has 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 modification of one or more nucleotides. The term "mRNA" means "messenger RNA" and refers to a "transcript" that is produced by using a DNA template and codes for a peptide or protein. Typically, mRNA includes 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 the present 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 in vitro transcription kits available commercially. In some aspects of the present disclosure, the RNA, preferably the 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 the sequences. In certain embodiments, sequence identity is calculated based on the full length of two given SEQ ID NOs, or a portion thereof. The portion may mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NOs, or any other specified percentage. The term "identity" may also mean the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.

[0080] In certain embodiments, the methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.

[0081] "Substantial homology" or "substantial similarity," when referring to a nucleic acid or a fragment thereof, is meant to indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95-99% nucleotide sequence identity of the sequences.

[0082] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount," e.g., of a composition comprising a polynucleotide disclosed herein, refer to an amount sufficient to effect beneficial or desired results, including clinical results, when administered to a subject, including a human; e.g., "effective amount" or synonyms thereto will depend on the context in which it is applied.

[0083] The amount of a given therapeutic agent or composition will correspond to such an amount and will vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and / or weight) or host being treated, etc.

[0084] The term "half-life" refers to the period of time required for half of the activity, amount or number of a molecule to be eliminated. In the context of this disclosure, the half-life of an RNA refers to the stability of said RNA. The "occurrence" or "progression" of a disease refers to the initial manifestation and / or subsequent progression of a disease. The occurrence of a disease can be detected and evaluated using standard clinical techniques well known in the art. However, occurrence also refers to progression, which may be unpredictable. As used herein, occurrence or progression refers to the biological course of a symptom. Occurrence includes emergence, recurrence, and onset. As used herein, the onset or occurrence of a target disease or disorder includes initial onset and / or recurrence.

[0085] Nucleotide expressing IL-12 The present disclosure is directed to polynucleotides (e.g., isolated polynucleotides) that include nucleic acid molecules that encode IL-12 proteins. As described herein, the polynucleotides disclosed herein include one or more properties such that they differ (e.g., structurally and / or functionally) from naturally occurring reference polynucleotides. For example, as further described elsewhere in this disclosure, for example, nucleic acid molecules encoding IL-12 proteins (e.g., IL-12α subunit and / or IL-12β subunit) are codon-optimized (i.e., synthetic).

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

[0087] In some aspects, the modification may be located in the sugar moiety of the nucleotide. In some aspects, the modification may be located in the phosphate backbone of the nucleotide.

[0088] In some embodiments, for example, when precise timing of protein production is desired, it is desirable for modified nucleic acids introduced into cells to be degraded within the cell. Thus, in some embodiments, the nucleotide sequence encoding IL-12 contains a degradation domain, which can be acted on in a directed manner within the cell.

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

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

[0091] In some embodiments, the nucleotide sequence encoding IL-12 is circularized or concatemerized to generate a translation-competent molecule to support the interaction between polyA binding protein and 5' end binding protein. The mechanism of circularization or concatemerization can occur through at least three different routes: 1) chemical route, 2) enzymatic route, and 3) ribozyme catalytic route. 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 brought together, form a new covalent link between the 5' and 3' ends of the molecule. The 5' end can contain an NHS-ester reactive group and the 3' end can contain a 3'-amino terminal nucleotide, such that in 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 the second pathway, T4 RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid to form a new phosphorodiester linkage. In an example reaction, 1 μg of nucleic acid molecule is incubated for 1 hour at 37 °C with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. The ligation reaction can occur in the presence of a split oligonucleotide that can base pair with both the juxtaposed 5' and 3' regions to assist in 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 a nucleic acid molecule to the 3' end of a nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, a group I intron, hepatitis delta virus, a hairpin ribozyme, or may be selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.

[0095] In some embodiments, multiple separate nucleic acids, IL-12-encoding nucleotide sequences or primary constructs can be linked 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, the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, can be provided to HepG2 cells in a 1:1 ratio to alter the fatty acid metabolism of the cells. This ratio can be controlled by chemically linking nucleic acids or modified RNAs using a 3'-azido terminal nucleotide on one nucleic acid or modified RNA species and a C5-ethynyl or alkynyl-containing nucleotide on the opposite nucleic acid or IL-12-encoding nucleotide sequence species. The IL-12-encoding nucleotide sequence is added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, Mass.) according to the manufacturer's protocol. After addition of the 3'-modified nucleotide, the two nucleic acids or IL-12-encoding nucleotide sequences can be combined in aqueous solution, with or without copper, to form a new covalent linkage via a click chemistry mechanism that has been described in the literature.

[0096] In some embodiments, more than two polynucleotides can be linked together using functionalized linker molecules. For example, functionalized saccharide molecules can be chemically modified to contain multiple chemically reactive groups (SH-, NH2-, N3, etc.) that react with the cognate moieties on 3'-functionalized mRNA molecules (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 direct control of the stoichiometric ratio of the conjugated nucleic acid or mRNA.

[0097] In some aspects, to further enhance protein production, the IL-12 encoding nucleotide sequences, polynucleotides or primary constructs of the present disclosure may be coupled to other polynucleotides, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPE G]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specified cell types such as cancer cells, endothelial cells or bone cells, hormones and hormone receptors, non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, or drugs.

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

[0099] In some aspects, the primary construct is designed to encode one or more polypeptides of interest, or fragments thereof. A polypeptide of interest can include, but is not limited to, an entire polypeptide, multiple polypeptides or fragments of a polypeptide that can be encoded independently by one or more nucleic acids, multiple nucleic acids, fragments or variants of any of the aforementioned nucleic acids. As used herein, the term "polypeptide of interest" refers to any polypeptide selected to be encoded in the primary construct of the present disclosure. As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) most often linked together by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the encoded polypeptide is smaller than about 50 amino acids, such that the polypeptide is referred to as a peptide. When the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. Polypeptides can be single molecules or multi-molecular complexes such as dimers, trimers or tetramers. They can also include single chains or multi-chain polypeptides such as antibodies or insulin, which may be associated or linked. The most common disulfide linkage is 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 molecules whose amino acid sequence differs from that of a native or reference sequence. Amino acid sequence variants can have substitutions, deletions and / or insertions at certain positions in the amino acid sequence compared to the native or reference sequence. Usually, 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 (homology) to the native or reference sequence.

[0101] Thus, polynucleotides encoding polypeptides of interest that contain substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences are included within the scope of the present disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added (e.g., at the N-terminus or C-terminus) to the peptide sequences of the present disclosure. Sequence tags can be used for purifying or localizing peptides. Lysines can be used to increase the solubility of peptides or to allow biotinylation. Alternatively, amino acid residues located at the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be deleted as necessary to provide a truncated sequence. Alternatively, certain amino acids (e.g., C-terminus or N-terminus residues) can be deleted depending on the use of the sequence, for example, the expression of the sequence as part of a larger sequence that is soluble or 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 within the scope of the polypeptide of interest of this disclosure.For example, any protein fragment (meaning a polypeptide sequence that is otherwise identical but at least one amino acid residue shorter than the reference polypeptide sequence) of a reference protein that is longer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or 100 amino acids in length is provided herein.In another example, any protein that includes 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 according to the present disclosure.In certain aspects, the polypeptide utilized according to the present disclosure includes 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 aspects, the nucleotide sequence encoding IL-12 comprises a modified 5'-cap, a half-life extending 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; 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)-m3’-O 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-methylpseudouridine, m7G(5')ppp(5')(2'OMeA)pG, and combinations thereof.

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

[0106] The 5' cap structure of native mRNA is involved in nuclear export, increases mRNA stability, and binds mRNA cap-binding protein (CBP), which is responsible for mRNA stability and translational competence in cells through the association of CBP with poly(A)-binding protein to form mature circular mRNA species. The cap further assists in the removal of the 5' proximal intron during mRNA splicing.

[0107] Endogenous mRNA molecules can be 5'-end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or anteterminal transcribed nucleotide at the 5' end of the mRNA can also be 2'-O-methylated, as appropriate. 5'-decapping by hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0108] According to the present disclosure, the 5' end cap can include an endogenous cap or a cap analog. According to the present disclosure, the 5' end cap can 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' end 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] Non-limiting additional caps include the 5' caps disclosed in WO / 2017 / 201350, published November 23, 2017, which is incorporated by reference herein.

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

[0112] In some embodiments, the half-life extending moiety comprises Fc. In some embodiments, the half-life extending moiety comprises albumin or a fragment thereof.

[0113] In some 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 linked nucleosides, an AU-rich element (ARE), a post-transcriptional modulator, and combinations thereof.

[0114] In some embodiments, the regulatory element further comprises a polyA region. In some embodiments, the IL-12-encoding nucleotide sequence of the present 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 polyA tail. In some embodiments, terminal groups on the polyA tail can be incorporated for stabilization. In other embodiments, the polyA tail comprises a des-3' hydroxyl tail. During RNA processing, long stretches of adenine nucleotides (polyA tails) can be added to polynucleotides such as mRNA molecules to increase stability.

[0115] Immediately after transcription, the 3' end of the transcript can be cleaved to free the 3' hydroxyl. PolyA polymerase then adds a chain of adenine nucleotides to the RNA. A process called polyadenylation adds a polyA tail that can be between about 80 and about 250 residues long, including, for example, about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.

[0116] A polyA tail can also be added after the construct is exported from the nucleus.

[0117] According to the present disclosure, the terminal group on the polyA tail can be incorporated for stabilization. The polynucleotides of the present disclosure can include des-3' hydroxyl tails. They can also include structural moieties or 2'-O methyl modifications taught by Junjie Li, et al. (Current Biology, Vol. 15, 1501-1507, August 23, 2005, the contents of which are incorporated herein by reference in their entirety). For further polyA tails, see also WO / 2017 / 201350, published November 23, 2017, which is incorporated herein by reference.

[0118] In some aspects, the nucleotide sequence encoding IL-12 comprises 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 starts at the transcription initiation site and continues to, but does not include, the start codon, while the 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence of the regulatory role that UTR plays in terms of the stability of nucleic acid molecules and translation. The regulatory properties of UTR can be incorporated into the RNA (e.g., modified RNA) of the present disclosure to enhance the stability of the molecule. Certain properties can also be incorporated to ensure the controlled downregulation of transcripts when they are misdirected to undesirable organ sites.

[0120] 5'UTR and translation start Natural 5'UTRs have properties that play a role in translation initiation. They possess signatures such as the Kozak sequence, which is commonly known to be involved in the process by which the ribosome initiates the translation of many genes. The Kozak sequence has the 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 5'UTR or 3'UTR to regulate gene expression.For example, the elongation factor EIF4A2, which binds to secondary structured elements in 5'UTR, is required for microRNA-mediated suppression (Meijer HA et al., Science, 2013, 340, 82-85, the entirety of which is incorporated herein by reference).Different secondary structures in 5'UTR can be incorporated into adjacent regions to stabilize or selectively destabilize mRNA in specific tissues or cells.

[0122] By manipulating the characteristics typically found in abundantly expressed genes of specific target organs, the stability and protein production of the nucleic acid or mRNA of the present disclosure can be enhanced. For example, the introduction of the 5'UTR of liver-expressed mRNA such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII can be used to enhance the expression of nucleic acid molecules such as mmRNA in hepatic cell lines or the liver. Similarly, use of 5'UTRs from other tissue-specific mRNAs to improve expression in that tissue 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, introns or portions of intron sequences can be incorporated into the adjacent regions of the nucleic acids or mRNAs of the present disclosure. The incorporation of intron sequences can increase protein production and mRNA levels.

[0124] In some embodiments of the present disclosure, at least one fragment of the IRES sequence from GTX gene can be included in 5'UTR.As a non-limiting example, the fragment can be an 18 nucleotide sequence from the IRES of GTX gene.As another non-limiting example, the 18 nucleotide sequence fragment from the IRES sequence of GTX gene can be tandemly repeated in the 5'UTR of the polynucleotide described herein.The 18 nucleotide sequence can 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 replaced nucleotide(s) can be 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 more than 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 translational enhancer elements (TEEs) In some embodiments, the 5'UTR of the nucleotide sequence encoding IL-12 comprises at least one translation enhancer polynucleotide, translation enhancer element, or translation 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, the RNA (e.g., modified RNA) having at least one TEE in the 5'UTR comprises a cap in the 5'UTR. In some embodiments, the at least one TEE can be located in the 5'UTR of the 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 herein as "TEE") refers to a sequence that increases the amount of a polypeptide or protein produced from an mRNA.

[0128] In one embodiment, a TEE is a conserved element in a UTR that can promote translation activity of a nucleic acid, such as, but not limited to, cap-dependent or cap-independent translation. The conservation of these sequences has been previously shown by Panek et al. (Nucleic Acids Research, 2013, 1-10; incorporated herein by reference in its entirety) across 14 species, including humans.

[0129] In some aspects, a nucleotide sequence encoding IL-12 has at least one TEE that has 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 to that disclosed in U.S. Application No. 2014 / 0147454, the entirety of which is incorporated herein by reference. In some aspects, the RNA (e.g., modified RNA) may be modified as described in any of the following publications: U.S. Patent Publication Nos. US20090226470, US20070048776, US20130177581, and US20110124100; International Patent Publication Nos. WO1999024595, WO2012009644, WO2009075886, and WO2007025008; European Patent Publication Nos. EP2610341A1 and EP26 The present 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 to a TEE described in U.S. Pat. No. 10340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, or U.S. Pat. No. 7,183,395.

[0130] In some embodiments, the 5'UTR of the nucleotide sequence encoding IL-12 can comprise 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 more than 60 TEE sequences. In some embodiments, the TEE sequences in the 5'UTR of the RNA (e.g., modified RNA) are the same or different TEE sequences. In some embodiments, the TEE sequences are repeated once, twice, or more than three times in a pattern such as ABABAB, or AABBABBAABB, 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 spacer separating the two TEE sequences comprises other sequences known in the art that regulate the translation of an RNA (e.g., a modified RNA), such as, but not limited to, the miR sequences described herein (e.g., miR binding sites and miR seeds). In some embodiments, each spacer used to separate the two TEE sequences comprises a different miR sequence or a component of a miR sequence (e.g., a miR seed sequence).

[0132] In some aspects, the TEE used in the 5'UTR of the IL-12 encoding nucleotide sequence of the present disclosure is an IRES sequence, such as, but not limited to, those described in U.S. Pat. No. 7,468,275 and International Patent Publication No. WO2001055369, each of which is incorporated by reference in its entirety.

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

[0134] In some embodiments, the 3'UTR of a nucleotide sequence encoding IL-12 can comprise 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 more than 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 once, twice, or more than three times in a pattern such as ABABAB, or AABBABBAABB, 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 comprises a spacer separating the 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 can comprise 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, in the 3'UTR.

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

[0137] Incorporation of microRNA binding sites In some embodiments, the nucleotide sequence encoding IL-12 further comprises a sensor sequence.Sensor sequences include, for example, microRNA binding sites, transcription factor binding sites, structured mRNA sequences and / or motifs, and artificial binding sites engineered to act as pseudo-receptors of endogenous nucleic acid binding molecules.Non-limiting examples of polynucleotides that comprise at least one sensor sequence are described in US Application No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

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

[0139] MicroRNAs (or miRNAs) are 19-25 nucleotide long non-coding RNAs that bind to the 3'UTR of a nucleic acid molecule and downregulate gene expression by either reducing the stability of the nucleic acid molecule or inhibiting translation. In some embodiments, the RNA (e.g., modified RNA) comprises one or more microRNA target sequences, microRNA sequences, or microRNA seeds. Such sequences can correspond to any known microRNA, 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. Application No. 2014 / 0147454, which is incorporated herein by reference in its entirety.

[0140] The microRNA sequence comprises a "seed" region, i.e., the sequence of the region from positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed comprises positions 2-8 or 2-7 of the mature microRNA. In some embodiments, the microRNA seed comprises 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite position 1 of the microRNA. In some embodiments, the microRNA seed comprises 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), where the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite position 1 of the microRNA. See, e.g., Grimson A, Farh K, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul. 6; 27(1):91-105. The base of microRNA seed has perfect complementarity with target sequence.Provided that the microRNA in question is available, the microRNA target sequence can be engineered into the 3'UTR of the nucleic acid or mRNA of the present disclosure to target the molecule for degradation or reduced translation.This process reduces the risk of off-target effect during nucleic acid molecule delivery.The identification of microRNAs, microRNA target regions, and their expression patterns and roles in biology 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 references therein; each of which is incorporated herein by reference in its entirety).

[0141] For example, miR-122, a microRNA that is abundant in the liver, can inhibit the expression of a gene of interest when one or more target sites of miR-122 are engineered into the 3'UTR of a modified nucleic acid, enhanced modified RNA, or ribonucleic acid. The introduction of one or more binding sites for different microRNAs can be engineered to further reduce the longevity, stability, and protein translation of the modified nucleic acid, 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" can follow the rules of conventional Watson-Crick hybridization or can reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.

[0142] Conversely, for purposes of the IL-12-encoding nucleotide sequences of the present disclosure, the microRNA binding sites in which they naturally occur can be engineered out (i.e., removed) to increase protein expression in a particular tissue. 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 contains at least one miRNA binding site in the 3'UTR to direct cytotoxic or cytoprotective mRNA therapeutics to specific cells, such as, but not limited to, normal and / or cancerous cells (e.g., HEP3B or SNU449).

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

[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, granuocytes, natural killer cells, and the like. 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 myeloid dendritic cells. It has been demonstrated in the art that immune responses to exogenous nucleic acid molecules can be blocked by adding miR-142 binding sites to the 3'UTR of delivered gene constructs, allowing for 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 its entirety).

[0146] Many microRNA expression studies have been carried out in the art to profile the differential expression of microRNAs in various cancer cells / tissues and other diseases.Some microRNAs are abnormally overexpressed in certain cancer cells, while others are underexpressed.For example, microRNAs have been shown to be involved in the expression of microRNAs 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, U.S. Patent No. 8,389,210); asthma and inflammation (U.S. Patent No. 8,415,096); prostate cancer (US2013 / 0053 264); hepatocellular carcinoma (WO2012 / 151212, US2012 / 0329672, WO2008 / 054828, U.S. 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 carcinoma (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 98, WO2007 / 081740, US2012 / 0214699), leukemias and lymphomas (WO2008 / 073915, US2009 / 0092974, US2012 / 0316081, US2012 / 0283310, WO2010 / 018563, the contents of each of which are incorporated by reference in their entirety herein).

[0147] At least one microRNA site can be engineered into the 3'UTR of the nucleotide sequence encoding IL-12. In some embodiments, 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, or more microRNA sites can be engineered 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 the modified nucleic acid mRNA can reduce the degree of expression in certain cell types (e.g., hepatocytes, myeloid cells, endothelial cells, cancer cells, etc.).

[0148] In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR, about halfway 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 engineered near the 5' end of the 3'UTR and about halfway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is engineered near the 3' end of the 3'UTR and about halfway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR and about halfway between the 5' and 3' ends of the 3'UTR. In some embodiments, the microRNA site is engineered near the 5' end of the 3'UTR and near the 3' end of the 3'UTR.

[0149] In some embodiments, modified messenger RNA comprises a microRNA binding region site that has 100% identity with known seed sequence or has less than 100% identity with seed sequence.Seed sequence can be partially mutated to reduce microRNA binding affinity, thus resulting in reduced down-modulation of its mRNA transcript.In essence, the degree of match or mismatch between target mRNA and microRNA seed can act as a rheostat to finely adjust the ability of microRNA to modulate protein expression.In addition, mutations in non-seed regions of microRNA binding site can also affect the ability of microRNA to modulate protein expression.

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

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

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

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

[0154] In some embodiments, the first region of the linked nucleosides and / or at least one flanking region comprises at least one RBD. In some embodiments, the first region of the linked nucleosides comprises an RBD associated with a splicing factor and at least one flanking region comprises an RBD for a stability and / or translation factor.

[0155] Other regulatory elements in the 3'UTR In addition to microRNA binding sites, other regulatory sequences in the 3'-UTR of natural 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 can 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-richness, 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 that encode regulators of development and inflammation 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, the RNA (e.g., modified mRNA) is auxotrophic. As used herein, the term "auxotrophic" refers to an mRNA that contains at least one property that induces, promotes, or induces degradation or inactivation of the 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 to be translated, such as a specific tissue or organ or cellular environment. Cues including temperature, pH, ionic strength, water content, and the like are also contemplated.

[0157] 3'UTR and AU-rich elements 3'UTRs are known to have stretches of adenosines and uridines embedded in them. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al, 1995). Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules that contain this type of ARE include GM-CSF and TNF-a. Class III AREs are less 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. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule results in HuR binding and therefore stabilization of the message in vivo.

[0158] The introduction, removal or modification of AU-rich elements (AREs) in the 3'UTR can be used to modulate the stability of the nucleic acid or mRNA of the present disclosure. When engineering a particular nucleic acid or mRNA, one or more copies of AREs can be introduced to make the nucleic acid or mRNA of the present disclosure more unstable, thereby suppressing the translation and decreasing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase the translation and production of the resulting protein. Transfection experiments can be performed in the relevant cell system using the nucleic acid or mRNA of the present disclosure, and protein production can be assayed at various times after transfection. For example, cells can be transfected with different ARE engineered molecules, and the proteins produced can be assayed about 6 hours, about 12 hours, about 24 hours, about 48 hours, and / or about 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 comprises a triple helix at the 3' end of the modified nucleic acid, the nucleotide sequence encoding the enhanced IL-12, or the ribonucleic acid. In some embodiments, the 3' end of the nucleotide sequence encoding IL-12 comprises a triple helix alone or in combination with a polyA tail.

[0160] In some embodiments, the nucleotide sequence encoding IL-12 comprises at least a first and a second U-rich region, a conserved stem-loop region between the first and the second region, and an A-rich region. In some embodiments, the first and the second U-rich region 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 nuclear (PAN) RNA (see Wilusz et al., Genes & Development 2012 26:2392-2407; the entire contents of which are incorporated herein by reference).

[0161] Stem Loop In some embodiments, the nucleotide sequence encoding IL-12 includes a stem loop, such as, but not limited to, a histone stem loop. In some embodiments, the stem loop is a nucleotide sequence that is about 25 or about 26 nucleotides in length, such as, but not limited to, SEQ ID NOs: 7-17 described in International Patent Publication No. WO2013103659, the entirety of which is incorporated herein by reference. The histone stem loop can be located 3' to the coding region (e.g., the 3' end of the coding region). As a non-limiting example, the stem loop can be located at the 3' end of the nucleic acid described herein.

[0162] In some embodiments, IL-12-encoding nucleotide sequences that include histone stem loops can be stabilized by the addition of at least one chain-terminating nucleoside. Without wishing to be bound by theory, the addition of at least one chain-terminating nucleoside can slow down the degradation of the nucleic acid and thus increase the half-life of the nucleic acid.

[0163] In some embodiments, the chain-terminating nucleoside is one described in International Patent Publication No. 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, a 2',3'-dideoxynucleoside, such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or an -O-methyl nucleoside.

[0164] In some embodiments, the nucleotide sequence encoding IL-12 comprises a histone stem loop, a polyA tail sequence, and / or a 5' cap structure. In some embodiments, the histone stem loop precedes and / or follows the polyA tail sequence. The nucleic acid comprising the histone stem loop and the polyA tail sequence can comprise a chain-terminating nucleoside as described herein.

[0165] In some embodiments, the nucleotide sequence encoding IL-12 comprises a histone stem loop and a 5' cap structure, which may include, but is 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 protein (CBP), which is responsible for mRNA stability and translational competence in cells through the association of CBP with poly(A)-binding protein to form 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'-end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue of the mRNA and the 5'-terminal transcribed sense nucleotide. This 5'-guanylic acid cap can then be methylated to generate an N7-methyl-guanylic acid residue. The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide at the 5' end of the mRNA can also be 2'-O-methylated as needed. 5'-cap removal by hydrolysis and cleavage of the guanylic acid cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0168] The modifications to RNA of the present disclosure can generate a non-hydrolyzable cap structure that prevents cap removal, thus increasing the half-life of the mRNA. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester linkage, modified nucleotides can be used during the capping reaction. For example, 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 phosphorothioate linkages in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can be used.

[0169] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-end and / or pre-5'-terminal nucleotide of an mRNA at the 2'-hydroxyl group of the sugar ring (as mentioned above).Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

[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 the natural (i.e., endogenous, wild-type, or physiological) 5'-cap, but retain cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule.

[0171] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, where one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which 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 a capped nucleic acid molecule (e.g., an mRNA or mmRNA). The N7- and 3'-O-methylated guanine provide the terminal portion of the capped nucleic acid molecule (e.g., an mRNA or mmRNA).

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

[0173] In some aspects, the cap is a dinucleotide cap analog modified with boranophosphate or phosphoroselenoate groups at different phosphate positions, such as those described in U.S. Patent No. 8,519,110, the contents of which are incorporated herein by reference in their entirety.

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

[0175] Although cap analogs allow for simultaneous capping of nucleic acid molecules in in vitro transcription reactions, up to about 20% of the transcripts remain uncapped. This and the difference in the structure of the cap analog from the endogenous 5'-cap structure of the nucleic acid produced by the endogenous cellular transcription machinery can result in reduced translational competence and reduced cellular stability. Thus, in some aspects, the methods provided herein (see, for example, Examples 1-3) can increase the capping efficiency of the nucleotides expressing the IL-12 produced as described herein. In some embodiments, the methods provided herein cap 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%, about 100% of the polynucleotides. In some embodiments, the methods provided herein cap at least about 50% of the polynucleotides. In some embodiments, at least about 60% of the polynucleotides are capped. In some embodiments, at least about 70% of the polynucleotides are capped. In some embodiments, at least about 80% of the polynucleotides are capped. In some embodiments, at least about 85% of the polynucleotides are capped. In some embodiments, at least about 90% of the polynucleotides are capped. In some embodiments, at least about 95% of the polynucleotides are capped. In some embodiments, about 100% of the polynucleotides are capped. In some embodiments, at least about 80% to about 100% of the polynucleotides are capped.

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

[0177] In some embodiments, the RNA can be produced, for example, by in vitro transcription, and a 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme. In some embodiments, the IL-12-encoding nucleotide sequence is post-transcriptionally capped using an enzyme to generate a more authentic 5'-cap structure. As used herein, the phrase "more authentic" refers to a property that closely reflects or mimics an endogenous or wild-type property, either structurally or functionally. That is, a "more authentic" property is a better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure, or is superior in one or more respects to the corresponding endogenous, wild-type, natural or physiological property, as compared to the synthetic properties or analogs of the prior art, etc. Non-limiting examples of more authentic 5' cap structures of the present disclosure are those that have, among other things, enhanced binding of cap-binding proteins, increased half-life, reduced susceptibility 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 enzyme and recombinant 2'-O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5' terminal nucleotide of an mRNA and a guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5' terminal nucleotide of the mRNA contains a 2'-O-methyl. This cap results in greater translational competence and cellular stability, as well as reduced activation of cellular 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'-end cap includes an endogenous cap or a cap analog. In some embodiments, the 5'-end cap includes 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 comprises a 5'-5' triphosphate linkage. In some embodiments, the 5' cap comprises a 5'-5' triphosphate linkage that includes a triphosphate modification. In some embodiments, the 5' cap comprises a 2'-O or 3'-O-ribose methylated nucleotide. In some embodiments, the 5' cap comprises a modified guanosine or adenosine nucleotide. In some embodiments, the 5' cap comprises 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 comprises a modified 5' cap. Modifications in the 5' cap can increase the stability of the mRNA, can increase the half-life of the mRNA, and can increase the efficiency of mRNA translation. In some embodiments, the modified 5' cap comprises one or more of the following modifications: modification at the 2' and / or 3' positions of the capped guanosine triphosphate (GTP), replacement of the oxygen of the sugar ring (resulting in a carbocyclic ring) with a methylene moiety (CH2), modification at the triphosphate bridge portion of the cap structure, or modification at the nucleobase (G) portion.

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

[0182] IRES sequence In some embodiments, the nucleotide sequence encoding IL-12 contains an internal ribosome entry site (IRES). IRES, which was first identified as a characteristic picornavirus RNA, plays an important role in initiating protein synthesis in the absence of a 5' cap structure. IRES can act as the sole ribosome binding site or can serve as one of multiple ribosome binding sites of an mRNA. A nucleic acid or mRNA containing more than one functional ribosome binding site can code for several peptides or polypeptides that are translated independently by ribosomes ("polycistronic nucleic acid molecule"). When a nucleic acid or mRNA is provided with an IRES, a second translatable region is optionally provided in addition. Examples of IRES sequences that can be used in accordance with the present disclosure include, but are not limited to, those derived from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), foot and mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0183] Terminal structure modification: polyA tail During RNA processing, a long chain of adenine nucleotides (poly-A tail) is usually added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3' end of the transcript is cleaved to free the 3' hydroxyl. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. A process called polyadenylation adds the poly-A tail, which is between 100 and 250 residues long.

[0184] In some embodiments, the length of the 3' tail is greater than about 30 nucleotides in length. In some embodiments, the polyA tail is greater than about 35 nucleotides in length. 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 poly-AG 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 poly-A 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 discovered that the poly-AG quartet results in protein production that is at least 75% equivalent to that seen using a 120-nucleotide poly-A tail alone.

[0186] In some embodiments, the nucleotide sequence encoding IL-12 comprises a polyA tail and is stabilized by the addition of a chain-terminating nucleoside. In some embodiments, the nucleotide sequence encoding IL-12 having a polyA tail further comprises a 5' cap structure.

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

[0188] In some aspects, nucleotide sequences encoding IL-12 containing poly-A tails or poly-AG quartets are stabilized by the addition of oligonucleotides terminating in 3'-deoxynucleosides, 2',3'-dideoxynucleosides 3'-O-methylnucleosides, 3'-O-ethylnucleosides, 3'-arabinosides, and other modified nucleosides known in the art and / or described herein.

[0189] Modified Nucleosides In some embodiments, the nucleotide sequence encoding IL-12 includes one or more modified nucleosides. In some embodiments, the one or more modified nucleosides include 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 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-cytidine, 5-amino-cytidine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-amino-cytidine, 5 ... These include xo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, and combinations thereof.

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

[0191] In some aspects, the nucleotide sequence encoding IL-12 comprises the nucleotide sequence encoding IL-12 described in U.S. Application No. 2014 / 0147454, International Application WO2018160540, International Application WO2015 / 196118, or International Application WO2015 / 089511, which are incorporated by reference herein in their entireties.

[0192] Cytotoxic nucleosides In some embodiments, the nucleotide sequence encoding IL-12 comprises one or more cytotoxic nucleosides. For example, cytotoxic nucleosides can be incorporated into a polynucleotide, such as a bifunctional nucleotide sequence or mRNA encoding IL-12. Cytotoxic nucleoside anticancer drugs 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] As anticancer drugs, several cytotoxic nucleoside analogs are in clinical use or are the subject of clinical trials. Examples of such analogs include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidene cytidine (DMDC), cladribine, clofarabine, 5-azacytidine, 4'-thio-aracytidine, cyclopentenyl cytosine 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 can have side effects typical of cytotoxic agents, including, but not limited to, little or no specificity for tumor cells over proliferating normal cells.

[0194] Some prodrugs of cytotoxic nucleoside analogues 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 ester). In general, these prodrugs can be converted to active drugs mainly in the liver and systemic circulation, and may show little or no selective release of active drugs 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" has been claimed by Fujiu et al. (U.S. Patent No. 4,966,891) and is 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 yield 5'-deoxy-5-fluorocytidine.

[0195] A series of cytarabine N4-carbamates have been reported by Fadl et al (Pharmazie. 1995, 50, 382-7, the entirety of which is incorporated herein by reference), and these compounds were designed to be converted to cytarabine in the liver and plasma. WO2004 / 041203, the entirety of which is incorporated herein by reference, discloses prodrugs of gemcitabine, some of which are N4-carbamates. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine, and were intended to provide gemcitabine by hydrolytic 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 by reference in its entirety) describe acetal derivatives of 1-(3-C-ethynyl-β-D-ribo-pentopharanosyl)cytosine that, upon bioreduction, give rise to intermediates that require further hydrolysis under acidic conditions to yield cytotoxic nucleoside compounds.

[0196] Cytotoxic nucleotides, which may be chemotherapeutic agents, include, but are not limited to, pyrazolo[3,4-D]-pyrimidines, 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 ribonucleotides, 5-bromodeoxyuridine, 2-chloro-purine, and inosine, or combinations thereof.

[0197] Code Sequence In some embodiments of the present disclosure, the nucleotide sequence encoding IL-12 comprises a sequence encoding an interleukin (IL)-12 molecule. In some embodiments, the IL-12 molecule comprises 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 heterodimeric cytokine with multiple biological effects on the immune system. It is composed of two subunits, p35 (also known as alpha subunit) and p40 (also known as 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 grammatical variants thereof) can 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 is 219 amino acids long. The wild-type human IL-12 p40 protein is 328 amino acids long. The amino acids of the wild-type human IL-12 protein are further provided below in Table 1.

[0200] In some aspects, an IL-12 protein (e.g., encoded by a 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-12 alpha subunit and / or an IL-12 beta subunit. In some embodiments, the IL-12 alpha 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-12 β 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] As described herein, the nucleic acid molecules of the present disclosure are codon-optimized. Thus, in some embodiments, the nucleotide sequence encoding the IL-12 protein disclosed herein (e.g., IL-12 p35 subunit, IL-12 p40 subunit, or heterodimeric IL-12 p70) differs from the wild-type nucleotide sequence (e.g., SEQ ID NO: 185 or SEQ ID NO: 186).

[0204] In some aspects, the nucleic acid molecules described herein encode an IL-12 β subunit and have a sequence identity at least about 75%, at least about 76%, or similar to any one of the sequences set forth in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. The present invention 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. In certain embodiments, the nucleotide sequence comprises 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 the IL-12 β subunit sequence of any of the constructs provided in Table 1.

[0205] In some embodiments, IL-12 A nucleic acid molecule encoding a 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 set forth in SEQ ID NO:51; or (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% identical to the sequence set forth in SEQ ID NO:52. , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ 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 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; (iv) 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 set forth in SEQ ID NO:54;(v) 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 set forth in SEQ ID NO:55; (vi) 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 (vii) 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 set forth in SEQ ID NO:57; (viii) 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 set forth in SEQ ID NO:58. 98%, at least 99%, or 100% identical; (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 set forth in SEQ ID NO: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%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:65, 69, or 74. (xi) 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 set forth in SEQ ID NO:66, 70, or 75; (xii) is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:62; (xiii) is at least 99% or 100% identical to the sequence set forth in SEQ ID NO:63;or (xiv) comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:64;

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

[0207] In some aspects, the nucleic acid molecules described herein encode an IL-12 p35 subunit and have a sequence identity of at least about 77%, at least about 78%, or at least about 80% identical to any one of the sequences set forth 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. In some embodiments, the nucleotide sequence includes a nucleotide sequence that is 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 the nucleotide sequence of the present invention. In certain embodiments, the nucleotide sequence 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 the IL-12 alpha subunit sequence of any of the constructs provided in Table 1.

[0208] In some embodiments, IL-12 A nucleic acid molecule encoding a 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 set forth in SEQ ID NO:101; or (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%, at least (iii) 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% identical to the sequence set forth in SEQ ID NO:103; %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:104; (iv) 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 set forth in SEQ ID NO:104;(v) 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 set forth in SEQ ID NO:105; (vi) 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% identical to the sequence set forth in SEQ ID NO:106. , at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:107; (vii) 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 set forth in SEQ ID NO:107; (viii) at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% (ix) 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 set forth in SEQ ID NO: 109; (x) 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%, at least 100% identical to the sequence set forth in SEQ ID NO: 115, 119, or 124. (xi) 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 set forth in SEQ ID NO:116, 120, or 125; (xii) is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:112; (xiii) is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:113;or (xiv) comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO:114;

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

[0210] In some embodiments, the nucleic acid molecule encoding the IL-12 p40 subunit and the nucleic acid molecule encoding the IL-12 p35 subunit can be conjugated to each other. For example, in some embodiments, the present 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-12 α subunit and the IL-12 β 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 an IL-12 β subunit has a sequence identity at least about 75%, at least about 76%, or at least about the sequence set forth in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. 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 the second nucleic acid molecule encoding an IL-12 α subunit has at least about 77 identical sequences to the sequence set forth 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 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 the nucleotide sequence of the present invention.

[0212] In some embodiments, the first nucleic acid molecule encoding an IL-12 β 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 about 100% identical to the IL-12 β subunit sequence of any of the constructs provided in Table 1. the second nucleic acid molecule encoding an IL-12 α 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 the IL-12 α subunit sequence of any 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 set forth in SEQ ID NO:51. and / or the second nucleic acid molecule comprises 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 set forth in SEQ ID NO:101;(ii) 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 set forth in SEQ ID NO:52, and and / or the second 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 set forth in SEQ ID NO:102;(iii) 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 set forth in SEQ ID NO:53, and / or the second 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 9 (iv) 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 set forth 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 set forth in SEQ ID NO:104;(v) 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 set forth 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 set forth in SEQ ID NO:105. (vi) 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 set forth in SEQ ID NO:56; (vii) 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 set forth in SEQ ID NO: 57; 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 set forth in SEQ ID NO: 106; 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 set forth in SEQ ID NO: 107;(viii) 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 set forth 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 set forth in SEQ ID NO:108; (ix) 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 set forth in SEQ ID NO:59, 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 set forth in SEQ ID NO:59. (x) 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 set forth in SEQ ID NO: 109; (x) 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 set forth 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 set forth in SEQ ID NO: 115, 119, or 124;(xi) 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 set forth in SEQ ID NO: 66, 70, or 75, 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 set forth in SEQ ID NO: 116, 120, or 125; (xii) the first nucleic acid molecule comprises a nucleotide sequence that is at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 62. (xiii) the first nucleic acid molecule comprises a nucleotide sequence that is at least 99% or 100% identical to the sequence set forth 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 set forth in SEQ ID NO: 113; or (xiv) the first nucleic acid molecule comprises a nucleotide sequence that is at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 65, 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 set forth in SEQ ID NO: 115.

[0214] In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:51 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:101. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:52 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:102. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:53 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:103. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:54 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:104. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:55 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:105. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:56 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:106. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:57 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:107. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:58, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:118. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:59, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:119. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:65, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:115. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:66, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:116. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:67, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:117. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:68, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:118. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:69, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:119. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 70 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 120. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 71 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 121.In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 72 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 122. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 73 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 123. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 74 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 124. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 75 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 125. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 62 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 112. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 63 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 113. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 64 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 114. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 60 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 110. In some embodiments, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 61 and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 111. In some embodiments, the first nucleic acid molecule comprises the IL-12 β subunit sequence of any of the constructs provided in Table 1 and the second nucleic acid molecule comprises the IL-12 α subunit sequence of any 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., isolated polynucleotides comprising nucleic acid molecules encoding IL-12 (e.g., IL-12α subunit and / or IL-12β subunit), comprise 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 distinct from the IL-12 protein (e.g., IL-12α subunit and / or IL-12β subunit) encoded by the nucleic acid molecule disclosed herein. Such heterologous moieties may be genetically fused, conjugated, and / or otherwise associated with the IL-12 protein. For example, in some embodiments, the heterologous moiety may be fused to the 3' end of the IL-12α subunit. In some embodiments, the heterologous moiety may be conjugated to the 3' end of the IL-12α subunit via a linker (e.g., a GS linker). In some embodiments, the heterologous moiety may 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-12 β subunit via a linker (eg, a GS linker).

[0216] In some embodiments, the heterologous moiety comprises a half-life extending moiety. The term "half-life extending moiety" refers to a pharma- ceutically acceptable moiety, domain, or molecule covalently linked ("conjugated" or "fused") to an IL-12 protein (e.g., IL-12α subunit and / or IL-12β subunit) encoded by a nucleic acid molecule of the present disclosure, either directly or through a linker, optionally through a non-naturally encoded amino acid, that prevents or mitigates chemical modifications that reduce in vivo proteolysis or other activity of the IL-12 protein, increases half-life, and / or improves or alters other pharmacokinetic or biophysical properties, including but not limited to, increasing the rate of absorption, reducing toxicity, improving solubility, reducing protein aggregation, increasing the biological activity and / or target selectivity of the IL-12 protein, increasing manufacturability, and / or reducing immunogenicity of the IL-12 protein, as compared to a reference compound, such as an unconjugated or unfused form of the IL-12 protein.

[0217] In the context of this disclosure, the term "fused" or "fusion" refers to at least two polypeptide chains (e.g., encoded by a nucleic acid molecule described herein) that 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 term "conjugate" or "conjugation" refers to two molecular entities (e.g., two polypeptides, or a polypeptide and a polymer such as PEG) that are chemically linked.

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

[0219] In some embodiments, the heterologous moiety comprises lumican. Lumican binds to collagen, which is abundantly and ubiquitously expressed in tumors. In certain embodiments, lumican can be conjugated to IL-12 protein (e.g., IL-12α subunit and / or IL-12β subunit) to improve the targeting of IL-12 protein to tumors (e.g., by preventing and / or reducing IL-12 protein from entering systemic circulation), thereby reducing the toxicity of IL-12 protein. Additional disclosure (e.g., sequences) regarding lumican that can be used is provided elsewhere in this disclosure.

[0220] In some embodiments, the heterologous moiety encodes a cytokine, a chemokine, or a growth factor other than IL-12. Cytokines are known in the art, and the term itself refers to a generalized grouping of small proteins secreted by certain cells in the immune system and have effects on other cells. Cytokines are known to enhance cellular immune responses, and as used herein, can 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 nearby responsive cells, typically white blood cells, to the site of infection. Non-limiting examples of chemokines include CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10. Growth factors are known in the art, and the term itself may be interchangeable with the term cytokine. As used herein, the term "growth factor" refers to a naturally occurring substance that can signal between cells and stimulate cell growth. Although cytokines can be growth factors, certain types of cytokines can also have inhibitory effects on cell growth, thus distinguishing the two terms. Non-limiting examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility stimulating factor, bone morphogenetic protein (BMP), ciliary neurotrophic factor (CNTF), leukemia inhibitory 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), epidermal growth factor (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 (FGF15), Fibroblast growth factor 15 (FGF16), Fibroblast growth factor 16 (FGF17), Fibroblast growth factor 17 (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), Fibroblast growth factor 24 (FGF24), Fibroblast growth factor 25 (FGF25), Fibroblast growth factor 26 (FGF26), Fibroblast growth factor 27 (FGF27), Fibroblast growth factor 28 (FGF28), Fibroblast growth factor 29 (FGF29), Fibroblast growth factor 30 (FGF30), Fibroblast growth factor 31 (FGF31), Fibroblast growth factor 32 (FGF32), Fibroblast growth factor 33 (FGF33), Fibroblast growth factor 34 (FGF34), Fibroblast growth factor 35 (FGF35), Fibroblast growth factor 36 (FGF36), Fibroblast Follicular growth factor 14 (FGF14), fibroblast growth factor 15 (FGF15), fibroblast growth factor 16 (FGF16), fibroblast growth factor 17 (FGF17), 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 line-derived neurotrophic factor (GDNF), neurturin, parsley, Peresphin, Artemin, Growth Differentiation Factor-9 (GDF9), Hepatocyte Growth Factor (HGF), Hepatoma-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), Migration Stimulating Factor (MSF), Macrophage Stimulating Protein (MSP), Myostatin (GDF-8), Neuregulin 1 (NR G1), neuregulin 2 (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 aspects, the polynucleotides (e.g., isolated polynucleotides) of the present disclosure further comprise a nucleic acid molecule encoding a leader sequence. As used herein, the term "leader sequence" refers to a sequence located at the amino terminus of a precursor form of a protein. The leader sequence is cleaved during maturation. In certain aspects, the leader sequence comprises a signal peptide. The term "signal peptide" refers to a leader sequence that ensures entry of the protein into the secretory pathway. Additional description of leader sequences is provided, for example, in US2007 / 0141666A1, which is incorporated herein by reference in its entirety. In some aspects, a leader sequence that may be used in the present disclosure comprises the amino acid sequence MRVPAQLLGLLLLWLPGARCA (SEQ ID NO: 180). In some aspects, a nucleic acid molecule encoding such a leader sequence comprises a sequence set forth in any one of SEQ ID NOs: 26-50. In some aspects, a nucleic acid molecule encoding a leader sequence comprises a sequence encoding any of the leader sequences of the constructs provided in Table 1.

[0222] As is apparent from the above disclosure, in some embodiments, a polynucleotide (e.g., an isolated polynucleotide) of the present disclosure comprises multiple nucleic acid molecules. For example, in certain embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises (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. As described herein, in some embodiments, such a polynucleotide further comprises one or more additional properties described herein. For example, in some embodiments, the polynucleotides described herein comprise (from 5' to 3') (1) a 5'-cap, (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 polynucleotides described herein comprise (from 5' to 3') (1) a 5'-cap, (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 description of exemplary constructs is provided below.

[0223] In some aspects, (i) the first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence set forth in SEQ ID NO:40; (ii) the second nucleic acid molecule (i.e., encoding the IL-12 β subunit) comprises the sequence set forth in SEQ ID NO:65; (iii) the third nucleic acid molecule (i.e., encoding the linker) comprises the sequence set forth in SEQ ID NO:90; and (iv) the fourth nucleic acid molecule (i.e., encoding the IL-12 α subunit) comprises the sequence set forth in SEQ ID NO:115. Thus, in certain aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises the sequence set forth in SEQ ID NO:15. In some aspects, a polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (5' to 3') (1) a 5'-cap (or cap analog), (2) a first nucleotide sequence comprising the sequence set forth in SEQ ID NO:40 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:65 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:90 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:41; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:66; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:91; and (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:116. Thus, in certain aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises the sequence set forth in SEQ ID NO:16. In some aspects, a polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (5' to 3') (1) a 5'-cap (or cap analog), (2) a first nucleotide sequence comprising the sequence set forth in SEQ ID NO:41 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:66 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:91 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 42; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 67; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 92; and (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 117. Thus, in certain aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises the sequence set forth in SEQ ID NO: 17. In some aspects, a polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (5' to 3') (1) a 5'-cap (or cap analog), (2) a first nucleotide sequence comprising the sequence set forth in SEQ ID NO:42 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:67 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:92 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 43; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 68; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 93; and (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 118. Thus, in certain aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises the sequence set forth in SEQ ID NO: 18. In some aspects, a polynucleotide comprises one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (5' to 3') (1) a 5'-cap (or cap analog), (2) a first nucleotide sequence comprising the sequence set forth in SEQ ID NO:43 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:68 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:93 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth 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 aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence; (ii) a second nucleic acid molecule encoding an IL-12 beta 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-12 alpha 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 extending moiety (e.g., human serum albumin). As described herein, in some aspects, such polynucleotides further comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide described herein includes (from 5' to 3') (1) a 5'-cap, (2) a first nucleotide sequence encoding a leader sequence, (3) a second nucleotide sequence encoding an IL-12 β subunit, (4) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (5) a fourth nucleotide sequence encoding an IL-12 α subunit, (6) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (7) a sixth nucleotide sequence encoding a half-life extending moiety (e.g., human serum albumin), and (8) a poly(A) tail. In some aspects, the polynucleotides described herein include (from 5' to 3') (1) a 5'-cap, (2) a 5'-UTR, (3) a promoter, (4) a first nucleotide sequence encoding a leader sequence, (5) a second nucleotide sequence encoding an IL-12 β subunit, (6) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (7) a fourth nucleotide sequence encoding an IL-12 α subunit, (8) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (9) a sixth nucleotide sequence encoding a heterologous moiety (e.g., albumin), (10) a 3'-UTR, and (11) a poly(A) tail. Additional description of such exemplary constructs is provided below.

[0228] In some aspects, (i) the first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence set forth in SEQ ID NO:26; (ii) the second nucleic acid molecule (i.e., encoding the IL-12β subunit) comprises the sequence set forth in SEQ ID NO:51; (iii) the third nucleic acid molecule (i.e., encoding the first linker) comprises the sequence set forth in SEQ ID NO:76; (iv) the fourth nucleic acid molecule (i.e., encoding the IL-12α subunit) comprises the sequence set forth in SEQ ID NO:101; (v) the fifth nucleic acid molecule (i.e., encoding the second linker) comprises the sequence set forth in SEQ ID NO:126; and (vi) the sixth nucleic acid molecule (i.e., encoding the half-life extending moiety) comprises the sequence set forth in SEQ ID NO:147. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:1. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:26 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:51 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:76 (i.e., a first GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:101 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:126 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:27; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:52; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:77; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:102; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:127; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:148. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:2. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:27 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:52 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:77 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:102 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:127 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:28; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:53; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:78; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:103; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:128; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:149. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:3. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:28 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:53 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:78 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:103 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 comprises the sequence set forth in SEQ ID NO:29; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:54; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:79; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:104; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:129; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:150. Thus, in certain embodiments, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:4. In some embodiments, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:29 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:54 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:79 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:104 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:129 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 30; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 55; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 80; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 105; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 130; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 151. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 5. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:30 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:55 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:80 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:105 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:130 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:31; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:56; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:81; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:106; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:131; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:152. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:6. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:31 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:56 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:81 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:106 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:131 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 32; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 57; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 82; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 107; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 132; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 153. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 7. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:32 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:57 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:82 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:107 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:132 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (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 aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 8. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:58 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:83 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:108 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:133 (i.e., a 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 "H2 constructs."

[0236] In some aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 34; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 59; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 84; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 109; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 134; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 155. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 9. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:34 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:59 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:84 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:109 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:134 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 37; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 62; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 87; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 112; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 137; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 158. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 12. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some aspects, a polynucleotide provided herein comprises (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:37 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:62 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:87 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:112 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:137 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 38; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 63; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 88; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 113; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 138; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 159. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 13. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some aspects, a polynucleotide provided herein comprises (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:38 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:63 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:88 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:113 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:138 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 39; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 64; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 89; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 114; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 139; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 160. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 14. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some aspects, a polynucleotide provided herein comprises (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:39 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:64 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:89 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:114 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:139 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 44; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 69; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 94; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 119; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 140; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 161. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 19. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:44 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:69 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:94 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:119 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:140 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth in SEQ ID NO:161 (i.e., human serum albumin), and (8) a poly(A) tail. An example of such a polynucleotide is described herein as a "B1 construct."

[0241] In some aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 45; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 70; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 95; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 120; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 141; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 162. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 20. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:45 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:70 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:95 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:120 (i.e., an IL-12α subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:141 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 46; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 71; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 96; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 121; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 142; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 163. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 21. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:46 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:71 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:96 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:121 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:142 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth in SEQ ID NO:163 (i.e., human serum albumin), and (8) a poly(A) tail. An example of such a polynucleotide is described herein as a "B3 construct."

[0243] In some aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 47; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 72; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 97; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 122; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 143; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 164. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 22. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:47 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:72 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:97 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:122 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:143 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 35; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 60; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 85; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 110; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 135; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 156. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 10. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:35 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:60 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:85 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:110 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:135 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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 aspects, (i) the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 36; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 61; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 86; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 111; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 136; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 157. Thus, in certain aspects, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 11. In some aspects, the polynucleotides comprise one or more additional properties described herein. For example, in some embodiments, a polynucleotide provided herein comprises (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:36 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:61 (i.e., an IL-12 beta subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:86 (i.e., a GS linker), (5) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., an IL-12 alpha subunit), (6) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:136 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising the sequence set forth 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, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises (5' to 3') (i) a first nucleic acid molecule encoding a leader sequence; (ii) a second nucleic acid molecule encoding an IL-12 beta 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-12 alpha 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 extending moiety (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 lumican. As described herein, in some embodiments, such polynucleotides further comprise one or more additional properties described herein. In some aspects, a polynucleotide described herein includes (from 5' to 3') (1) a 5'-cap, (2) a first nucleotide sequence encoding a leader sequence, (3) a second nucleotide sequence encoding an IL-12 β subunit, (4) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (5) a fourth nucleotide sequence encoding an IL-12 α subunit, (6) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (7) a sixth nucleotide sequence encoding a heterologous moiety (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 moiety (e.g., lumican), and (12) a poly(A) tail.In some embodiments, the polynucleotides described herein include (from 5' to 3') (1) a 5'-cap, (2) a 5'-UTR, (3) a promoter, (4) a first nucleotide sequence encoding a leader sequence, (5) a second nucleotide sequence encoding an IL-12 β subunit, (6) a third nucleotide sequence encoding a first linker (e.g., a GS linker), (7) a fourth nucleotide sequence encoding an IL-12 α subunit, (8) a fifth nucleotide sequence encoding a second linker (e.g., a GS linker), (9) a sixth nucleotide sequence encoding a heterologous 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. Additional description of such exemplary constructs is provided below.

[0247] In some embodiments, (i) the first nucleic acid molecule (i.e., encoding the leader sequence) comprises the sequence set forth in SEQ ID NO:48; (ii) the second nucleic acid molecule (i.e., encoding the IL-12 β subunit) comprises the sequence set forth in SEQ ID NO:73; (iii) the third nucleic acid molecule (i.e., encoding the first linker) comprises the sequence set forth in SEQ ID NO:98; (iv) the fourth nucleic acid molecule (i.e., encoding the IL-12 α subunit) comprises the sequence set forth in SEQ ID NO:123; (v) the fifth nucleic acid molecule (i.e., encoding the second linker) comprises the sequence set forth in SEQ ID NO:144; (vi) the sixth nucleic acid molecule (i.e., encoding the half-life extending moiety) comprises the nucleic acid sequence set forth in SEQ ID NO:165; (vii) the seventh nucleic acid molecule (i.e., encoding the third linker) comprises the sequence set forth in SEQ ID NO:168; and (viii) the eighth nucleic acid molecule (i.e., encoding lumican) comprises the sequence set forth in SEQ ID NO:171. Thus, in certain aspects, a polynucleotide (e.g., an isolated polynucleotide) described herein comprises the sequence set forth in SEQ ID NO: 23. In some aspects, the polynucleotide comprises one or more additional properties 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:48 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:73 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:98 (i.e., a first GS linker), (5) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:123 (i.e., an IL-12β subunit), (6) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (7) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:112 (i.e., a GS linker), (8) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (9) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (10) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:112 (i.e., a GS linker), (11) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (12) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (13) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (14) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:111 (i.e., a GS linker), (15) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:11 (5) a fourth nucleotide sequence comprising a GS linker (GlcNAc-12α subunit), (6) a fifth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 144 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 165 (i.e., human serum albumin), (8) a seventh nucleotide sequence comprising a sequence set forth in SEQ ID NO: 168 (i.e., a third GS linker), (9) an eighth nucleotide sequence comprising a sequence set forth 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 comprises the sequence set forth in SEQ ID NO: 49; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 74; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 99; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 124; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 145; (vi) the sixth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 166; (vii) the seventh nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 169; (viii) the eighth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 172. Thus, in certain embodiments, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO: 24. In some embodiments, the polynucleotides comprise one or more additional properties 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:49 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:74 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:99 (i.e., a first GS linker), (5) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:124 (i.e., an IL-12β subunit), (6) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:116 (i.e., a GS linker), (7) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:118 (i.e., a GS linker), (8) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:126 (i.e., a GS linker), (9) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (10) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (11) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (12) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (13) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (14) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:128 (i.e., a GS linker), (15) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:12 (5) a fourth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 145 (i.e., a second GS linker), (6) a fifth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 145 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 166 (i.e., a human serum albumin), (8) a seventh nucleotide sequence comprising a sequence set forth in SEQ ID NO: 169 (i.e., a third GS linker), (9) an eighth nucleotide sequence comprising a sequence set forth 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 comprises the sequence set forth in SEQ ID NO:50; (ii) the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:75; (iii) the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:100; (iv) the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:125; (v) the fifth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:146; (vi) the sixth nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO:167; (vii) the seventh nucleic acid molecule comprises the sequence set forth in SEQ ID NO:170; (viii) the eighth nucleic acid molecule comprises the sequence set forth in SEQ ID NO:173. Thus, in certain embodiments, the polynucleotides (e.g., isolated polynucleotides) described herein comprise the sequence set forth in SEQ ID NO:25. In some embodiments, the polynucleotides comprise one or more additional properties 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:50 (i.e., a leader sequence), (3) a second nucleotide sequence comprising the sequence set forth in SEQ ID NO:75 (i.e., an IL-12β subunit), (4) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:100 (i.e., a first GS linker), (5) a third nucleotide sequence comprising the sequence set forth in SEQ ID NO:125 (i.e., an IL-12β subunit), (6) a fourth nucleotide sequence comprising the sequence set forth in SEQ ID NO:130 (i.e., a first GS linker), (7) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:140 (i.e., a second GS linker), (8) a fifth nucleotide sequence comprising the sequence set forth in SEQ ID NO:150 (i.e., a first GS linker), (9) a fifth GS linker, (10) a fifth GS linker, (11) a fifth GS linker, (12) a sixth GS linker, (13) a sixth GS linker, (14) a sixth GS linker, (15) a sixth GS linker, (16) a sixth GS linker, (17) a sixth GS linker, (18) a sixth GS linker, (19) a sixth GS linker, (20) a sixth GS linker, (21) a sixth GS linker, (22) a seventh GS linker, (23) a seventh GS linker, (24) a seventh GS linker, (25) a seventh GS linker, (26) a seventh GS linker, (27) a seventh GS linker, (28) a seventh GS linker, ( (5) a fourth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 146 (i.e., a second GS linker), (6) a fifth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 146 (i.e., a second GS linker), (7) a sixth nucleotide sequence comprising a sequence set forth in SEQ ID NO: 167 (i.e., a human serum albumin), (8) a seventh nucleotide sequence comprising a sequence set forth in SEQ ID NO: 170 (i.e., a third GS linker), (9) an eighth nucleotide sequence comprising a sequence set forth 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 aspects, the present disclosure relates to the delivery of biologically active molecules (e.g., IL-12 protein) to cells. In certain aspects, the delivery can occur in vivo (e.g., by administering a polynucleotide described herein to a subject) or ex vivo (e.g., by culturing a polynucleotide described herein with a cell in vitro). In some aspects, the delivery of a polynucleotide described herein (e.g., an isolated polynucleotide) can be performed using any suitable delivery system known in the art. In certain aspects, the delivery system is a vector. Thus, in some aspects, the present disclosure provides a vector comprising a polynucleotide of the present disclosure. Suitable vectors that can 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 comprising nucleic acid molecules encoding IL-12 protein) are delivered using lipid nanoparticles. Thus, in some embodiments, the present disclosure relates to polynucleotides (e.g., RNA) expressing IL-12 encapsulated by lipid nanoparticles, compositions thereof, and the use of the compositions to treat subjects having or suspected of having cancer.

[0252] "Lipid nanoparticle" (LNP), as used herein, refers to a vesicle, e.g., a spherical vesicle with a continuous lipid bilayer. Lipid nanoparticles can be used in methods to deliver pharmaceutical therapies to targeted locations. Non-limiting examples of LNPs include liposomes, bolaamphiphiles, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), and unilamellar membrane structures (e.g., archaeosomes and micelles).

[0253] In some embodiments, the lipid nanoparticles comprise one or more lipids. Lipid, as used herein, refers to a group of organic compounds including, but not limited to, esters of fatty acids, and in some embodiments, is characterized by being insoluble in water but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids", including fats and oils and waxes; (2) "complex lipids", including phospholipids and glycolipids; and (3) "derived lipids", such as steroids. Non-limiting examples of lipids include triglycerides (e.g., tristearin), diglycerides (e.g., glycerol bahenate), 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 LNP comprise cationic lipids. In some embodiments, one or more lipids in the LNP comprises a lipidoid, e.g., TT3. Thus, in some embodiments, any of the polynucleotides described herein (e.g., comprising a sequence set forth in any one of SEQ ID NOs: 1-25, 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 comprises a sequence set forth in SEQ ID NO: 1, 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 a sequence set forth in SEQ ID NO: 2, 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 set forth in SEQ ID NO:3, 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:4, 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:5, 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:6, 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:7, 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:8, 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:9, 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:10, 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:11, 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 set forth in SEQ ID NO:12, 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: 13, 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: 14, 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: 15, 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: 16, 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: 17, 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: 18, 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: 19, 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: 20, 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 set forth in SEQ ID NO:21, 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: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 the present disclosure include, but are not limited to, N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); Lipofectamine; 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA); Dioctadecyldimethylammonium (DODMA), Distearyldimethylammonium. (DSDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N-N-distearyl-N,N-dimethylammonium bromide (DDAB); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0255] In some embodiments of the present disclosure, the lipid, e.g., lipidoid, is TT3. TT3, as used herein, can form lipid nanoparticles for delivery of various biologically active agents to cells. In addition, the present disclosure also demonstrates that unloaded TT3-LNP 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 activation of dendritic cells (DCs) and the resulting activation of specific T cell responses. In some embodiments of the present disclosure, the cells undergoing immunogenic cell death are tumor cells. Immunogenic tumor cell death can induce an effective anti-tumor immune response. In some embodiments of the present disclosure, the lipid nanoparticles include TT3-LNPs (TT3-LNP-modRNA) that encapsulate a nucleotide sequence (modRNA) that encodes IL-12 that encodes only a reporter gene. The nucleotide sequence encoding IL-12 can synergize with TT3-LNP to induce higher levels of ICD in tumor cells compared to TT3-LNP alone. In some embodiments of the present disclosure, lipid nanoparticles include TT3-LNPs encapsulating modRNA encoding IL-12 molecules. IL-12, an immunomodulatory cytokine, induces a strong immune response against local tumors. The combination of TT3-LNP, modRNA and IL-12 expression is not only effective in synergistically inhibiting tumor cells at the site, but also induces a systemic anti-tumor immune response to kill distant tumor cells and prevent tumor recurrence.

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

[0257] In some embodiments of the present disclosure, the cationic lipid is lipofectamine. As used herein, lipofectamine is a common transfection reagent manufactured and sold by Invitrogen, 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 transfection payloads, such as modRNA. RNA-containing liposomes (positively charged on their surface) can fuse with the negatively charged plasma membrane of living cells due to neutral co-lipids that mediate the fusion of liposomes with the cell membrane, allowing nucleic acid cargo molecules to cross the cytoplasm for replication or expression.

[0258] In some embodiments of the present disclosure, the LNPs are primarily composed of cationic lipids along with other lipid components. These typically include, but are not limited to, other lipid molecules belonging to the phosphatidylcholine (PC) class (e.g., 1,s-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), sterols (e.g., cholesterol), and polyethylene glycol (PEG)-lipid conjugates (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-2000 (DSPE-PEG2000) and C14-PEG2000). Table 2 shows the formulations of exemplary LNPs, TT3-LNPs, and DOTAP-LNPs. [Table 2]

[0259] In some embodiments, the LNP comprises C14-PEG2000. In certain embodiments, the C14-PEG2000 comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DMPE-PEG2000), or both. As described herein (see, e.g., Example 3), in some embodiments, the C14-PEG2000 (or other lipid components disclosed herein) can be embedded in the LNP prior to encapsulation of the polynucleotide. In some embodiments, the C14-PEG2000 (or other lipid components disclosed herein) can be added to the LNP after encapsulation of the polynucleotide. For example, in certain embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprising a nucleic acid molecule encoding an IL-12 protein (e.g., IL-12α and / or IL-12β subunit) is encapsulated into an LNP, and then C14-PEG2000 (other lipid components disclosed herein) is attached to the LNP, e.g., using micelles.

[0260] The particle size of lipid nanoparticles can affect drug release rate, biodistribution, mucoadhesion, cellular uptake of water and buffer exchange into the interior of the nanoparticle, and protein diffusion. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of about 30 to about 500 nm, about 50 to about 400 nm, about 70 to about 300 nm, about 100 to about 200 nm, about 100 to about 175 nm, or about 100 to about 160 nm. In some embodiments of the present disclosure, the diameter of the LNP is in the range of 100 to 160 nm. In some embodiments of the present disclosure, the diameter of the LNP can be about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm, about 117 nm, about 118 nm, about 119 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, or about 160 nm. In certain embodiments, the lipid nanoparticle has a diameter of about 140 nm.

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

[0262] In some embodiments, the present disclosure relates to polynucleotides (e.g., mRNA) encapsulated by lipid nanoparticles (LNPs). In some embodiments of the present disclosure, the mass ratio between the lipids and the polynucleotide (e.g., mRNA) of the LNPs ranges from about 1:2 to about 15:1. In some embodiments, the mass ratio between the lipids and the polynucleotide (e.g., mRNA) ranges from about 1:2, about 1:1.9, about 1:1.8, about 1:1.7, about 1:1.6, about 1:1.5, about 1:1.4, about 1:1.3, about 1:1.2, about 1:1.1, about 1:1, about 1.1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2 ... 1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1, about 5:1, about 5.5:1, about 6:1, about 6.5:1, about 7:1, about 7.5:1, about 8:1, about 8.5:1, about 9:1, about 9.5:1, about 10:1, about 10.5:1, about 11:1, about 11.5:1, about 12:1, about 12.5:1, about 13:1, about 13.5:1, about 14:1, about 14.5:1, or about 15:1. In some embodiments of the present disclosure, the mass ratio between lipid and polynucleotide (e.g., mRNA) is about 10:1.

[0263] Pharmaceutical Compositions In some aspects, the present disclosure relates to pharmaceutical compositions comprising the polynucleotide, vector and / or lipid nanoparticle described herein. In some aspects of the present disclosure, the pharmaceutical composition further comprises a pharma- ceutically 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 aspects, the carrier can stabilize the active ingredient. 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] The pharmaceutical composition used for in vivo administration must be sterile.This can be easily achieved, for example, by filtration through sterile filtration membrane.Lipid nanoparticles can be placed in a container with sterile access port, for example, intravenous solution bag or vial with a stopper that can be pierced by hypodermic injection needle.

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

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

[0267] In some aspects of the present disclosure, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, buffer, excipient, salt, or stabilizer in the form of a lyophilized formulation or an aqueous solution. See, e.g., 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 nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); serum albumin, gelatin. or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or 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 non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0268] In some aspects, the pharmaceutical compositions described herein include lipid nanoparticles that can be prepared by methods known in the art, such as those described in 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. Pat. Nos. 4,485,045 and 4,544,545, which are incorporated herein by reference in their entirety. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556, which is incorporated herein by reference in its entirety. In some aspects, liposomes can be produced by reverse phase evaporation using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with desired diameters.

[0269] In some embodiments of the present disclosure, pharmaceutical compositions are formulated in sustained release form.Suitable examples of sustained release preparations include semipermeable matrix of solid hydrophobic polymers containing lipid nanoparticles, and this matrix is ​​in the form of molded article, such as film or microcapsule.Examples of sustained release matrix include, but are not limited to, polyester, hydrogel (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymer of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, such as LUPROM DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0270] In some embodiments, suitable surfactants include, but are not limited to, non-ionic 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 composition having a surfactant comprises between 0.05-5% surfactant. In some embodiments, the composition comprises 0.1 and 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, can be added if necessary.

[0271] In some aspects, the pharmaceutical composition is in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral, or rectal administration, or administration by inhalation or insufflation.

[0272] To prepare solid compositions such as tablets, the main active ingredient can be mixed with pharmaceutical carriers, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogeneous mixture of the compound of the present disclosure or its non-toxic pharmaceutically acceptable salts. When these preformulation compositions are referred to as homogeneous, it is meant that the active ingredient is uniformly dispersed throughout the composition, so that the composition can be easily further divided into similarly effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation composition is then further divided into unit dosage forms of the type described above containing about 0.1 to about 500 mg of the active ingredient of the present disclosure. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides the benefit of extended action. For example, a tablet or pill can comprise an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer that serves to resist disintegration in the stomach and allows the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including some polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0273] Suitable emulsions can be prepared using commercially available fat emulsions such as INTRALIPIDS™, LIPOSYN™, INFONUTROL™, LIPOFUNDIN™, and LIPIPHYSAN™. The active ingredient may be dissolved in a premixed emulsion composition or may be dissolved in 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 appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions typically contain up to about 20% oil, e.g., between about 5 and about 20%. The fat emulsion can contain fat droplets having a suitable size and can have a pH ranging from about 5.5 to about 8.0.

[0274] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as set forth above. In some aspects, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

[0275] Compositions in pharmaceutically acceptable solvents can be nebulized by using gas.Nebulized solutions can be inhaled directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent or intermittent positive pressure breathing machine.Solution, suspension or powder compositions can be administered from a device that delivers the formulation in an appropriate manner.

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

[0277] In some aspects, an effective amount of any of the compositions described herein is administered to a subject in need thereof via a suitable route, for example, intratumoral administration, intravenous administration (e.g., as a bolus or by continuous infusion over a period of time), intramuscular, intraperitoneal, intracerebospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, inhalation, or local route. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be nebulized, and lyophilized powders can be nebulized after reconstitution. In some aspects, the pharmaceutical compositions described herein are aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and crushed powders. In some aspects, the pharmaceutical compositions described herein are formulated for intratumoral injection. In some aspects, the pharmaceutical compositions described herein are administered to a subject via a local route, for example, injected into a local site, such as the site of a tumor or an infection. In some aspects, the subject is a human.

[0278] As is evident from the present disclosure, in some embodiments, the compositions described herein are administered to a subject in an effective amount to provide 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 suffering from cancer, and the therapeutic effect includes reduced tumor burden, reduced 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). Effective amounts will vary according to the particular condition being treated, the severity of the condition, individual patient parameters including age, health, size, sex and weight, duration of treatment, the nature of the combination therapy (if any), the particular route of administration, and similar factors within the expertise of the medical practitioner, as will be recognized by those skilled in the art.

[0279] Empirical considerations such as half-life generally contribute to the determination of dosage.Frequency of administration can be determined and adjusted during treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or recovery and / or delay of target disease / disorder.Alternatively, sustained continuous release formulation of the composition 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 aspects of the present disclosure, the treatment is a single injection of the composition disclosed herein. In some aspects, the single injection is administered intratumorally to a subject in need thereof.

[0281] In some aspects of the present disclosure, dosages for the compositions described herein can be empirically determined in individuals given one or more administrations of the composition (e.g., lipid nanoparticles described herein). In some aspects, individuals are given increasing dosages of the compositions described herein. To evaluate the effectiveness of the compositions herein, disease / disorder indicators can be tracked. For repeated administration over several days or longer, depending on the condition, in some aspects, treatment is sustained until desired symptom suppression occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms.

[0282] In some embodiments of the present disclosure, the dosing frequency is about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per 5 weeks, about once per 6 weeks, about once per 7 weeks, about once per 8 weeks, about once per 9 weeks, or about once per 10 weeks; or about once per month, about once per 2 months, or about once per 3 months, or longer.The dosing regimen (e.g., dosage and / or dosing frequency) of the compositions (e.g., lipid nanoparticles) described herein used can be varied over time.

[0283] In some embodiments of the present disclosure, the methods include administering one or more doses of a composition described herein to a subject in need thereof.

[0284] The appropriate dosage of a composition (e.g., lipid nanoparticles described herein) depends on the particular 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, the clinician can administer a composition disclosed herein until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in tumor burden, a reduction in cancer cells, or increased immune activity. Administration of one or more compositions described herein can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of a composition described herein can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0285] As used herein, alleviating the target disease / disorder includes delaying the onset or progression of the disease or reducing the severity of the disease. Alleviating the disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means to prolong, prevent, slow down, retard, stabilize, and / or delay the progression of the disease. This retardation can be of various lengths of time, depending on the disease being treated and / or the medical history of the subject. A method of retarding or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the probability of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame when compared to when the method is not used. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0286] In some embodiments, the compositions described herein are administered to a subject in need thereof in an amount sufficient to reduce tumor burden or cancer cell growth in vivo by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more. In some embodiments, the compositions described herein are administered in an amount effective to increase immune activity by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.

[0287] In some embodiments, administering a composition (e.g., a polynucleotide, vector, lipid nanoparticle, or pharmaceutical composition 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-fold, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 50-fold, or more, compared to the immune activity of a reference subject (e.g., a subject before administration of the composition, or a corresponding subject that was not responsive to administration of the composition).

[0288] In some embodiments, the subject is a human who has cancer, is suspected to have cancer, or is at risk for cancer.In some embodiments, the cancer is selected from the group consisting of melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, gastric cancer, and various types of head and neck cancer, including squamous cell head and neck cancer.In some embodiments, the cancer can be melanoma, lung cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, or Hodgkin's lymphoma.

[0289] The subject with target disease or disorder can be identified by routine medical examination, such as clinical examination, organ function test, CT scan or ultrasound.The subject suspected of having target disease or disorder may show one or more symptoms of disease or disorder.The subject at risk for disease or disorder can be the subject with one or more risk factors associated with the disease or disorder.The subject at risk for disease or disorder can also be identified by routine medical practice.

[0290] In some embodiments, the compositions described herein are co-administered with at least one additional suitable therapeutic agent. In some embodiments, the at least one additional suitable therapeutic agent includes anti-cancer agents, anti-viral agents, anti-bacterial agents, or other agents that serve to enhance and / or complement the immunostimulatory effects of the compositions described herein (e.g., lipid nanoparticles). Further examples of additional therapeutic agents that can be used in combination with the compositions described herein include chemotherapeutic agents, targeted anti-cancer therapies, oncolytic agents, cytotoxic agents, immune-based therapies, cytokines, surgical procedures, radiation treatments, 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 in a sequential manner, 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 in a substantially simultaneous manner.

[0291] It will be recognized by those skilled in the art that any combination of the compositions described herein and another anti-cancer agent (e.g., chemotherapeutic agent) can be used in any order to treat cancer.The combinations described herein can be selected based on several factors, including, but 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 alleviating the side effects of another agent in the combination.For example, the combination therapy described herein can reduce any of the side effects associated with each individual member of the combination, such as the side effects associated with anti-cancer agents.

[0292] In some embodiments, the other anti-cancer therapeutic agent is chemotherapy, radiation therapy, surgery, immunotherapy, or a combination thereof. In some embodiments, the chemotherapy agent is carboplatin, cisplatin, docetaxel, gemcitabine, nab-paclitaxel, pemetrexed, vinorelbine, or a combination thereof. In some embodiments, the radiation therapy is ionizing radiation, gamma radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, whole body radioisotope, radiosensitizer, or a combination thereof. In some embodiments, the surgery therapy is curative surgery (e.g., tumor removal surgery), preventive surgery, laparoscopic surgery, laser surgery, or a combination thereof. In some embodiments, the immunotherapy is adoptive cell transfer, therapeutic cancer vaccine, or a combination thereof.

[0293] In some embodiments, the chemotherapeutic agent is a platinating agent, such as carboplatin, oxaliplatin, cisplatin, nedaplatin, satraplatin, lobaplatin, triplatin, tetranitrate, picoplatin, prolindac, aroplatin, and other derivatives; topoisomerase I inhibitors, such as camptothecin, topotecan, irinotecan / SN38, rubitecan, belotecan, and other derivatives; topoisomerase II inhibitors, such as etoposide (VP-16), daunorubicin, doxorubicin agents (e.g., doxorubicin, doxorubicin HCl, doxorubicin doxorubicin and its salts or analogs in liposomes), mitoxantrone, aclarubicin, epirubicin, idarubicin, amrubicin, amsacrine, pirarubicin, valrubicin, zorubicin, teniposide, and other derivatives; antimetabolites, such as the folic acid family (methotrexate, pemetrexed, raltitrexed, aminopterin, and related); purine antagonists (thioguanine, fludarabine, cladribine, 6-mercaptopurine, pentostatin, clofarabine, and and related products); pyrimidine antagonists (cytarabine, floxuridine, azacitidine, tegafur, carmofur, capacitabine, gemcitabine, hydroxyurea, 5-fluorouracil (5FU), and related products); alkylating agents, such as nitrogen mustards (e.g., cyclophosphamide, melphalan, chlorambucil, mechlorethamine, ifosfamide, trofosfamide, prednimustine, bendamustine, uramustine, estramustine, and related products); nitrosogens rea (e.g., carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, and related); triazenes (e.g., dacarbazine, altretamine, temozolomide, and related); alkylsulfonates (e.g., busulfan, mannosulfan, treosulfan, and related); procarbazine; mitobronitol, and aziridines (e.g., carboquone, triaziquone, thiotepa, triethylenemalamine, and related);Antibiotics, such as hydroxyurea, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and other derivatives); anthracenediones (e.g., mitoxantrone and related); Streptomyces family (e.g., bleomycin, mitomycin C, actinomycin, plicamycin); ultraviolet light; and combinations thereof.

[0294] In some embodiments, the other anti-cancer therapeutic agent is an antibody. Antibodies (preferably monoclonal antibodies) achieve their therapeutic effect on cancer cells through various mechanisms. They can have direct effects in causing apoptosis or programmed cell death. They can block components of signal transduction pathways, such as growth factor receptors, effectively arresting tumor cell proliferation. In cells expressing monoclonal antibodies, they can cause anti-idiotypic antibody formation. Indirect effects include recruiting cytotoxic cells, such as monocytes and macrophages. This type of antibody-mediated cell killing is called antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies also bind complement, resulting in direct cytotoxicity known as complement-dependent cytotoxicity (CDC). Combining surgical procedures with immunotherapeutic agents 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 anti-cancer antibodies and potential antibody targets (in brackets) that can be used in combination with the present invention: abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30 TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1),Capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatox (EpCAM), cizutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRA...

Claims

1. 1. An isolated polynucleotide comprising a nucleic acid molecule encoding a beta subunit of the IL-12 protein ("IL-12β"), said nucleic acid molecule having a sequence identity at least about 75%, at least about 75%, or at least about 75% identical to a sequence set forth in SEQ ID NO:65, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:

75. 6%, 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 the nucleotide sequence of the polynucleotide.

2. 2. The isolated polynucleotide of claim 1, wherein the nucleic acid molecule encoding the 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 set forth in SEQ ID NO:

65.

3. 1. An isolated polynucleotide comprising a nucleic acid molecule encoding the alpha subunit of the IL-12 protein ("IL-12α"), said nucleic acid molecule being set forth in SEQ ID NO:115, 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: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. and 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 a sequence of interest.

4. 4. The isolated polynucleotide of claim 3, wherein the nucleic acid molecule encoding the 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 set forth in SEQ ID NO:

115.

5. An isolated polynucleotide comprising a first nucleic acid molecule and a second nucleic acid molecule, the first nucleic acid molecule encodes the beta subunit of the IL-12 protein ("IL-12β") and has at least about 75%, at least about 76%, or at least about 77% similar to the sequence set forth in SEQ ID NO:65, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75; 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; the second nucleic acid molecule encodes the alpha subunit of the IL-12 protein ("IL-12α") and has a sequence set forth in SEQ ID NO:115, 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: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. 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 the sequence, An isolated polynucleotide.

6. 6. The isolated polynucleotide of claim 5, wherein 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 set forth in SEQ ID NO:65, 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 set forth in SEQ ID NO:

115.

7. 7. The isolated polynucleotide of claim 5 or 6, further comprising a third nucleic acid molecule encoding a linker connecting the first nucleic acid molecule and the second nucleic acid molecule.

8. 8. The isolated polynucleotide of claim 7, wherein the linker comprises a (GS) linker.

9. 9. The isolated polynucleotide of claim 7 or 8, wherein the third nucleic acid molecule encoding the linker comprises the sequence shown in SEQ ID NO:

90.

10. 10. The isolated polynucleotide of any one of claims 1 to 9, further comprising an additional nucleic acid molecule encoding a half-life extending moiety, wherein said half-life extending moiety comprises Fc, albumin or a fragment thereof, an albumin binding moiety, PAS, HAP, transferrin or a fragment thereof, XTEN, or any combination thereof.

11. 11. The isolated polynucleotide of any one of claims 1 to 10, further comprising an additional nucleic acid molecule encoding a leader sequence.

12. 12. The isolated polynucleotide of claim 11, wherein the additional nucleic acid molecule encoding a leader sequence comprises the sequence set forth in SEQ ID NO:

40.

13. 1. 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 a beta subunit of an IL-12 protein ("IL-12β"), (iii) a third nucleic acid molecule encoding a first linker, and (iv) a fourth nucleic acid molecule encoding an alpha subunit of an IL-12 protein ("IL-12α"), a. the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO:40; b. the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO:65; c. the third nucleic acid molecule comprises the sequence set forth in SEQ ID NO:90; d. the fourth nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 115; An isolated polynucleotide.

14. An isolated polynucleotide comprising the sequence shown in SEQ ID NO:

15.

15. (a) m 2 7,2'-O Gpp s pGRNA, m 7 GpppG, m 7 Gpppppm 7 G, m 2 (7,3'-O) GpppG, m 2 (7,2'-O) GppspG(D1), m 2 (7,2'-O) GppspG(D2), m 2 7,3'-O Gppp(m 1 2'-O )ApG, (m 7 G-3'mppp-G; which may 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'-O G(5')ppp(5')G, 7mG(5')ppp(5')N,pN2p, 7mG(5')ppp(5')NlmpNp, 7mG(5')-ppp(5')NlmpN2 5'-caps including 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-methylpseudouridine, m7G(5')ppp(5')(2'OMeA)pG, or combinations thereof; (b) a regulatory element comprising at least one translational enhancer element (TEE), a translational initiation sequence, at least one microRNA binding site or its seed, a 3′ tailing region of linked nucleosides, an AU-rich element (ARE), a post-transcriptional modulator, a 5′ UTR, a 3′ UTR, or a combination thereof; (c) a 3′ tailing region of the linked nucleosides, comprising a poly A tail, a poly A-G quartet, or a stem-loop sequence; (d) 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-guanosine, O6-methyl-guanosine, at least one modified nucleoside comprising 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, or a combination thereof; or (e) any combination of (a) to (d) The isolated polynucleotide of any one of claims 1 to 14, further comprising:

16. 16. The isolated polynucleotide of any one of claims 1 to 15, which is capable of autonomous replication.

17. 17. The isolated polynucleotide of claim 16, which is a self-amplifying replicon RNA.

18. 18. The isolated polynucleotide of claim 17, wherein the self-amplifying replicon RNA is derived from an alphavirus, the alphavirus comprising Venezuelan equine encephalitis virus, Semliki Forest virus, Sindbis virus, or a combination thereof.

19. A vector comprising the isolated polynucleotide of any one of claims 1 to 18.

20. A lipid nanoparticle comprising: (i) an isolated polynucleotide according to any one of claims 1 to 19; and (ii) one or more lipids.

21. The lipid nanoparticle of claim 20, wherein the one or more lipids comprise a cationic lipid or a lipidoid.

22. The lipid nanoparticle of claim 20 or 21, wherein the one or more lipids are N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3).

23. The lipid nanoparticle of any one of claims 20 to 22, comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, C14-PEG2000, or any combination thereof.

24. The lipid nanoparticle of claim 23, wherein the C14-PEG2000 is embedded in the LNP or the C14-PEG2000 is added after the isolated polynucleotide is encapsulated in the lipid nanoparticle.

25. The lipid nanoparticle described in any one of claims 20 to 24, wherein the lipid nanoparticle has a diameter of about 30 to 500 nm, and the lipid and the isolated polynucleotide (e.g., modified RNA) have a mass ratio of about 1:2 to about 15:

1.

26. A pharmaceutical composition comprising an isolated polynucleotide according to any one of claims 1 to 18, a vector according to claim 19, or a lipid nanoparticle according to any one of claims 20 to 25, and a pharma- ceutically acceptable carrier.

27. A cell comprising an isolated polynucleotide according to any one of claims 1 to 18, a vector according to claim 19, or a lipid nanoparticle according to any one of claims 20 to 25, wherein the cell is an in vitro cell, an ex vivo cell, or an in vivo cell.

28. A method for making a polynucleotide, comprising enzymatically or chemically synthesizing an isolated polynucleotide according to any one of claims 1 to 18.

29. 27. A composition comprising the isolated polynucleotide of any one of claims 1 to 18, the vector of claim 19, or the lipid nanoparticle of any one of claims 20 to 25, for use in an in vivo or ex vivo method of producing IL-12 protein, said method comprising contacting a cell with said isolated polynucleotide, said vector, or said lipid nanoparticle.

30. A composition comprising an isolated polynucleotide according to any one of claims 1 to 18, a composition comprising a vector according to claim 19, a composition comprising a lipid nanoparticle according to any one of claims 20 to 25, or a pharmaceutical composition according to claim 26, for treating cancer.

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