Adeno-associated viral vector compositions and methods of use

Abstract

Described herein are nucleic acids and recombinant viral vectors comprising the nucleic acids, where the nucleic acids encode a payload of interest (e.g., interferon).

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of and priority to U.S. Provisional Application Nos. 63 / 496,916 and 63 / 502,546, filed April 18, 2023 and May 16, 2023, respectively, the entire disclosures of which are hereby incorporated by reference in their entireties for all purposes. SUMMARY OF THE DISCLOSURE

[0002] In certain embodiments, described herein are recombinant adeno-associated viral (rAAV) vectors for use in delivering a payload of interest (e.g., a therapeutic payload) to a subject in need thereof and which exhibit improved efficacy and / or reduced immunogenicity.

[0003] In certain embodiments, also described herein are rAAV vectors comprising a coding sequence for a payload of interest (e.g., a therapeutic polypeptide), wherein CpG dinucleotides within the coding sequence have been reduced, depleted, and / or methylated, resulting in certain beneficial therapeutic properties. In some embodiments, the coding sequence comprising one or more modified CpG dinucleotides (e.g., CpG dinucleotide reduction, CpG dinucleotide depletion, and / or CpG dinucleotide methylation) encodes a therapeutic polypeptide, as described herein. In some embodiments, the coding sequence comprising one or more modified CpG dinucleotides encodes an interferon (e.g., any interferon known in the art, e.g., those described herein), as described herein.

[0004] In certain embodiments, also described herein are methods of treating a disease or disorder in a subject in need thereof comprising administering a modified rAAV vector as described herein. In some embodiments, the present disclosure provides methods of treating a cancer in a subject comprising administering a modified rAAV vector as described herein. In some embodiments, the cancer is a brain cancer. In some embodiments, the cancer is an eye cancer. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a glioma, such as a grade III or IV glioma (glioblastoma). In some embodiments, the cancer is a glioblastoma. In some embodiments, the cancer is a high-grade glioma, metastatic brain tumor, or uveal melanoma. In some embodiments, administration of the rAAV vector comprising a cytokine (e.g., any of the cytokines delivered by the rAAV vectors described herein) is local to the tumor of the subject having the cancer. In some embodiments, administration of the rAAV vector comprising a cytokine to the subject having the cancer is systemic (e.g., intravenous). Moreover, the rAAV vector compositions and methods listed herein provide long-term cytokine production (e.g., by vectorization, robust expression, and the like), high specificity of payload delivery (e.g., by localized administration), and minimal risk of systemic toxicity (e.g., by low-dose administration of self-limiting AAV genomes).

[0005] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a CAG promoter; and b) a polynucleotide encoding a payload of interest, the polynucleotide comprising reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to a parental equivalent.

[0006] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a CAG promoter; b) a small gene comprising a splice modulator binding site; c) a polynucleotide encoding a payload of interest, the polynucleotide comprising reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to a parental equivalent.

[0007] In some embodiments, the payload of interest is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a cytokine or an interleukin. In some embodiments, the therapeutic polypeptide is a cytokine. In some embodiments, the cytokine is a colony stimulating factor (CSF), a transforming growth factor, a tumor necrosis factor, an interleukin, or an interferon. In some embodiments, the cytokine is an interferon. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more compared to the parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more compared to the parental equivalent. In some embodiments, the CpG dinucleotides are depleted.

[0008] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a CAG promoter; b) a polynucleotide encoding an interferon; and c) a polynucleotide comprising a WPRE comprising SEQ ID NO: 33.

[0009] In some embodiments, the CAG promoter comprises a cytomegalovirus (CMV) immediate early enhancer element, a promoter element, and a splice acceptor element. In some embodiments, the CMV early enhancer element is derived from a wild-type CMV enhancer. In some embodiments, the CMV immediate early enhancer element is truncated relative to the wild-type CMV enhancer. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 26. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide sequence as set forth in SEQ ID NO: 26. In some embodiments, the CMV early enhancer element comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 27. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 27. In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide sequence as set forth in SEQ ID NO: 27. In some embodiments, the promoter element is derived from a chicken beta-actin gene. In some embodiments, the promoter element comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 28. In some embodiments, the promoter element comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 52. In some embodiments, the promoter element comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, the promoter element comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 52. In some embodiments, the promoter element comprises a polynucleotide sequence as set forth in SEQ ID NO: 28. In some embodiments, the promoter element comprises a polynucleotide sequence as set forth in SEQ ID NO: 52. In some embodiments, the splice acceptor is derived from a rabbit beta-globin gene. In some embodiments, the splice acceptor comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 30. In some embodiments, the splice acceptor comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 53.In some embodiments, the splice acceptor comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 30. In some embodiments, the splice acceptor comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 53. In some embodiments, the splice acceptor comprises a polynucleotide sequence as set forth in SEQ ID NO: 30. In some embodiments, the splice acceptor comprises a polynucleotide sequence as set forth in SEQ ID NO: 53. In some embodiments, the interleukin is IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36. In some embodiments, the CSF is CSF1 (M-CSF), CSF2 (GM-CSF), or CSF3 (G-CSF). In some embodiments, the TGF is TGF-β1, TGF-β2, or TGF-β3. In some embodiments, the TNF is TNF-a, TNF-β, or LT-β. In some embodiments, the interferon is IFNa, IFNp, IFNy, IFNe, IFNk, IFNw, IFNl, or a variant or derivative thereof. In some embodiments, the interferon is human IFNa, IFNp, IFNy, IFNe, IFNk, IFNw, IFNl, or a variant or derivative thereof. In some embodiments, the interferon is human IFNp. In some embodiments, the human IFNp comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the human IFNp comprises an amino acid having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the human IFNp comprises an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the human IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the human IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.In some embodiments, the human IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the interferon is human IFNa. In some embodiments, the human IFNa comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, the human IFNa comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, the human IFNa comprises an amino acid sequence set forth as SEQ ID NO: 5. In some embodiments, the human IFNa is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the human IFNa is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the human IFNa is encoded by a polynucleotide set forth as SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the interferon is human IFNy. In some embodiments, the human IFNy comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9. In some embodiments, the human IFNy comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, the human IFNy comprises an amino acid sequence set forth as SEQ ID NO: 9. In some embodiments, the human IFNy is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the human IFNy is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the human IFNy is encoded by a polynucleotide set forth as SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the interferon is mouse IFNa, IFNp, IFNy, IFN£, IFN, IFN, IFN, or a variant or derivative thereof. In some embodiments, the interferon is mouse IFNp. In some embodiments, the mouse IFNp comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the mouse IFNp comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13.In some embodiments, the mouse IFNp comprises an amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the mouse IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the mouse IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the mouse IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 14. In some embodiments, the mouse IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, the mouse IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, the mouse IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 42. In some embodiments, the interferon is canine IFN a, IFNp, IFN g, IFN e, IFN K, IFN w, IFN l, or a variant or derivative thereof. In some embodiments, the interferon is canine IFNp. In some embodiments, the canine IFNp comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15. In some embodiments, the canine IFNp comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, the canine IFNp comprises an amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, the canine IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 16. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, the canine IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 43. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 60. In some embodiments, the canine IFNp is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 60. In some embodiments, the canine IFNp is encoded by a polynucleotide set forth as SEQ ID NO: 60.In some embodiments, the interferon is a rat IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, or a variant or derivative thereof. In some embodiments, the interferon is a rat IFN beta. In some embodiments, the rat IFN beta comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 61. In some embodiments, the rat IFN beta comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61. In some embodiments, the rat IFN beta comprises the amino acid sequence set forth as SEQ ID NO: 61. In some embodiments, the rat IFN beta is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 62. In some embodiments, the rat IFN beta is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 62. In some embodiments, the rat IFN beta is encoded by a polynucleotide set forth as SEQ ID NO: 62. In some embodiments, the rat IFN beta is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 63. In some embodiments, the rat IFN beta is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 63. In some embodiments, the rat IFN beta is encoded by a polynucleotide set forth as SEQ ID NO: 63. In some embodiments, the interferon is a guinea pig IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, or a variant or derivative thereof. In some embodiments, the interferon is a guinea pig IFN beta. In some embodiments, the guinea pig IFN beta comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, the guinea pig IFN beta comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, the guinea pig IFN beta comprises the amino acid sequence set forth as SEQ ID NO: 71. In some embodiments, the guinea pig IFN beta is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, the guinea pig IFN beta is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, the guinea pig IFN beta is encoded by a polynucleotide set forth as SEQ ID NO: 72. In some embodiments, the interferon-encoding polynucleotide comprises a reduced number of CpG dinucleotides and / or an increased methylation of CpG dinucleotides as compared to a parental equivalent.In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated. In some embodiments, the methylation of CpG dinucleotides is decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are depleted. In some embodiments, the rAAV vector further comprises a first ITR sequence and a second ITR sequence. In some embodiments, the rAAV vector comprises, from 5’ to 3’: a) a first ITR sequence; b) a promoter; c) a polynucleotide encoding a payload of interest; and d) a second ITR sequence. In some embodiments, the rAAV vector comprises, from 5’ to 3’: a) a first ITR sequence; b) a promoter; c) a minigene; d) a polynucleotide encoding a payload of interest; and e) a second ITR sequence. In some embodiments, the first ITR sequence and / or the second ITR sequence is truncated compared to their corresponding wild-type ITR sequence. In some embodiments, the first ITR sequence and / or the second ITR sequence is truncated at the 5’ end or the 3’ end by at least about 5 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by 20 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by 20 nucleotides.In some embodiments, the first ITR sequence and / or the second ITR sequence comprises an ITR sequence derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof. In some embodiments, the first ITR sequence and / or the second ITR sequence is an AAV2 ITR sequence or is derived from an AAV2 ITR sequence. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 36-41. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 36-41. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a polynucleotide sequence as set forth in SEQ ID NO: 36-41. In some embodiments, the rAAV vector further comprises at least one regulatory element. In some embodiments, the regulatory element is selected from the group consisting of a promoter, an enhancer, a terminator sequence, an mRNA stability sequence, a sequence that allows for an internal ribosome entry site (IRES) for a bicistronic mRNA, an intron, a synthetic intron, a sequence that inhibits viral recognition, a sequence necessary for transduction into a cell, and a polyadenylation sequence. In some embodiments, the regulatory element is a promoter. In some embodiments, the promoter is selected from the group consisting of a mini-promoter, an inducible promoter, a constitutive promoter, and a derivative thereof.In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synapsin, GFAP, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is a CAG promoter. In some embodiments, the sequence that allows for internal ribosome entry site (IRES) of a bicistronic mRNA is a WPRE. In some embodiments, the WPRE is a wild-type WPRE. In some embodiments, the WPRE comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 32. In some embodiments, the WPRE comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 32. In some embodiments, the WPRE comprises a polynucleotide as set forth in SEQ ID NO: 32. In some embodiments, the WPRE is a modified WPRE. In some embodiments, the modified WPRE comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 33. In some embodiments, the modified WPRE comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 33. In some embodiments, the modified WPRE comprises an amino acid sequence as set forth in SEQ ID NO: 33. In some embodiments, the polyadenylation sequence is selected from the group consisting of SV40, hGH, bGH, and rbGlob. In some embodiments, the polyadenylation sequence is an SV40 sequence. In some embodiments, the SV40 sequence comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 19. In some embodiments, the SV40 sequence comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 19. In some embodiments, the SV40 sequence comprises a polynucleotide as set forth in SEQ ID NO: 19. In some embodiments, the polyadenylation sequence is a bGH sequence. In some embodiments, the bGH sequence comprises a polynucleotide having at least 80% sequence identity to SEQ ID NO: 34. In some embodiments, the bGH sequence comprises a polynucleotide having at least 90% sequence identity to SEQ ID NO: 34. In some embodiments, the bGH sequence comprises a polynucleotide sequence as set forth in SEQ ID NO: 34. In some embodiments, the rAAV vector further comprises a minigene located 5' to the polynucleotide encoding the payload of interest. In some embodiments, the minigene encodes a splice modulator binding site. In some embodiments, the splice modulator binding site is located in an exon and / or an intron.In some embodiments, the splice modulator binding site comprises one or more sequences required for spliceosome binding. In some embodiments, the splice modulator binding site comprises a donor site sequence, a branch site, and an acceptor site. In some embodiments, the minigene encodes an in-frame translational stop codon. In some embodiments, the polynucleotide encoding the payload of interest further comprises a translational stop codon. In some embodiments, the polynucleotide encoding the payload of interest does not comprise a start codon. In some embodiments, the polynucleotide encoding the payload of interest does not comprise an in-frame open reading frame. In some embodiments, the minigene is regulated by a small molecule splicing modifier. In some embodiments, the small molecule splicing modifier is a sudemycin, LMI070, RG7916, or RG7800. In some embodiments, the small molecule splicing modifier is selected from the group consisting of: , and In some embodiments, the minigene comprises exon 6, exon 7, and exon 8 of SMN2 and the splicing modulator binding site is recognized by LMI070. In some embodiments, the minigene comprises at least about 80% sequence identity to SEQ ID NO: 31. In some embodiments, the minigene comprises at least about 90% sequence identity to SEQ ID NO: 31. In some embodiments, the minigene comprises a polynucleotide as set forth in SEQ ID NO: 31. In some embodiments, the minigene comprises at least about 80% sequence identity to SEQ ID NO: 51. In some embodiments, the minigene comprises at least about 90% sequence identity to SEQ ID NO: 51. In some embodiments, the minigene comprises a polynucleotide as set forth in SEQ ID NO: 51. In some embodiments, the minigene is regulated by a disease state of a cell. In some embodiments, the disease state is cancer. In some embodiments, the cancer is glioblastoma, metastatic brain tumor, or uveal melanoma. In some embodiments, the minigene is regulated by a cell type or tissue type. In some embodiments, the polynucleotide comprising the minigene and the polynucleotide encoding the payload of interest are linked by a polynucleotide sequence encoding a cleavable peptide. In some embodiments, the cleavable peptide is a substrate for a self-cleaving peptide, a drug-sensitive protease, or an endogenous endoprotease. In some embodiments, the rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof. In some embodiments, the rAAV vector further comprises an antibiotic resistance gene.In some embodiments, the antibiotic resistance gene confers resistance to an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or a derivative thereof. In some embodiments, the antibiotic resistance gene confers resistance to kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, giomycin, or a derivative thereof. In some embodiments, the antibiotic resistance gene confers resistance to kanamycin. In some embodiments, the antibiotic resistance gene comprises a nucleic acid sequence having at least about 80% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 44, or SEQ ID NO: 55. In some embodiments, the antibiotic resistance gene comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 44, or SEQ ID NO: 55. In some embodiments, the antibiotic resistance gene comprises a nucleic acid as set forth in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 44, or SEQ ID NO: 55. In some embodiments, the antibiotic resistance gene comprises reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to a parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to a parental equivalent. In some embodiments, the CpG dinucleotides are depleted. In some embodiments, the rAAV vector further comprises an origin of replication. In some embodiments, the origin of replication is selected from the group consisting of pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, pBluescript, and combinations or derivatives thereof.

[0010] In certain embodiments, described herein are methods of treating cancer in a subject in need thereof comprising: a) administering a rAAV vector as described herein; and b) administering a small molecule splicing modifier. In some embodiments, the subject is a human. In some embodiments, the administration is administration to the central nervous system. In some embodiments, the administration is administration to the brain. In some embodiments, the administration is administration to the cerebral ventricles. In some embodiments, the administration is by convection enhanced delivery (CED). In some embodiments, the administration is by intratumoral injection, intracranial injection, intracerebral injection, intracerebroventricular injection, intraparenchymal injection, or injection into the cerebrospinal fluid (CSF) via the ventricular system, the cistema magna, or the intrathecal space. In some embodiments, the small molecule splicing modifier is a sudemycin, LMI070, RG7916, or RG7800. In some embodiments, the small molecule splicing modifier is selected from the group consisting of: , and .

[0011] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a first ITR set out as SEQ ID NO: 40 or SEQ ID NO: 41; b) a CAG promoter set out as any one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a polynucleotide encoding a CpG depleted human interferon set out as SEQ ID NO: 4; d) a WPRE sequence set out as SEQ ID NO: 33; e) a bGH poly-adenine set out as SEQ ID NO: 34; and f) a second ITR set out as SEQ ID NO: 40 or SEQ ID NO: 41.

[0012] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a first ITR set out as SEQ ID NO: 40 or SEQ ID NO: 41; b) a CAG promoter set out as any one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a polynucleotide encoding a CpG depleted human interferon set out as SEQ ID NO: 4; d) a WPRE sequence set out as SEQ ID NO: 33; e) a SV40 poly-adenine set out as SEQ ID NO: 19; and f) a second ITR set out as SEQ ID NO: 40 or SEQ ID NO: 41.

[0013] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR comprising the sequence set forth in any one of SEQ ID NOs: 36-39; b) a CAG promoter set forth in any one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a polynucleotide encoding a CpG depleted human interferon set forth in SEQ ID NO: 4; d) a WPRE sequence set forth in SEQ ID NO: 33; e) a bGH polyadenylate set forth in SEQ ID NO: 34; and f) a second ITR sequence set forth in any one of SEQ ID NOs: 36-39.

[0014] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR set forth in SEQ ID NO: 40 or SEQ ID NO: 41; b) a CAG promoter set forth in any one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a polynucleotide encoding a human interferon set forth in SEQ ID NO: 2 or SEQ ID NO: 3; d) a WPRE sequence set forth in SEQ ID NO: 33; e) a SV40 polyadenylate set forth in SEQ ID NO: 19; and f) a second ITR set forth in SEQ ID NO: 40 or SEQ ID NO: 41.

[0015] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR set forth in SEQ ID NO: 40 or SEQ ID NO: 41; b) a CAG promoter set forth in any one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a minigene set forth in SEQ ID NO: 31 or SEQ ID NO: 51; d) a polynucleotide encoding a CpG depleted human interferon set forth in SEQ ID NO: 4; e) a WPRE sequence set forth in SEQ ID NO: 33; f) a SV40 polyadenylate set forth in SEQ ID NO: 19; and g) a second ITR set forth in SEQ ID NO: 40 or SEQ ID NO: 41.

[0016] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG depleted human interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0017] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG depleted human interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0018] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG depleted human interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0019] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG depleted human interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0020] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG depleted human interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0021] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a first ITR sequence; b) a CAG promoter; c) a minigene as set forth in SEQ ID NO: 31 or SEQ ID NO: 51; d) a coding sequence for a CpG depleted human interferon; e) a WPRE sequence; f) a SV40 polyadenylation sequence; and g) a second ITR sequence.

[0022] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5’ to 3’: a) a first ITR sequence; b) a CAG promoter; c) a minigene as set forth in SEQ ID NO: 31 or SEQ ID NO: 51; d) a coding sequence for a CpG depleted human interferon; e) a WPRE sequence; f) a bGH polyadenylation sequence; and g) a second ITR sequence. BRIEF DESCRIPTION OF DRAWINGS

[0023] The disclosure can be more completely understood in reference to the following drawings.

[0024] Figure 1A An exemplary workflow depicting the mechanism of the rAAV described herein.

[0025] Figure 1B An exemplary inducible AAV expression system plasmid map depicting the various component elements used throughout the AAV expression system in the specific embodiments.

[0026] Figure 2 An exemplary constitutive AAV expression system plasmid map depicting the various component elements used throughout the AAV expression system in the specific embodiments.

[0027] Figure 3 Data from in vitro validation of inducible GFP fluorescence is depicted.

[0028] Figure 4 In vitro kinetic data for inducible GFP fluorescence obtained by viral dose escalation is depicted.

[0029] Figures 5A-5AD An exemplary plasmid vector map of plasmids 1-30 is depicted. Figure 5A An exemplary plasmid, plasmid #1, is depicted, which includes a CAG promoter sequence, a CpG depleted human interferon beta (hIFNp) gene sequence, a polyadenylation signal sequence derived from SV40 (SV40 pA), and an ampicillin resistance (AmpR) gene. Figure 5BAn exemplary plasmid, plasmid #2, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, an SV40 pA, and an AmpR gene. Figure 5C An exemplary plasmid, plasmid #3, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, a bovine growth hormone polyadenylation signal (bGH pA), and a kanamycin resistance (KanR) gene. Figure 5D An exemplary plasmid, plasmid #4, is depicted that includes a CAG promoter sequence, a CpG depleted hIFN beta gene sequence, a bGH pA, and a KanR gene. Figure 5E An exemplary plasmid, plasmid #5, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a KanR gene. Figure 5F An exemplary plasmid, plasmid #6, is depicted that includes a CAG promoter sequence, a CpG depleted hIFN beta gene sequence, a bGH pA, and a KanR gene. Figure 5G An exemplary plasmid, plasmid #7, is depicted that includes a CAG promoter sequence, a wild type hIFN beta gene sequence, an SV40 pA, and an AmpR gene. Figure 5H An exemplary plasmid, plasmid #8, is depicted that includes a CAG promoter sequence, a CpG depleted hIFN beta gene sequence, a bGH pA, and a KanR gene. Figure 5I An exemplary plasmid, plasmid #9, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a KanR gene. Figure 5J An exemplary plasmid, plasmid #10, is depicted that includes a CAG promoter sequence, a CpG depleted hIFN beta gene sequence, a bGH pA, and a KanR gene. Figure 5K An exemplary plasmid, plasmid #11, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a KanR gene. Figure 5L An exemplary plasmid, plasmid #12, is depicted that includes a CAG promoter sequence, a CpG depleted hIFN beta gene sequence, a bGH pA, and a CpG depleted KanR gene. Figure 5M An exemplary plasmid, plasmid #13, is depicted that includes a CAG promoter sequence, a Xon, a hIFN beta gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a CpG depleted KanR gene. Figure 5NAn exemplary plasmid, plasmid #14, is depicted that includes a CAG promoter sequence, a CpG-depleted hIFN beta gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5O An exemplary plasmid, plasmid #15, is depicted that includes a CAG promoter sequence, Xon, a hIFN beta gene sequence that is CpG-depleted and lacks an ATG codon, a bGH pA, and a CpG-depleted KanR gene. Figure 5P An exemplary plasmid, plasmid #16, is depicted that includes a CAG promoter sequence, an enhanced green fluorescent protein (eGFP) gene sequence, a bGH pA, and a KanR gene. Figure 5Q An exemplary plasmid, plasmid #17, is depicted that includes a CAG promoter sequence, Xon, an eGFP gene sequence that lacks an ATG codon, a bGH pA, and a KanR gene. Figure 5R An exemplary plasmid, plasmid #18, is depicted that includes a CAG promoter sequence, an eGFP gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5S An exemplary plasmid, plasmid #19, is depicted that includes a CAG promoter sequence, Xon, an eGFP gene sequence that lacks an ATG codon, a bGH pA, and a CpG-depleted KanR gene. Figure 5T An exemplary plasmid, plasmid #20, is depicted that includes a CAG promoter sequence, an mCardinal gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5U An exemplary plasmid, plasmid #21, is depicted that includes a CAG promoter sequence, an mCardinal gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5V An exemplary plasmid, plasmid #22, is depicted that includes a CAG promoter sequence, Xon, an mCardinal gene sequence that lacks an ATG codon, a bGH pA, and a CpG-depleted KanR gene. Figure 5W An exemplary plasmid, plasmid #23, is depicted that includes a CAG promoter sequence, Xon, an mCardinal gene sequence that lacks an ATG codon, a bGH pA, and a CpG-depleted KanR gene. Figure 5X An exemplary plasmid, plasmid #24, is depicted that includes a CAG promoter sequence, a CpG-depleted hIFN beta gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5Y An exemplary plasmid, plasmid #25, is depicted that includes a CAG promoter sequence, an mCardinal gene sequence, a bGH pA, and a CpG-depleted KanR gene. Figure 5ZAn exemplary plasmid, plasmid #26, is depicted, which includes a CAG promoter sequence, a mouse interferon beta (mIFNp) gene sequence, a bGH pA, and a CpG depleted KanR gene. Figure 5AA An exemplary plasmid, plasmid #27, is depicted, which includes a CAG promoter sequence, a rat interferon beta (rIFNp) gene sequence, a bGH pA, and a CpG depleted KanR gene. Figure 5AB An exemplary plasmid, plasmid #28, is depicted, which includes a CAG promoter sequence, a canine interferon beta (cIFNp) gene sequence, a bGH pA, and a CpG depleted KanR gene. Figure 5AC An exemplary plasmid, plasmid #29, is depicted, which includes a CAG promoter sequence, Xon, a hIFNp gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a CpG depleted KanR gene. Figure 5AD An exemplary plasmid, plasmid #30, is depicted, which includes a CAG promoter sequence, Xon, a hIFNp gene sequence that is CpG depleted and lacks an ATG codon, a bGH pA, and a CpG depleted KanR gene.

[0030] Figure 6 An exemplary workflow for producing rAAV vector plasmids used to produce rAAV vectors as disclosed herein is depicted.

[0031] Figure 7 A sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) stained with Coomassie blue is depicted, evaluating the purity of exemplary AAV preparations when 1e11 vg of the corresponding AAV preparation was loaded into a well on a 10% protein gel and electrophoresed for protein separation.

[0032] Figures 8A-8C Data from in vitro assessment of payload production efficiency is depicted. Figures 8A-8B Relative production efficacy of various AAV preparations is depicted, expressed as the ratio of vg titer (vg / mL) of each AAV preparation to the vg titer of a reference stock (AAV produced using plasmid #1 (A) or plasmid #12 (B)). The plasmid used to produce the corresponding AAV is identified on the X-axis. Figure 8A Data from in vitro assessment of payload production efficiency is depicted. Figure 8B Data from in vitro assessment of payload production efficiency is depicted. Figure 8C Vg titers obtained from 5 individual 1 liter (L) shake flasks of suspended cells using plasmid #1 are depicted.

[0033] Figures 9A-9E Data from in vitro assessment of payload expression is depicted. Figure 9APayload expression measured for AAV prep #6, AAV prep #5, AAV prep #8, and AAV prep #9, each produced at a different vendor with a different process, is depicted. All preparations were produced with plasmid #1, which carries a human INF beta (hIFN beta) payload and is packaged within the same AAV capsid serotype. Figure 9B Payload expression measured for AAV prep #1, AAV prep #9, AAV prep #10, and AAV prep #11 (produced from plasmid #7, plasmid #1, plasmid #12, and plasmid #14, respectively) of various AAV genomes produced at one vendor using the same process and packaged within the same AAV capsid serotype is depicted. Figure 9C Payload expression measured for AAV prep #6, AAV prep #9, AAV prep #10, AAV prep #2, AAV prep #13, and AAV prep #16 (produced from plasmid #1, plasmid #1, plasmid #12, plasmid #7, plasmid #24, and plasmid #25, respectively) with different AAV genomes expressing hIFN beta (except AAV prep #16, which contains a reporter payload (mCardinal) and serves as a negative control) is depicted. All other preparations, except AAV prep #6, were performed at the same vendor using the same process. Figure 9D Payload expression of AAV prep #3, AAV prep #6, AAV prep #14, and AAV prep #15 (produced from plasmid #7, plasmid #1, plasmid #24, and plasmid #24, respectively) is depicted. Both AAV prep #14 and AAV prep #15 utilize plasmid #24, but are produced at 2 different vendors. Figure 9E Payload expression measured for AAV prep #6 and AAV prep #17 (produced using plasmid #1 and plasmid #26 and expressing human and mouse IFN beta, respectively) packaged in the same AAV serotype is depicted. For all graphs, data represent mean ± SD of technical replicates. Abbreviations: MOI, multiplicity of infection; prep, preparation.

[0034] Figure 10 In vitro measurement of cytokine payload activity measured for six exemplary AAVs expressing hIFN beta (AAV prep #6, AAV prep #9, AAV prep #10, AAV prep #2, and AAV prep #13), one AAV expressing a reporter payload (mCardinal; AAV prep #12), and media (NC) serving as a negative control is depicted. Values represent mean ± SD of 8 technical replicates.

[0035] Figure 11Depicted is an in vitro assessment of reduced cancer cell viability following exposure to the human IFNβ-expressing AAV expression system described herein (AAV-hIFNβ). As depicted, relative cell viability is expressed as a percentage at day 6 following exposure to low, medium, or high doses of AAV-hIFNβ or AAV-GFP. Staurosporine (STS) was used as a positive killing control, and data are expressed as mean ± SD with n = 4 technical replicates.

[0036] Figure 12 Depicted is the level of hIFNβ secreted in vitro from transduced GBM cells as measured with an ELISA. Values represent the mean ± SD of 4 technical replicates.

[0037] Figure 13 Depicted is an exemplary process and assay for manufacturing the AAV constructs described herein. DETAILED DESCRIPTION

[0038] In the United States, cancer continues to be the second most common cause of death after heart disease. In 2023, an estimated 1.9 million new cases of cancer (about 5,370 cases per day) and 609,820 cancer deaths (about 1,670 deaths per day) are expected to occur in the United States. Treatment options for many cancers (e.g., glioblastoma, metastatic brain tumors, and uveal melanoma) remain largely limited to invasive and / or cytotoxic methods, including resection, radiation, chemotherapy, and combinations thereof. Even common immune-based oncology drugs, such as cytokine therapies, exhibit negative features, including toxicity due to systemic administration, short half-life, and lack of specificity. Moreover, methods for delivering therapies adequately to cancerous tissue remain problematic.

[0039] There is a need for improved therapies for treating cancer (e.g., glioblastoma, metastatic brain tumors, uveal melanoma), including those with improved features, such as lower toxicity, long-term, stable, and persistent expression, improved delivery (e.g., local delivery to tumors), and low-dose cytokine therapies. Provided herein are compositions and methods involving rAAV delivery of a payload of interest (e.g., a cytokine such as interferon).

[0040] DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. Generally, the nomenclature and techniques described herein follow those used in the art of immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization, which are well known and commonly used. Units of measure not otherwise defined conform to The International System of Units (SI), NIST Special Publication 330, 2019 Edition.

[0041] Unless the context clearly dictates otherwise, as used herein, all numerical values or numerical ranges include the whole integers within or encompassing the range or values or fractions of the integers within or encompassing the range. Thus, for example, reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, or 1.5 fold, etc., 2.1, 2.2, 2.3, 2.4, or 2.5 fold, etc., and so on.

[0042] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated items.

[0043] As used herein, the term "adeno-associated viral vector" or "AAV vector" refers to a vector derived from an adeno-associated viral serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16. An AAV vector can have all or a portion of one or more AAV wild-type genes, e.g., Rep and / or Cap genes, deleted, but retains functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences facilitate rescue, replication, and packaging of the AAV virion. Thus, an AAV vector is defined herein as containing at least those sequences required in cis for replication and packaging of the virus (e.g., functional ITRs). The ITR sequence need not be a wild-type polynucleotide sequence so long as it provides for functional rescue, replication, and packaging, and in some embodiments is altered, e.g., by insertion, deletion, or substitution of nucleotides.

[0044] The term "adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the region flanking each end of the AAV genome that functions together in cis as an origin of DNA replication and as a packaging signal for the virus. As used herein, an "AAV ITR" does not necessarily comprise a wild-type polynucleotide sequence, which is altered, e.g., by insertion, deletion or substitution of nucleotides, in some embodiments. Additionally, the AAV ITR is derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, among others. Furthermore, the 5' and 3' ITRs flanking a selected polynucleotide sequence in an AAV vector need not be identical or derived from the same AAV serotype or isolate, so long as they function as intended, e.g., to permit a desired therapeutic or genome editing effect. Additionally, AAV ITR modifications to the D element can encode or facilitate different configurations of the AAV genome, single-stranded AAV genome (i.e., ssAAV), or self-complementary AAV genome (i.e., scAAV).

[0045] As used herein, the term "about," in reference to a number or a range of numbers, is understood to mean the number ± 10% of the number, or, in reference to a range, the lower limit is 10% less and the upper limit is 10% greater than what is recited.

[0046] As used herein, "CpG dinucleotide," "CpG site," or "CpG" refers to a region in a nucleic acid (e.g., DNA or RNA) in which a cytosine nucleotide occurs next to a guanine nucleotide along the length of the linear nucleic acid sequence of nucleotides, e.g., -C-phospho-G-, with the cytosine and guanine separated by only one phosphate, or with the cytosine on the 5' side of the guanine nucleotide.

[0047] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and, in some embodiments, refer to any mammalian subject in whom diagnosis, treatment, or therapy is desired, particularly a human. A "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sports animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require that the mammal be under medical supervision or housed in a facility.

[0048] "Percent identity," "% identity," or "sequence identity" refers to the extent to which two sequences (nucleotide or amino acid) have identical residues at a position in the alignment. For example, "a nucleotide sequence is X% identical to SEQ ID NO: Y" means that the nucleotide sequence is X% identical to SEQ ID NO: Y and is detailed as X% of the residues in the nucleotide sequence are identical to the corresponding residues of the sequence disclosed in SEQ ID NO: Y. A sequence that is said to be X% identical to a reference sequence can contain more nucleotide or amino acid residues than specified in the reference sequence, but must contain the sequence corresponding to the reference sequence. In most cases, the sequence in question will contain the sequence corresponding to all of the specified reference sequence. Typically, a computer program is applied to make such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0049] “Polynucleotide” or “nucleic acid” are used interchangeably herein and refer to a chain of nucleotides of any length and include DNA or RNA. In some embodiments, the nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs (e.g., increased methylated CpG dinucleotides as described). If present, modifications to the nucleotide structure are imparted before or after the chain is assembled. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, the polynucleotide is further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “capping”; substitution of one or more of the naturally occurring nucleotides with an analog; internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates) and charged linkages (e.g., phosphorothioates, phosphorodithioates); those containing prosthetic groups such as, for example, biotin, phenaziny, phosphodiesterase substrates; those with intercalators (e.g., acrida ine, psoralen); those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals); those containing alkylators; those with modified linkages (e.g., alpha anomeric nucleotides) and unmodified forms of polynucleotides. In some embodiments, any of the normally occurring hydroxyl groups on the sugars are replaced, for example, by phosphonate groups, phosphate groups, standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or to be conjugated to the solid support. In some embodiments, the 5’ and 3’ terminal OH are replaced by groups such as, for example, phosphate groups, biotin, phenaziny, phosphodiesterase substrates; acrida ine, psoralen; metals, radioactive metals, boron, oxidative metals; alkylators; alpha anomeric nucleotides and unmodified forms of polynucleotides. Other hydroxyls can also be derivatized to standard protecting groups. In some embodiments, the polynucleotide further comprises an analog form of ribose or deoxyribose sugar, including, for example, 2’-O-methyl-, 2’-O-allyl, 2’-fluoro-ribose or 2’-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose, furanose, octulosonate, acyclic analogs, and abasic nucleotide analogs such as methyl riboside. In some embodiments, one or more phosphodiester linkages are replaced by alternative linking groups.These alternative linkages include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NRi (“amidate”), P(O)R, P(O)OR’, CO, or CH2 (“formacetal”), where each R or R’ is independently H or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl group optionally containing ether (-O-) linkages. Not all linkages in a polynucleotide need be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA. In some embodiments, the nucleic acids or polynucleotides disclosed herein have reduced CpG dinucleotides compared to a parental equivalent. In some embodiments, the nucleic acids or polynucleotides disclosed herein have depleted CpG dinucleotides (i.e., all CpG dinucleotides have been modified so as to no longer be CpG dinucleotides or all CpG dinucleotides have been deleted) compared to a parental equivalent. In some embodiments, the nucleic acids or polynucleotides disclosed herein have increased methylated CpG dinucleotides.

[0050] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable to mammals, particularly human or animal patients.

[0051] A “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector derived from AAV and comprising one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one AAV ITR. In some embodiments, such rAAV vectors are replicated and packaged into viral particles when introduced into a host cell having a suitable helper polynucleotide or virus (or which expresses a suitable helper function) and expressing AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector is referred to as a “pre-vector” which is “rescued” by replication and encapsidation in the presence of AAV packaging functions and a suitable helper function.

[0052] As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance or composition (e.g., an AAV vector as described herein) that partially or completely relieves, ameliorates, alleviates, suppresses, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment can be of a subject who does not exhibit signs of the relevant disease, disorder, and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment can be of a subject who exhibits one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0053] As used herein, "operably linked," "operable linkage," "operatively linked," or grammatical equivalents thereof refer to an arrangement of genetic elements, e.g., promoters, enhancers, polyadenylation sequences, etc., in which the operation (e.g., movement or activation) of a first genetic element has some effect on a second genetic element. The effect on the second genetic element can be, but is not necessarily, the same type of operation as the first genetic element. For example, two genetic elements are operably linked if movement of the first element results in activation of the second element. For example, a regulatory element (which can include a promoter and / or enhancer sequence) is operably linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues can be present between the regulatory element and the coding region as long as the functional relationship is maintained.

[0054] The terms "peptide," "polypeptide," and "protein" as used herein refer to a chain of amino acids. The term protein as used herein also means a macromolecule comprising one or more chains of amino acids, and in some embodiments, is a fragment or domain of a protein, or a full-length protein. Further, as used herein, the term protein refers to a linear chain of amino acids or to a chain of amino acids that has been processed and folded into a functional protein. Protein structure is classified into four different levels: (1) primary structure—referring to the sequence of amino acids in a polypeptide chain, (2) secondary structure—referring to regular local sub-structures on the polypeptide backbone, such as alpha-helices and beta-sheets, (3) tertiary structure—referring to the three-dimensional structure of monomeric and multimeric protein molecules, and (4) quaternary structure—referring to the aggregated three-dimensional structure comprising two or more individual polypeptide chains operating as a single functional unit. The use of peptide or polypeptide herein does not mean that the chain of amino acids is not a protein (i.e., a chain of amino acids with secondary, tertiary, or quaternary structure).

[0055] The term "wild type" or "wild-type" as used herein refers to a form of an organism, strain, gene, nucleic acid, vector, or vector component that naturally occurs in nature as distinguished from a mutant or variant form.

[0056] Recombinant adeno-associated viral (rAAV) vectors of the disclosure Cytokines are regulators of innate and adaptive immunity, enabling cells of the immune system to communicate. There has been considerable interest in exploiting cytokines for the treatment of cancer due to the ability of the immune system to recognize and destroy cancer cells. However, current cytokine therapies exhibit negative features, including toxicity due to systemic administration, short half-life, and lack of specificity. There is a need for improved cytokine therapies for the treatment of cancer (e.g., glioblastoma, metastatic brain tumors, uveal melanoma).

[0057] Provided herein are compositions and methods related to rAAV delivery of a cytokine payload of interest that allow for targeted, low-dose, direct delivery to tumors and tumor microenvironments. Local tumor delivery and low-dose of rAAV can result in a reduction of local and systemic toxicity, for example, by bypassing neutralizing antibodies that arise during systemic administration of rAAV. In addition, the rAAV described herein have a longer half-life that enables long-term, durable, and stable expression, and are also self-limiting in that payload activity generally ceases with tumor death. The compositions and methods described herein can also provide reduced non-specific inflammation by being immunologically and to vector integrity optimized.

[0058] An exemplary workflow of the mechanism of the rAAV described herein is found in Figure 1AThe rAAV described herein allows for local delivery to the tumor (1), which in turn leads to expression of the engineered cytokine in tumor cells and direct tumor cell lysis. As a result, inflammatory cytokines are released, triggering an innate immune response (3) and activation of macrophages and natural killer cells to clear the tumor and released AAV antigens, followed by an adaptive immune response (4). As a result, the tumor is rapidly cleared with reduced local and systemic toxicity.

[0059] In certain embodiments, described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a promoter; b) a polynucleotide encoding a payload of interest; and c) a polynucleotide comprising a regulatory element. In some embodiments, the regulatory element is an inducible regulatory element (e.g., an inducible system, such as a splicing modulator system and / or a promoter). In some embodiments, the regulatory element is a constitutively active regulatory element.

[0060] In certain embodiments, also described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for an interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0061] In certain embodiments, also described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a promoter; and b) a polynucleotide encoding a payload of interest, which comprises reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides as compared to a parental equivalent.

[0062] In certain embodiments, also described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG-depleted interferon; d) a WPRE sequence; e) a SV40 polyadenylation sequence; and f) a second ITR sequence.

[0063] In certain embodiments, also described herein is a recombinant adeno-associated viral (rAAV) vector comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a coding sequence for a CpG-depleted interferon; d) a WPRE sequence; e) a bGH polyadenylation sequence; and f) a second ITR sequence.

[0064] In certain embodiments, also described herein are recombinant adeno-associated viral (rAAV) vectors comprising, from 5' to 3': a) a promoter; b) a small gene comprising a splice modulator binding site; and c) a polynucleotide encoding a payload of interest, the polynucleotide comprising reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent.

[0065] In certain embodiments, also described herein are recombinant adeno-associated viral (rAAV) vectors comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a small gene as set forth in SEQ ID NO: 31; d) a coding sequence for a CpG depleted human interferon; e) a WPRE sequence; f) a SV40 polyadenylation sequence; and g) a second ITR sequence.

[0066] In certain embodiments, also described herein are recombinant adeno-associated viral (rAAV) vectors comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a small gene as set forth in SEQ ID NO: 51; d) a coding sequence for a CpG depleted interferon; e) a WPRE sequence; f) a SV40 polyadenylation sequence; and g) a second ITR sequence.

[0067] In certain embodiments, also described herein are rAAV vectors comprising, from 5' to 3': a) a promoter; b) a regulatory element comprising a splice modulator binding site; and c) a polynucleotide encoding a payload of interest, the polynucleotide comprising reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent.

[0068] In certain embodiments, also described herein are rAAV vectors comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a regulatory element as set forth in SEQ ID NO: 31; d) a coding sequence for a CpG depleted interferon; e) a WPRE sequence; f) a bGH polyadenylation sequence; and g) a second ITR sequence.

[0069] In certain embodiments, also described herein are rAAV vectors comprising, from 5' to 3': a) a first ITR sequence; b) a CAG promoter; c) a regulatory element as set forth in SEQ ID NO: 51; d) a coding sequence for a CpG depleted interferon; e) a WPRE sequence; f) a bGH polyadenylation sequence; and g) a second ITR sequence.

[0070] CpG dinucleotide modification In certain embodiments, described herein are recombinant adeno-associated viral (rAAV) vectors comprising reduced CpG dinucleotides compared to a parent equivalent. In some embodiments, the rAAV vector comprises increased methylation of CpG dinucleotides compared to a parent equivalent. In some embodiments, the rAAV vector comprises depleted CpG dinucleotides compared to a parent equivalent. In some embodiments, the rAAV vector comprises reduced CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, a CpG dinucleotide is said to be depleted or reduced if one or more of its nucleotides is substituted with one or more different nucleotides such that it is no longer a CpG dinucleotide sequence. In some embodiments, if a CpG dinucleotide of a rAAV comprising a coding sequence (e.g., a coding sequence of a regulatory element, a gene (e.g., a transgene), an antibiotic resistance gene, etc.) is substituted with one or more different nucleotides, the one or more different nucleotides are selected such that the function of the coding sequence is preserved. In other words, if the coding sequence is a regulatory element such as a promoter or enhancer, the function of the promoter or enhancer is preserved, or if the coding sequence encodes a polypeptide, the polypeptide amino acid sequence and / or the polypeptide function is preserved.

[0071] In some embodiments, the rAAV vector comprises a reduced number of CpG dinucleotides compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the CpG dinucleotides are reduced in the range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0072] In some embodiments, the rAAV vector comprises depleted CpG dinucleotides as compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by deletion of certain CpG dinucleotides. In some embodiments, the CpG dinucleotides are reduced by substitution of one or more nucleotides within the CpG dinucleotide to produce a sequence that is not a CpG dinucleotide. In some embodiments, the CpG dinucleotides are depleted by deletion of all CpG dinucleotides. In some embodiments, the CpG dinucleotides are depleted by substitution of one or more nucleotides within each CpG dinucleotide to produce a sequence that is not a CpG dinucleotide.

[0073] In some embodiments, the rAAV vector comprises increased methylation of CpG dinucleotides compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%.

[0074] In some embodiments, CpG dinucleotides are modified (e.g., reduced, depleted, and / or increased in methylation) across the entire length of a provided rAAV vector. In some embodiments, CpG dinucleotides are modified only in one or more critical portions or elements of a provided rAAV vector (e.g., in regulatory elements, polynucleotides comprising coding sequences, etc.). The present disclosure provides, inter alia, the insight that certain polynucleotides encoding payloads of interest (e.g., polynucleotides encoding one or more interferons, such as IFN ) exhibit superior therapeutic outcomes (e.g., increased efficacy, lower toxicity, etc.) when the CpG dinucleotides are modified within the coding sequence of the payload of interest. Thus, in one non-limiting example, a rAAV vector provided by the present disclosure comprises a polynucleotide encoding an interferon, wherein the coding sequence of the interferon comprises modified CpG dinucleotides (e.g., reduced, depleted, and / or increased in methylation) compared to a parental equivalent, but other elements in the flanking nucleic acids do not have modified CpG dinucleotides. In another non-limiting example, a rAAV vector provided by the present disclosure comprises a polynucleotide encoding an interferon, wherein the coding sequence of the interferon comprises modified CpG dinucleotides (e.g., reduced, depleted, and / or increased in methylation) compared to a parental equivalent, and one or more other polynucleotides in the rAAV vector comprise modified CpG dinucleotides (e.g., promoters, antibiotic resistance genes, minigenes, and / or any polynucleotide element described herein).

[0075] Minigene regulation In some embodiments, the regulatory element of the present disclosure is an inducible regulatory element (e.g., an inducible system, such as a splice modulator system and / or a promoter). In some embodiments, the inducible regulatory element is a minigene. In some embodiments, the regulatory element is a constitutive regulatory element.

[0076] In some embodiments, described herein are recombinant adeno-associated viral (rAAV) vectors comprising a minigene comprising a splice modulator binding site. In some embodiments, the minigene is located 5’ to a polynucleotide encoding a payload of interest. In some embodiments, the rAAV described herein comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) exons, introns, pseudexons, fragments thereof, or combinations thereof.

[0077] In some embodiments, the minigene comprises a minimal gene fragment comprising at least one exon and a control region or splice modulator site that is necessary for the minimal gene fragment to regulate expression of a downstream polynucleotide sequence. In some embodiments, the minigene comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) exons, introns, pseudexons, fragments thereof, or combinations thereof. In many embodiments, the minigene splice modulator site controls the splicing outcome of the RNA transcript encoded by the minigene. In some embodiments, the splice modulator site directs the RNA transcript towards a particular splicing outcome based on the presence of factors in a particular cellular or tissue context (e.g., splicing outcome 1 occurs if the minigene is present in a liver cell, splicing outcome 2 occurs if the minigene is present in a retinal cell, splicing outcome 3 occurs if the minigene is present in a kidney cell, etc.). In some embodiments, the splice modulator site directs the RNA transcript towards a particular splicing outcome due to the binding of a splice modulator to the splice modulator site (e.g., at one or more nucleic acid sequences). In some embodiments, the splice modulator is a polypeptide, a nucleic acid, or a small molecule. In some embodiments, the minigene is used to regulate expression of a downstream polynucleotide sequence by engineering the minigene to only allow (or activate) expression of the downstream polynucleotide sequence in the presence of a splice modulator and / or in the context of a particular cell / tissue type.

[0078] In some embodiments, the minigene encodes an in-frame translational stop codon. In some embodiments, alternative splicing of the minigene transcript removes the in-frame translational stop codon. In some embodiments, alternative splicing of the minigene transcript modifies the transcript such that the stop codon is deleted or eliminated, a start codon or initiation codon is introduced, an open reading frame is restored, or a missing portion of a protein is provided. In some embodiments, alternative splicing of the minigene transcript allows for transcription and translation of a downstream polynucleotide (e.g., a polynucleotide encoding a payload of interest), as described herein.

[0079] In some embodiments, the polynucleotide downstream of the minigene (e.g., a polynucleotide encoding a payload of interest, as described herein) comprises a translational stop codon. In some embodiments, the polynucleotide downstream of the minigene does not comprise a start codon. In some embodiments, the polynucleotide downstream of the minigene does not comprise an open reading frame.

[0080] In some embodiments, the minigene and the polynucleotide encoding a payload of interest are linked by a cleavable peptide. In some embodiments, the cleavable peptide is a substrate for a self-cleaving peptide, a drug-sensitive protease, or an endogenous endoprotease.

[0081] In some embodiments, the minigene comprises the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 31.

[0082] In some embodiments, the minigene comprises the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 51.

[0083] In some embodiments, the minigene is modulated by a small molecule splicing modifier. In some embodiments, the small molecule splicing modifier is sudemycin (FR901464, pladiellin B), LMI070, RG7916, or RG7800, and derivatives thereof.

[0084] In some embodiments, the small molecule splicing modifier is LMI070 (CAS No.: 1562338-42-4), having the following structure:

[0085] In some embodiments, the small molecule splicing modifier is RG7916 (Roche / PTC / SMAF, 7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[l,2-b]pyrazin-6-yl)-4H-pyrido[l,2- a]pyrimidin-4-one) (CAS No.: 1825352-65-5), having the following structure:

[0086] In some embodiments, the small molecule splicing modifier is RG7800 (Roche) (CAS No.: 1449598-06-4), having the following structure:

[0087] In some embodiments, the small molecule splicing modulator is an analog of RG7916 or RG7800.

[0088] In some embodiments, the small molecule splicing modulator is a sudemycin selected from the group consisting of (5',Z)-5-(((lR,4R)-4-((2JE',4JE)-5-((3R,55')-7,7-dimethyl-l,6-dioxaspiro[2.5]octan-5-yl)-3-methylpent-2,4-dien-l-yl)cyclohexyl)amino)-5- oxopent-3-en-2-ylcarbamate and (5',Z)-5-(((lR,4R)-4-((2JE',4JE)-5-((3R,55')-7,7-dimethyl-l,6-dioxaspiro[2.5]octan-5-yl)-3-methylpent-2,4-dien-l-yl)cyclohexyl)amino)-5- oxopent-3-en-2-yl dimethylcarbamate.

[0089] In some embodiments, the small molecule splicing modulator is a pladiellin compound. One example of a pladiellin compound is (8E,12E,14E)-7-((4-cycloheptylpiperazin-l-yl)carbonyl)oxy-3,6,16,21-tetrahydroxy-6,10,12,16,20-pentamethyl-18,19-epoxy- tricos-8,12,14-trien-11-one, also known as E7107, which is a semisynthetic derivative of the natural product pladiellin D.

[0090] In some embodiments, the small gene is regulated by a disease state of a cell. In some embodiments, the disease state is cancer. In some embodiments, the cancer is glioblastoma.

[0091] In some embodiments, the small gene is regulated by a cell type or tissue type.

[0092] Payload of interest In certain embodiments, disclosed herein are polynucleotides encoding a payload of interest. In some embodiments, the payload of interest is a therapeutic agent, such as a therapeutic polypeptide.

[0093] In some embodiments, a therapeutic agent is any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, an appropriate population is a population of model organisms. In some embodiments, an appropriate population is defined by various criteria, such as a particular age group, gender, genetic background, pre-existing clinical conditions, and the like. In some embodiments, a therapeutic agent is a substance that can be used to reduce, ameliorate, alleviate, suppress, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is an agent that has been or needs to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription to be administered to humans. In some embodiments, a therapeutic agent is a therapeutic polypeptide or a therapeutic polynucleotide. In some embodiments, a therapeutic polypeptide is a cytokine (e.g., an interferon).

[0094] In some embodiments, a therapeutic polypeptide is a cytokine. In some embodiments, a cytokine is a colony-stimulating factor (CSF), a transforming growth factor (e.g., transforming growth factor-beta), a tumor necrosis factor (e.g., tumor necrosis alpha), an interleukin, or an interferon. In some embodiments, a cytokine is an interferon.

[0095] In some embodiments, the interleukin (IL) is interleukin-1 alpha (IL-1a), interleukin-1 beta (IL-1b), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-17 (IL-17), interleukin-18 (IL-18), interleukin-19 (IL-19), interleukin-20 (IL-20), interleukin-21 (IL-21), interleukin-22 (IL-22), interleukin-23 (IL-23), interleukin-24 (IL-24), interleukin-25 (IL-25), interleukin-26 (IL-26), interleukin-27 (IL-27), interleukin-28 (IL-28), interleukin-29 (IL-29), interleukin-30 (IL-30), interleukin-31 (IL-31), interleukin-32 (IL-32), interleukin-33 (IL-33), interleukin-34 (IL-34), interleukin-35 (IL-35), or interleukin-36 (IL-36).

[0096] In some embodiments, the colony stimulating factor (CSF) is CSF1 (M-CSF), CSF2 (GM-CSF), or CSF3 (G-CSF).

[0097] In some embodiments, the transforming growth factor (TGF) is TGF-beta 1, TGF-beta 2, or TGF-beta 3.

[0098] In some embodiments, the tumor necrosis factor (TNF) is TNF-alpha, TNF-beta, or LT-beta.

[0099] In some embodiments, the interferon (IFN) is a Type I IFN, Type II IFN, or Type III IFN. In some embodiments, the IFN is IFN alpha (IFNa), IFN beta (IFNp), IFN gamma (IFNy), IFN epsilon (IFNe), IFN kappa (IFNk), IFN omega (IFNw), IFN lambda (IFNl), IFN chi (IFNc), IFN xi (IFNxi), IFN tau (IFNt), IFN delta (IFNd), IFN nu (IFNv), IFN zeta (IFNz), IFN alfa, and derivatives thereof. In some embodiments, the interferon is IFNa, IFNp, IFNy, or a combination thereof. In some embodiments, the interferon is IFNa, IFNp, IFNy, IFNe, IFNk, IFNw, IFNl, IFNc, IFNxi, IFNt, IFNd, IFNv, IFNz, IFN alfa, or a variant or derivative thereof. In some embodiments, the IFN is IFN a-1, IFN a-2, IFN a-4, IFN a-5, IFN a-6, IFN a-7, IFN a-8, IFN a-10, IFN a-13, IFN a-14, IFN a-16, IFN a-17, IFN a-21, or a variant or derivative thereof. In some embodiments, the IFN is IFN-p1, IFN-p2, IFN-p3, or a variant or derivative thereof.

[0100] In some embodiments, the IFN is of a mouse, rat, equine, ruminant (e.g., sheep, cow, goat), primate (e.g., chimpanzee, baboon, gorilla, orangutan, monkey), dog, cat, pig, donkey, rabbit, fish, fly, human, non-primate placental mammal, or non-rodent placental mammal. In some embodiments, the IFN is human IFN beta. In some embodiments, the IFN is human IFN alpha, human IFN beta, human IFN gamma, human IFN omega, human IFN epsilon, human IFN kappa, human IFN tau, human IFN zeta, human IFN alfa, or a variant or derivative thereof. In some embodiments, the IFN is mouse IFN alpha, mouse IFN beta, mouse IFN gamma, or a variant or derivative thereof. In some embodiments, the IFN is mouse IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, IFN zeta, or a variant or derivative thereof. In some embodiments, the IFN is mouse IFN beta. In some embodiments, the IFN is canine IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, or a variant or derivative thereof. In some embodiments, the IFN is canine IFN beta, or a variant or derivative thereof. In some embodiments, the IFN is rat IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, or a variant or derivative thereof. In some embodiments, the IFN is rat IFN beta, or a variant or derivative thereof. In some embodiments, the IFN is guinea pig IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, or a variant or derivative thereof. In some embodiments, the IFN is guinea pig IFN beta, or a variant or derivative thereof. In some embodiments, the IFN is non-primate and non-rodent placental mammal IFN delta, or a variant or derivative thereof. In some embodiments, the IFN is placental mammal IFN epsilon, IFN kappa, or a variant or derivative thereof.

[0101] In some embodiments, the IFN is a subtype of IFN alpha. In some embodiments, the IFN alpha is IFN alpha 1, IFN alpha 2, IFN alpha 4, IFN alpha 5, IFN alpha 6, IFN alpha 7, IFN alpha 8, IFN alpha 10, IFN alpha 13, IFN alpha 14, IFN alpha 16, IFN alpha 17, IFN alpha 21, and derivatives thereof.

[0102] In some embodiments, the therapeutic polypeptide comprises one or more interferons. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, and IFNa-IFNp-IFNy. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, or IFNa-IFNp-IFNy, wherein the IFNs are mouse. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, or IFNa-IFNp-IFNy, wherein the IFNs are human. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, or IFNa-IFNp-IFNy, wherein the IFNs are canine. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, or IFNa-IFNp-IFNy, wherein the IFNs are rat. In some embodiments, the therapeutic polypeptide comprises IFNa, IFNp, IFNy, IFNa-IFNp, IFNa-IFNy, IFNp-IFNy, or IFNa-IFNp-IFNy, wherein the IFNs are guinea pig.

[0103] In some embodiments, the human interferon beta (hIFNp) has a sequence represented by UniProt / SwissProt database entry number P01574 (SEQ ID NO: 1). In some embodiments, the hIFNp is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 75% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 97% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 98% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having at least 99% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 2-4. In some embodiments, the hIFNp is encoded by a nucleic acid sequence having the nucleic acid sequence of any one of SEQ ID NOs: 2-4.

[0104] In some embodiments, the interferon is human IFNp. In some embodiments, the human IFNp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the human IFNp comprises the amino acid sequence set forth as SEQ ID NO: 1. In some embodiments, the human IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the human IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0105] In some embodiments, the human interferon alpha (hIFNa1) has a sequence represented by UniProt / SwissProt database entry number P01562 (SEQ ID NO: 5). In some embodiments, the hIFNa is encoded by a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 75% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 97% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 98% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having at least 99% sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 6-8. In some embodiments, the hIFNa is encoded by a nucleic acid sequence having the nucleic acid sequence of any one of SEQ ID NOs: 6-8.

[0106] In some embodiments, the interferon is a human IFNa. In some embodiments, the human IFNa comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, the human IFNa comprises the amino acid sequence set forth as SEQ ID NO: 5. In some embodiments, the human IFNa is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0107] In some embodiments, the human IFNα is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the human IFNα is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, the human IFNα is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, the human IFNα is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the human IFNα is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 6. In some embodiments, the human IFNα is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 7. In some embodiments, the human IFNα is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 8.

[0108] In some embodiments, human interferon gamma (hIFNy) has a sequence represented by UniProt / SwissProt database entry number P01579 (SEQ ID NO: 9). In some embodiments, hIFNy is encoded by a nucleic acid sequence of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 60% (e.g., 60%, 65%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a nucleic acid sequence in SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 75% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 97% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 98% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having at least 99% sequence identity to a nucleic acid sequence of any one of SEQ ID NO: 10-12. In some embodiments, hIFNy is encoded by a nucleic acid sequence having a nucleic acid sequence of any one of SEQ ID NO: 10-12.

[0109] In some embodiments, the interferon is human IFNy. In some embodiments, the human IFNy comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the human IFNy comprises the amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the human IFNy is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the human IFNy is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the human IFNy is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11. In some embodiments, the human IFNy is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, the human IFNy is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 10. In some embodiments, the human IFNy is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 11. In some embodiments, the human IFNy is encoded by a polynucleotide sequence as set forth in SEQ ID NO: 12.

[0110] In some embodiments, the interferon is mouse interferon β (mIFNβ). In some embodiments, mouse interferon β (mIFNβ) has the sequence represented by UniProt / SwissProt database entry P01575 (SEQ ID NO: 13). In some embodiments, mIFNβ is encoded by the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 60% (e.g., 60%, 65%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 75% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some embodiments, mIFNβ is encoded by a nucleic acid sequence having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 42. In some implementations, mIFNβ is encoded by a nucleic acid sequence having a nucleic acid sequence having SEQ ID NO: 14 or SEQ ID NO: 42.

[0111] In some embodiments, the interferon is mouse IFNp. In some embodiments, the mouse IFNp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the mouse IFNp comprises the amino acid sequence set forth as SEQ ID NO: 13. In some embodiments, the mouse IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the mouse IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42. In some embodiments, the mouse IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 14. In some embodiments, the mouse IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 42.

[0112] In some embodiments, the interferon is canine interferon beta (cIFNp). In some embodiments, the canine interferon beta (cIFNp) has a sequence represented by UniProt / UniProtKB database entry no. B6E116 (SEQ ID NO: 15). In some embodiments, the interferon is canine interferon beta (cIFNp) and is encoded by a nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 60% (e.g., 60%, 65%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60. In some embodiments, the cIFNp is encoded by a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 16, SEQ ID NO: 43, or SEQ ID NO: 60.

[0113] In some embodiments, the interferon is canine IFNp. In some embodiments, the canine IFNp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the canine IFNp comprises the amino acid sequence set forth as SEQ ID NO: 15. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 60. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 16. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 43. In some embodiments, the canine IFNp is encoded by a polynucleotide sequence set forth as SEQ ID NO: 60.

[0114] In some embodiments, the interferon is rat IFNp. In some embodiments, the rat IFNp comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 61. In some embodiments, the rat IFNp comprises the amino acid sequence set forth as SEQ ID NO: 61.

[0115] In some embodiments, the rat IFNβ is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 62. In some embodiments, the rat IFNβ is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 63. In some embodiments, the rat IFNβ is encoded by a polynucleotide sequence set forth as SEQ ID NO: 62. In some embodiments, the rat IFNβ is encoded by a polynucleotide sequence set forth as SEQ ID NO: 63.

[0116] In some embodiments, the interferon is guinea pig IFNβ. In some embodiments, the guinea pig IFNβ comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 71. In some embodiments, the guinea pig IFNβ comprises an amino acid sequence set forth as SEQ ID NO: 71.

[0117] In some embodiments, the guinea pig IFNβ is encoded by a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 72. In some embodiments, the guinea pig IFNβ is encoded by a polynucleotide sequence set forth as SEQ ID NO: 72.

[0118] In some embodiments, the polynucleotide encoding the interferon is codon-optimized. In some embodiments, the polynucleotide encoding the interferon is codon-optimized for expression of the interferon. Codon-optimization can be used to increase translation rate, or to produce a recombinant RNA transcript with desirable properties, such as higher expression efficiency compared to a transcript produced using an unoptimized sequence. In particular embodiments, the polynucleotide encoding the interferon is codon-optimized for expression in mammalian cells and human cells.

[0119] In some embodiments, provided herein are recombinant adeno-associated viral (rAAV) vectors comprising a polynucleotide encoding a payload of interest (e.g., a cytokine, such as any of the interferons described herein), wherein the coding sequence of the payload of interest comprises modified CpG dinucleotides (e.g., reduced, depleted, and / or increased methylation) compared to a parental equivalent. In one non-limiting example, a rAAV vector comprises a polynucleotide encoding an interferon (e.g., IFNa, IFNp, IFNy, IFNe, IFNk, IFNw, IFNl, IFNc, IFN, IFNt, IFNp, IFNv, IFNz, IFN alfa, or a variant or derivative thereof), wherein the coding sequence of the interferon comprises modified CpG dinucleotides (e.g., reduced, depleted, and / or increased methylation) compared to a parental equivalent. In some embodiments, the interferon-encoding polynucleotide comprises reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated. In some embodiments, the CpG dinucleotides are depleted.

[0120] In some embodiments, the polynucleotide encoding the payload of interest comprises a reduction in CpG dinucleotides compared to the parent equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parent equivalent. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the CpG dinucleotides are reduced in the range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0121] In some embodiments, the polynucleotide encoding the payload of interest comprises depleted CpG dinucleotides as compared to the parent equivalent.

[0122] In some embodiments, the CpG dinucleotides are reduced by deletion of certain CpG dinucleotides. In some embodiments, the CpG dinucleotides are reduced by substitution of one or more nucleotides within a CpG dinucleotide to produce a sequence that is not a CpG dinucleotide. In some embodiments, the CpG dinucleotides are depleted by deletion of all CpG dinucleotides. In some embodiments, the CpG dinucleotides are depleted by substitution of one or more nucleotides within each CpG dinucleotide to produce a sequence that is not a CpG dinucleotide.

[0123] In some embodiments, the polynucleotide encoding the payload of interest comprises an increase in methylation of CpG dinucleotides compared to the parent equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parent equivalent. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%.

[0124] In some embodiments, a polynucleotide encoding an interferon (e.g., mouse, human, canine, feline, ruminant, rat, guinea pig, primate, porcine, or ferret IFN alpha, IFN beta, IFN gamma, IFN epsilon, IFN kappa, IFN omega, IFN lambda, IFN chi, IFN xi, IFN tau, IFN delta, IFN nu, IFN zeta, IFN alfa, including variants or derivatives thereof) comprises a reduced number of CpG dinucleotides compared to the parental equivalent. In some embodiments, the number of CpG dinucleotides is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parental equivalent. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the number of CpG dinucleotides is reduced in a range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the CpG dinucleotides are reduced in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0125] In some embodiments, the polynucleotide encoding the interferon comprises depleted CpG dinucleotides compared to the parent equivalent.

[0126] In some embodiments, the CpG dinucleotides are reduced by deletion of certain CpG dinucleotides. In some embodiments, the CpG dinucleotides are reduced by substitution of one or more nucleotides within a CpG dinucleotide to produce a sequence that is not a CpG dinucleotide. In some embodiments, the CpG dinucleotides are depleted by deletion of all CpG dinucleotides. In some embodiments, the CpG dinucleotides are depleted by substitution of one or more nucleotides within each CpG dinucleotide to produce a sequence that is not a CpG dinucleotide.

[0127] In some embodiments, the polynucleotide encoding the interferon comprises an increase in methylation of CpG dinucleotides compared to the parent equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parent equivalent. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides is increased in a range of about 90% to about 95%, 96%, 97%, 98%, 99%, or 100%.

[0128] In some embodiments, the rAAV vectors described herein comprise one or more polynucleotides encoding one or more interferons. In some embodiments, the one or more polynucleotides encode a polypeptide that is separated by a self-cleaving peptide. For example, a first polynucleotide encoding a first interferon polypeptide and a second polynucleotide encoding a second interferon polypeptide are linked by a polynucleotide encoding a first self-cleaving peptide. Optionally, a sequence encoding a linker (e.g., Gly-Ser-Gly) is upstream (5’) of the sequence encoding the self-cleaving peptide. In some embodiments, a second polynucleotide encoding a second interferon polypeptide and a third polynucleotide encoding a third interferon polypeptide are linked by a polynucleotide encoding a second linker peptide and a polynucleotide encoding a second self-cleaving peptide. In some embodiments, the polynucleotide encoding the second self-cleaving peptide is upstream (3’) of the polynucleotide encoding the second linker peptide.

[0129] Suitable self-cleaving peptides include 2A self-cleaving peptides, such as a P2A self-cleaving peptide, a T2A self-cleaving peptide, a F2A self-cleaving peptide, or an E2A self-cleaving peptide. In some embodiments, the self-cleaving peptide is a P2A self-cleaving peptide and has the sequence of SEQ ID NO: 17.

[0130] In some embodiments, the first self-cleaving peptide and the second self-cleaving peptide are the same. For example, in some embodiments, both are P2A. In some embodiments, the first self-cleaving peptide and the second self-cleaving peptide are not the same. For example, in some embodiments, the first self-cleaving peptide is a P2A self-cleaving peptide and the second self-cleaving peptide is a T2A self-cleaving peptide. In some embodiments, the second self-cleaving peptide is a T2A self-cleaving peptide and has the sequence of SEQ ID NO: 18.

[0131] Vector and viral packaging In some embodiments, the polynucleotides of the present disclosure (e.g., polynucleotides encoding a payload of interest) are delivered by a vector. In some embodiments, the polynucleotides are delivered by a plasmid (e.g., a circular DNA molecule that can replicate autonomously within a cell), a cosmid (e.g., a pWE or sCos vector), an artificial chromosome, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a minicircle, a doggybone, a nanoplasmid, a P1-derived artificial chromosome (PAC), a phagemid, a phage derivative, a baculovirus shuttle vector (bacmid), or a virus. In some embodiments, the vector is selected from the list consisting of: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, pBluescript, pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1, pEF1a-tdTomato, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PURO-NH2-CMYC, pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, pSF-Tac, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pRI 101-AN DNA, pCambia2301, pTYB21, pKLAC2, pAc5.1 / V5-His A, and pDEST8.

[0132] In some embodiments, the plasmid comprises a polynucleotide and an antibiotic resistance gene (e.g., any of the antibiotic resistance genes described herein). In some embodiments, the plasmid further comprises one or more regulatory elements (e.g., any of those described herein). In some embodiments, one or more regulatory elements (e.g., a promoter, an enhancer, a WPRE, and / or a polyadenylation, etc.) are operably linked to one or more coding polynucleotides (e.g., polynucleotides encoding a payload of interest) in the plasmid.

[0133] In some embodiments, the polynucleotide of the present disclosure (e.g., a polynucleotide encoding a payload of interest) is delivered by a virus. In some embodiments, the virus is an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a poliovirus, a vaccinia virus, or a retrovirus. In some embodiments, the virus is an alphavirus. In some embodiments, the virus is a parvovirus. In some embodiments, the virus is an adenovirus. In some embodiments, the virus is an AAV. In some embodiments, the virus is a baculovirus. In some embodiments, the virus is a Dengue virus. In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a herpesvirus. In some embodiments, the virus is a poxvirus. In some embodiments, the virus is an anellovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a poliovirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0134] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSCl, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof. In some embodiments, the herpes virus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0135] In some embodiments, the AAV is AAV1 or a derivative thereof. In some embodiments, the AAV is AAV2 or a derivative thereof. In some embodiments, the AAV is AAV3 or a derivative thereof. In some embodiments, the AAV is AAV4 or a derivative thereof. In some embodiments, the AAV is AAV5 or a derivative thereof. In some embodiments, the AAV is AAV6 or a derivative thereof. In some embodiments, the AAV is AAV7 or a derivative thereof. In some embodiments, the AAV is AAV8 or a derivative thereof. In some embodiments, the AAV is AAV9 or a derivative thereof. In some embodiments, the AAV is AAV10 or a derivative thereof. In some embodiments, the AAV is AAV11 or a derivative thereof. In some embodiments, the AAV is AAV12 or a derivative thereof. In some embodiments, the AAV is AAV13 or a derivative thereof. In some embodiments, the AAV is AAV14 or a derivative thereof. In some embodiments, the AAV is AAV15 or a derivative thereof. In some embodiments, the AAV is AAV16 or a derivative thereof. In some embodiments, the AAV is AAV-rh8 or a derivative thereof. In some embodiments, the AAV is AAV-rh10 or a derivative thereof. In some embodiments, the AAV is AAV-rh20 or a derivative thereof. In some embodiments, the AAV is AAV-rh39 or a derivative thereof. In some embodiments, the AAV is AAV-rh74 or a derivative thereof. In some embodiments, the AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, the AAV is AAV-hu37 or a derivative thereof. In some embodiments, the AAV is AAV-Anc80 or a derivative thereof. In some embodiments, the AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, the AAV is AAV-7m8 or a derivative thereof. In some embodiments, the AAV is AAV-PHP-B or a derivative thereof. In some embodiments, the AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, the AAV is AAV-2.5 or a derivative thereof. In some embodiments, the AAV is AAV-2tYF or a derivative thereof. In some embodiments, the AAV is AAV-3B or a derivative thereof. In some embodiments, the AAV is AAV-LK03 or a derivative thereof. In some embodiments, the AAV is AAV-HSC1 or a derivative thereof. In some embodiments, the AAV is AAV-HSC2 or a derivative thereof. In some embodiments, the AAV is AAV-HSC3 or a derivative thereof. In some embodiments, the AAV is AAV-HSC4 or a derivative thereof. In some embodiments, the AAV is AAV-HSC5 or a derivative thereof. In some embodiments, the AAV is AAV-HSC6 or a derivative thereof. In some embodiments, the AAV is AAV-HSC7 or a derivative thereof.In some embodiments, the AAV is AAV-HSC8 or a derivative thereof. In some embodiments, the AAV is AAV-HSC9 or a derivative thereof. In some embodiments, the AAV is AAV-HSC10 or a derivative thereof. In some embodiments, the AAV is AAV-HSC11 or a derivative thereof. In some embodiments, the AAV is AAV-HSC12 or a derivative thereof. In some embodiments, the AAV is AAV-HSC13 or a derivative thereof. In some embodiments, the AAV is AAV-HSC14 or a derivative thereof. In some embodiments, the AAV is AAV-HSC15 or a derivative thereof. In some embodiments, the AAV is AAV-TT or a derivative thereof. In some embodiments, the AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, the AAV is AAV-Myo or a derivative thereof. In some embodiments, the AAV is AAV-NP40 or a derivative thereof. In some embodiments, the AAV is AAV-NP59 or a derivative thereof. In some embodiments, the AAV is AAV-NP22 or a derivative thereof. In some embodiments, the AAV is AAV-NP66 or a derivative thereof. In some embodiments, the AAV is AAV-HSC16 or a derivative thereof.

[0136] In some embodiments, the virus is HSV-1 or a derivative thereof. In some embodiments, the virus is HSV-2 or a derivative thereof. In some embodiments, the virus is VZV or a derivative thereof. In some embodiments, the virus is EBV or a derivative thereof. In some embodiments, the virus is CMV or a derivative thereof. In some embodiments, the virus is HHV-6 or a derivative thereof. In some embodiments, the virus is HHV-7 or a derivative thereof. In some embodiments, the virus is HHV-8 or a derivative thereof.

[0137] Regulatory element In some embodiments, the recombinant adeno-associated viral (rAAV) vectors of the present disclosure comprise one or more regulatory elements. In some embodiments, the one or more regulatory elements are operably linked to a polynucleotide comprising a coding sequence (e.g., a coding sequence for a polypeptide (e.g., a therapeutic polypeptide, a reporter polypeptide, etc.)). In some embodiments, the regulatory element facilitates a particular function (e.g., RNA splicing, translation initiation, translation termination, etc.) during and / or after transcription of the mRNA transcript. In some embodiments, the regulatory element facilitates the particular function when present on a DNA template. In some embodiments, the regulatory element facilitates the particular function when present on an RNA template. In some embodiments, the regulatory element is selected from the group consisting of a promoter, an enhancer, a terminator sequence, an mRNA stability sequence, a sequence that allows for an internal ribosome entry site (IRES) for a bicistronic mRNA, a sequence that inhibits viral recognition (e.g., by Toll-like or RIG-like receptors such as TLR7, TLR8, TLR9, MDA5, RIG1, and / or DAI), a sequence necessary for transduction into a cell, an intron, a synthetic intron, an exon, a synthetic exon, and a polyadenylation signal. In some embodiments, the rAAV vector comprises a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, a sequence that allows for an internal ribosome entry site (IRES) for a bicistronic mRNA, a polyadenylation signal sequence, or a combination thereof. In some embodiments, the sequence that allows for an internal ribosome entry site (IRES) for a bicistronic mRNA is a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In some embodiments, the rAAV comprises a promoter. In some embodiments, the rAAV vector comprises an enhancer. In some embodiments, the rAAV vector comprises an intron. In some embodiments, the rAAV vector comprises a synthetic intron. In some embodiments, the rAAV vector comprises a microRNA. In some embodiments, the rAAV vector comprises a linker. In some embodiments, the rAAV vector comprises a splicing element. In some embodiments, the rAAV vector comprises a polyadenylation signal sequence.

[0138] In some embodiments, the polyadenylation signal sequence is from SV40. In some embodiments, the polyadenylation signal sequence comprises the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the polyadenylation signal sequence is from bovine growth hormone (bGH). Examples of other suitable polyadenylation signals include synthetic polyadenylation signals, polyadenylation from human growth hormone (hGH), rabbit beta-globin (RGB), or modified RGB (mRGB).

[0139] In some embodiments, the polyadenylation sequence is selected from the group consisting of SV40, hGH, bGH, rbGlob, and derivatives and variants thereof. In some embodiments, the polyadenylation sequence is an SV40 sequence, or a derivative or variant thereof. In some embodiments, the SV40 sequence comprises a polynucleotide as set forth in SEQ ID NO: 19. In some embodiments, the polyadenylation sequence is a bGH sequence, or a derivative or variant thereof. In some embodiments, the bGH sequence comprises a polynucleotide sequence as set forth in SEQ ID NO: 34. In some embodiments, the polyadenylation sequence is a bGH sequence, or a derivative or variant thereof. In some embodiments, the polyadenylation sequence is a rbGlob sequence, or a derivative or variant thereof.

[0140] In some embodiments, the rAAV vector comprises a promoter. In some embodiments, the promoter is selected from the group consisting of a mini-promoter, an inducible promoter, a constitutive promoter, and derivatives thereof. In some embodiments, the constitutive promoter comprises a non-bacterial leader sequence. In some embodiments, the promoter is selected from the group consisting of CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synapsin, GFAP, CaMKII, GRK1, and derivatives thereof. In some embodiments, the promoter is a CMV promoter or a derivative thereof. In some embodiments, the promoter is a CBA promoter or a derivative thereof. In some embodiments, the promoter is an EF1a promoter or a derivative thereof. In some embodiments, the promoter is a CAG promoter or a derivative thereof. In some embodiments, the promoter is a PGK promoter or a derivative thereof. In some embodiments, the promoter is a TRE promoter or a derivative thereof. In some embodiments, the promoter is a U6 promoter or a derivative thereof. In some embodiments, the promoter is a UAS promoter or a derivative thereof. In some embodiments, the promoter is a T7 promoter or a derivative thereof. In some embodiments, the promoter is a Sp6 promoter or a derivative thereof. In some embodiments, the promoter is a lac promoter or a derivative thereof. In some embodiments, the promoter is an araBad promoter or a derivative thereof. In some embodiments, the promoter is a trp promoter or a derivative thereof. In some embodiments, the promoter is a Ptac promoter or a derivative thereof. In some embodiments, the promoter is a p5 promoter or a derivative thereof. In some embodiments, the promoter is a p19 promoter or a derivative thereof. In some embodiments, the promoter is a p40 promoter or a derivative thereof. In some embodiments, the promoter is a synapsin promoter or a derivative thereof. In some embodiments, the promoter is a GFAP promoter or a derivative thereof. In some embodiments, the promoter is a CaMKII promoter or a derivative thereof. In some embodiments, the promoter is a GRK1 promoter or a derivative thereof. In some embodiments, the promoter is a mini-promoter or a derivative thereof. In some embodiments, the promoter is an inducible promoter.

[0141] In some embodiments, the promoter is a CAG promoter. The CAG promoter is a synthetic promoter that is a hybrid of a cytomegalovirus (CMV) early enhancer element and a chicken β-actin (CBA) promoter, comprising (1) a CMV immediate early enhancer element, (2) a CBA promoter element including the first exon and first intron of the CBA gene, and (3) a splice acceptor element of the rabbit β-globin gene. In some embodiments, the CMV immediate early enhancer element is derived from a wild-type CMV enhancer. In some embodiments, the CMV immediate early enhancer element is truncated relative to the wild-type CMV enhancer. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 49. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 50. In some embodiments, the CAG promoter comprises a polynucleotide as listed in SEQ ID NO: 48. In some embodiments, the CAG promoter comprises a polynucleotide as listed in SEQ ID NO: 49. In some embodiments, the CAG promoter comprises a polynucleotide as listed in SEQ ID NO: 50. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 64.In some embodiments, the CAG promoter comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 65.

[0142] In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25.

[0143] In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26. In some embodiments, the CMV early enhancer element comprises the polynucleotide sequence as set forth in SEQ ID NO: 26.

[0144] In some embodiments, the CMV immediate early enhancer element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 27. In some embodiments, the CMV immediate early enhancer element comprises the polynucleotide sequence as set forth in SEQ ID NO: 27.

[0145] In some embodiments, the promoter element is derived from the chicken beta-actin gene. In some embodiments, the promoter element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28. In some embodiments, the promoter element comprises the polynucleotide sequence as set forth in SEQ ID NO: 28. In some embodiments, the promoter element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 52. In some embodiments, the promoter element comprises the polynucleotide sequence as set forth in SEQ ID NO: 52.

[0146] In some embodiments, the splice acceptor is derived from a rabbit beta-globin gene. In some embodiments, the splice acceptor refers to an intron comprising a splice acceptor polynucleotide sequence. In some embodiments, the splice acceptor comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 30. In some embodiments, the splice acceptor comprises a polynucleotide sequence as set forth in SEQ ID NO: 30. In some embodiments, the splice acceptor comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 53. In some embodiments, the splice acceptor comprises a polynucleotide sequence as set forth in SEQ ID NO: 53.

[0147] In some embodiments, the polynucleotide sequence that allows for internal ribosome entry site (IRES) of the bicistronic mRNA is a WPRE. In some embodiments, the WPRE sequence is a wild-type WPRE. In some embodiments, the WPRE comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32. In some embodiments, the WPRE comprises a nucleic acid sequence as set forth in SEQ ID NO: 32. In some embodiments, the WPRE sequence is a modified WPRE sequence. In some embodiments, the modified WPRE comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33. In some embodiments, the modified WPRE comprises an amino acid sequence as set forth in SEQ ID NO: 33.

[0148] Inverted terminal repeat sequence The inverted terminal repeat (ITR) sequences each generally comprise 145 bases (i.e., both the 5’ ITR and 3’ ITR sequences each comprise 145 bases).

[0149] In some embodiments, the recombinant adeno-associated viral (rAAV) vectors described herein further comprise a first ITR sequence and a second ITR sequence. In some embodiments, the rAAV vector comprises, from 5’ to 3’: a) a first ITR sequence; b) a promoter; c) a polynucleotide encoding a payload of interest; and d) a second ITR sequence. In some embodiments, the nucleic acid comprises, from 5’ to 3’: a) a first ITR sequence; b) a promoter; c) a minigene comprising a splice modulator site; d) a polynucleotide encoding a payload of interest; and e) a second ITR sequence.

[0150] In some embodiments, the first ITR sequence and / or the second ITR sequence is truncated compared to the corresponding wild-type ITR sequence or parental equivalent. In some embodiments, the first ITR sequence and / or the second ITR sequence is truncated at the 5’ end or the 3’ end by at least about 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, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 5 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 10 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 15 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 20 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 25 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by about 30 nucleotides. In some embodiments, the first ITR sequence is truncated at the 5’ end by 15 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 5 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 10 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 15 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 20 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 25 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by about 30 nucleotides. In some embodiments, the second ITR sequence is truncated at the 3’ end by 15 nucleotides. In some embodiments, the first ITR sequence is modified to facilitate formation of a self-complementary AAV genome (scAAV). In some embodiments, the second ITR sequence is modified to facilitate formation of a self-complementary AAV genome.

[0151] In some embodiments, the first ITR sequence and / or the second ITR sequence comprises an ITR derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, or a derivative thereof.

[0152] In some embodiments, the first ITR sequence and / or the second ITR sequence is an AAV2 ITR sequence or is derived from an AAV2 ITR sequence. In some embodiments, the first ITR sequence is or is derived from an AAV2 ITR sequence. In some embodiments, the second ITR sequence is or is derived from an AAV2 ITR sequence.

[0153] In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 36. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 38. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 39. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the first ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 41. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 36. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 37. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 38. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 39. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 40. In some embodiments, the second ITR sequence comprises the nucleic acid sequence as set forth in SEQ ID NO: 41.

[0154] Additional nucleic acid elements In some embodiments, the recombinant adeno-associated viral (rAAV) vectors described herein further comprise an antibiotic resistance gene, an origin of replication, an open reading frame, or a combination thereof. In some embodiments, the rAAV vector comprises an antibiotic resistance gene, an origin of replication, and an open reading frame.

[0155] In some embodiments, the rAAV vector further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes resistance to an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or a derivative thereof. In some embodiments, the rAAV vector comprises an antibiotic resistance gene for an aminoglycoside or a derivative thereof. In some embodiments, the rAAV vector comprises an antibiotic resistance gene for a beta-lactam or a derivative thereof. In some embodiments, the rAAV vector comprises an antibiotic resistance gene for a macrolide or a derivative thereof. In some embodiments, the rAAV vector comprises an antibiotic resistance gene that confers resistance to a tetracycline or a derivative thereof.

[0156] In some embodiments, the antibiotic resistance gene confers resistance to kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, giomycin, or a derivative thereof.

[0157] In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 44.

[0158] In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the ampicillin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 55.

[0159] In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20 or 21. In some embodiments, the kanamycin resistance gene comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 20 or 21.

[0160] In some embodiments, the antibiotic resistance gene is operably linked to a promoter. In some embodiments, the promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 45. In some embodiments, the promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 46. In some embodiments, the promoter comprises a polynucleotide sequence as set forth in SEQ ID NO: 47. In some embodiments, the promoter comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47.

[0161] In some embodiments, the antibiotic resistance gene (e.g., any of the antibiotic resistance genes described herein, e.g., a kanamycin resistance gene) comprises reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent.

[0162] In some embodiments, the methylation of CpG dinucleotides in the antibiotic resistance gene (e.g., any of the antibiotic resistance genes described herein, e.g., a kanamycin resistance gene) is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to a parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated.

[0163] In some embodiments, the antibiotic resistance gene (e.g., any of the antibiotic resistance genes described herein, e.g., a kanamycin resistance gene) comprises reduced CpG dinucleotides and / or increased methylation of CpG dinucleotides compared to a parental equivalent.

[0164] In some embodiments, the rAAV vector further comprises an origin of replication. In some embodiments, the origin of replication is derived from a plasmid, such as pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, pBluescript, and / or combinations or derivatives thereof. In some embodiments, the origin of replication is pMB1 or a derivative thereof. In some embodiments, the origin of replication is pBR322 or a derivative thereof. In some embodiments, the origin of replication is ColE1 or a derivative thereof. In some embodiments, the origin of replication is R6K or a derivative thereof. In some embodiments, the origin of replication is p15A or a derivative thereof. In some embodiments, the origin of replication is pSC101 or a derivative thereof. In some embodiments, the origin of replication is ColE2 or a derivative thereof. In some embodiments, the origin of replication is F1 or a derivative thereof. In some embodiments, the origin of replication is pUC or a derivative thereof. In some embodiments, the origin of replication is pBluescript or a derivative thereof.

[0165] In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a derivative thereof. In some embodiments, the WPRE is a mut6 variant of WPRE.

[0166] In some embodiments, the rAAV vectors described herein comprise a reporter sequence for co-expression, such as, but not limited to, lacZ, GFP (e.g., enhanced GFP (eGFP)), CFP, YFP, RFP, BFP, mCherry, mCardinal, firefly luciferase (fLuc), Renilla luciferase, NanoLuc luciferase (nLuc), and tdTomato. In some embodiments, the rAAV vectors comprise a selectable marker.

[0167] In some embodiments, the reporter sequence is mCardinal. In some embodiments, the mCardinal comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 56 or 57.

[0168] In some embodiments, the reporter sequence is eGFP. In some embodiments, the eGFP comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 58 or 59.

[0169] In some embodiments, the reporter sequence is fLuc. In some embodiments, the fLuc comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 66-68.

[0170] In some embodiments, the reporter sequence is nLuc. In some embodiments, the nLuc comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 69-70.

[0171] Recombinant viral vectors In some embodiments, a recombinant adeno-associated viral (rAAV) vector comprises any polynucleotide as described herein or any plasmid as described herein. In some embodiments, the viral vector is derived from an anellovirus. In some embodiments, the viral vector is derived from a parvovirus (e.g., AAV and bocaviruses), a retrovirus (e.g., retroviridae viral vectors), an adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), an anellovirus, an arenavirus, a coronavirus, a negative-strand RNA virus such as an orthomyxovirus (e.g., influenza virus), a rhabdovirus (e.g., rabies and vesicular stomatitis virus), a paramyxovirus (e.g., measles and Sendai), a positive-strand RNA virus such as a picornavirus and an alphavirus (e.g., poliovirus), and a double-stranded DNA virus, including adenoviruses, herpesviruses (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, human papilloma virus, human foamy virus, and hepatitis virus.Examples of retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus. Other examples are murine leukemiaviruses, murine sarcoma viruses, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentivirus.

[0172] Various recombinant viral vectors are contemplated herein. In some embodiments, the recombinant viral vector is a recombinant herpes virus, is a herpes simplex virus (HSV), such as HSV type 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus (HHV) type 6 (HHV-6), HHV-7, or HHV-8. In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2. In some embodiments, the HSV is VZV. In some embodiments, the HSV is EBV. In some embodiments, the HSV is CMV. In some embodiments, the HSV is HHV-6. In some embodiments, the HSV is HHV-7. In some embodiments, the HSV is HHV-8.

[0173] In some embodiments, the recombinant viral vector is an AAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16. In some embodiments, the recombinant AAV vector is an engineered AAV vector.

[0174] In some embodiments, the recombinant AAV is AAV1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV4 or a derivative thereof. In some embodiments, the recombinant AAV is AAV5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV16 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhlO or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhM4-l or a derivative thereof. In some embodiments, the recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-7m8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, the recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC4 or a derivative thereof.In some embodiments, the recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, the recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Myo or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC16 or a derivative thereof.

[0175] Recombinant AAV vector Recombinant adeno-associated viral vectors (rAAV) can be used for incorporation of a gene (e.g., a polynucleotide encoding a payload of interest) to facilitate introduction of the gene into a cell, such as a target cell. The present disclosure provides rAAV vectors comprising any of the nucleic acids described herein. Among other things, rAAV vectors comprising a nucleic acid encoding one or more payloads of interest are described herein. In some embodiments, the one or more payloads of interest encode one or more interferons, as described herein. In some embodiments, the rAAV vector comprises a nucleic acid encoding a single interferon polypeptide. In some embodiments, the rAAV vector comprises a bi- or tri-cistronic nucleic acid encoding more than one interferon polypeptide.

[0176] In some embodiments, the polynucleotides described herein encode one or more payloads of interest (e.g., a therapeutic polypeptide, such as a cytokine) to be delivered to a cell or tissue, as well as regulatory elements that control expression of the one or more payloads of interest. In some embodiments, the polynucleotides described herein encode one or more interferons to be delivered to a cell or tissue, as well as regulatory elements that control expression of the one or more interferons. Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation sequences, introns, synthetic introns, mRNA stability sequences (e.g., the woodchuck hepatitis virus posttranscriptional regulatory element; WPRE), sequences that allow for internal ribosome entry sites (IRES) of bicistronic mRNAs, sequences necessary for episome maintenance (e.g., ITRs), sequences that avoid or inhibit viral recognition by Toll-like or RIG-like receptors (e.g., TLR-7, TLR-8, TLR-9, MDA-5, RIG-1, and / or DAI), and / or sequences necessary for transduction into a cell.

[0177] In some embodiments, the rAAV vectors described herein comprise a CAG promoter operably linked to one or more polynucleotides encoding one or more interferon polypeptides. In some embodiments, the CAG promoter comprises a first segment comprising a cytomegalovirus (CMV) enhancer sequence, a second segment comprising a chicken beta-actin (CBA) gene promoter element, a third segment comprising a spacer sequence, and a fourth segment comprising a rabbit beta-globin splice acceptor. In some embodiments, the order of the 5’-to-3’ segments is first, second, third, and fourth.

[0178] In some embodiments, the CAG promoter comprises the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of contiguous nucleotides of any one of SEQ ID NOs: 22-23. In some embodiments, the CAG promoter comprises at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of contiguous nucleotides of any one of SEQ ID NOs: 22-23.In some embodiments, the CAG promoter comprises the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65. In some embodiments, the CAG promoter comprises about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 contiguous nucleotides of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65.In some embodiments, the CAG promoter comprises at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a contiguous sequence of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of any one of SEQ ID NOs: 48-50 and SEQ ID NOs: 64-65.

[0179] In some embodiments, the CMV enhancer is derived from human CMV. In some embodiments, the CMV enhancer comprises various repeat sequence elements. In some embodiments, the CMV enhancer comprises the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a nucleic acid sequence having the nucleic acid sequence of any one of SEQ ID NOs: 25-27. In some embodiments, the CMV enhancer comprises a contiguous nucleotide of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of any one of SEQ ID NOs: 25-27.In some embodiments, the CMV enhancer comprises at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a contiguous nucleotide sequence of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of any one of SEQ ID NOs: 25-27.

[0180] In some embodiments, the CAG promoter comprises chicken beta-actin (CBA) gene promoter elements. In some embodiments, the CBA gene promoter elements comprise a CBA gene promoter sequence, a CBA gene first exon, and a CBA gene first intron. In some embodiments, the CBA promoter comprises the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a contiguous nucleotide of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of SEQ ID NO: 28.In some embodiments, the CBA promoter comprises at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a contiguous nucleotide sequence of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of SEQ ID NO: 28. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter comprises a contiguous nucleotide of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 520, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of SEQ ID NO: 52.In some embodiments, the CBA promoter comprises at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to a contiguous nucleotide sequence of about 20, about 25, about 40, about 50, about 75, about 100, about 125, about 150, about 175, about 180, about 200, about 220, about 240, about 260, about 520, about 300, about 320, about 340, about 360, about 380, about 400, or about 240 nucleotides of SEQ ID NO: 52.

[0181] In some embodiments, the CAG promoter comprises a spacer sequence immediately 3' of the CBA promoter element. In some embodiments, the spacer contains an intronic element. In some embodiments, the spacer contains an exonic element. In some embodiments, the spacer sequence is about 5 to about 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides in length. In some embodiments, the spacer sequence is about 10 to about 12, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides in length. In some embodiments, the spacer sequence is about 20 to about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides in length. In some embodiments, the spacer sequence is about 50 to about 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides in length. In some embodiments, the spacer sequence is about 80 to about 100, 120, 140, 160, 180, 200, or 250 nucleotides in length. In some embodiments, the spacer sequence is at least 10 nucleotides in length. In some embodiments, the spacer sequence is at least 12 nucleotides in length. In some embodiments, the spacer sequence is at least 20 nucleotides in length. In some embodiments, the spacer sequence is at least 50 nucleotides in length. In some embodiments, the spacer sequence is at least 100 nucleotides in length. In some embodiments, the spacer sequence is at least 250 nucleotides in length. In some embodiments, the spacer sequence is 5 to 20 nucleotides in length. In some embodiments, the spacer is 10 to 20 nucleotides in length. In some embodiments, the spacer sequence is 10 to 20 nucleotides in length. In some embodiments, the spacer sequence is 10 to 15 nucleotides in length. In some embodiments, the spacer sequence is 250 to 350 nucleotides in length. In some embodiments, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 29.In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence is 350 to 1000 nucleotides in length. In some embodiments, the spacer sequence is 500 to 1000 nucleotides in length. In some embodiments, the spacer sequence is 600 to 1000 nucleotides in length. In some embodiments, the spacer sequence is 700 to 1000 nucleotides in length. In some embodiments, the spacer sequence is 800 to 1000 nucleotides in length. In some embodiments, the spacer sequence is 900 to 1000 nucleotides in length.

[0182] In some embodiments, the CAG promoter comprises a rabbit beta-globin splice acceptor. In some embodiments, the rabbit beta-globin splice acceptor comprises the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor comprises the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the CAG promoter comprises a rabbit beta-globin splice acceptor. In some embodiments, the rabbit beta-globin splice acceptor comprises the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 53.In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor comprises a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 53.

[0183] Useful rAAV vectors include those in which one or more naturally occurring AAV genes have been deleted in whole or in part, but functional flanking ITR sequences are retained. In some embodiments, the AAV ITR is of any serotype suitable for a particular application. In some embodiments, the nucleic acid does not comprise an ITR. In some embodiments, the AAV ITR is of any suitable AAV serotype, including any now known or later discovered serotype or any engineered, evolved, selected, or chimeric capsid serotype. In some embodiments, the ITR has a different length or configuration.

[0184] The capsid proteins of AAV make up the exterior, non-nucleic acid portion of the virion and are encoded by the AAV Cap gene. In some embodiments, the Cap gene encodes VP1, VP2, VP3, MAAP, AAP, or a combination thereof.

[0185] In some embodiments, provided herein are Rep, Cap, or other polynucleotides required to produce the rAAV of the disclosure. In some embodiments, the Rep, Cap, or other polynucleotides are delivered to the packaging host cell using any suitable genetic element (e.g., vector). In some embodiments, a single nucleic acid encoding all three capsid proteins (e.g., VP1, VP2, and VP3) is delivered to the packaging host cell in a single vector. In some embodiments, the nucleic acids encoding the capsid proteins are delivered to the packaging host cell by two vectors; a first vector comprising a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2) and a second vector comprising a second nucleic acid encoding a single capsid protein (e.g., VP3). In some embodiments, three vectors each comprising a nucleic acid encoding a different capsid protein are delivered to the packaging host cell.

[0186] In some embodiments, a single nucleic acid encoding more than one replication protein (e.g., Rep78, Rep68, Rep52, and Rep40) is delivered to the packaging host cell in a single vector. In some embodiments, the nucleic acids encoding the replication proteins are delivered to the packaging host cell by two vectors; a first vector comprising a first nucleic acid encoding one to three replication proteins and a second vector comprising a second nucleic acid encoding one to three replication proteins. In some embodiments, four vectors each comprising a nucleic acid encoding a different replication protein are delivered to the packaging host cell.

[0187] In some embodiments, a single nucleic acid encoding more than one adenovirus helper protein (e.g., E1A, E1B, E4, E2A, and VA RNA) is delivered to the packaging host cell in a single vector. In some embodiments, the nucleic acids encoding the adenovirus helper proteins are delivered to the packaging host cell by two vectors. In some embodiments, more than two vectors each comprising a nucleic acid encoding a different capsid protein are delivered to the packaging host cell. The selected genetic elements are delivered by any suitable method, including those described herein. Methods for constructing any embodiment of the disclosure include genetic engineering, recombinant engineering, and synthetic techniques.

[0188] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16.

[0189] In some embodiments, the recombinant AAV is AAV1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV4 or a derivative thereof. In some embodiments, the recombinant AAV is AAV5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV16 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhlO or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhM4-l or a derivative thereof. In some embodiments, the recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-7m8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, the recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC4 or a derivative thereof.In some embodiments, the recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, the recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Myo or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC16 or a derivative thereof.

[0190] In some embodiments, the rAAV vector is a pseudotyped rAAV vector. Pseudotyped vectors include, for example, AAV genome vectors of a given serotype pseudotyped with Cap gene products derived from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, etc.). For example, a representative pseudotyped vector is an AAV serotype 2 genome vector pseudotyped with Cap gene products derived from AAV serotype 9.

[0191] In some embodiments, the AAV has a mutation within the virion capsid that serves to more efficiently transduce a particular cell type than the unmutated capsid virion. In some embodiments, suitable AAV mutants have ligand insertions that serve to promote targeting of the AAV to a particular cell type. In some embodiments, construction and characterization of AAV capsid mutants including insert mutants, alanine scanning mutants, and epitope tag mutants are used.

[0192] In some embodiments, artificial AAV capsids are used. Such artificial capsids are generated by any suitable technique using selected AAV sequences (e.g., fragments of VP1 capsid proteins) combined with heterologous sequences obtained from a different selected AAV serotype, non-contiguous portions of the same AAV serotype, from a non- AAV viral source, or from a non-viral source. Artificial AAV serotypes are, without limitation, pseudotyped AAVs, chimeric AAV capsids, recombinant AAV capsids, engineered AAV capsids, or “humanized” AAV capsids.

[0193] Other rAAV virions used in the compositions and methods of the disclosure include, without limitation, those capsid chimeras generated by molecular breeding of viruses, as well as by exon shuffling, directed evolution, rational design, error-prone PCR, generated by computational algorithms, or generated by using artificial intelligence or machine learning.

[0194] In some embodiments, the capsid is modified to improve therapy. In some embodiments, the capsid is modified to obtain minimized immunogenicity and / or immune cloaking, better stability and / or particle persistence, efficient degradation, and / or accurate delivery of a heterologous coding sequence or a functional fragment or variant thereof to the nucleus. In some embodiments, the modification or mutation is a deletion, insertion, substitution, or any combination thereof, of an amino acid in the capsid polypeptide. In some embodiments, the capsid polypeptide comprises 1, 2, 3, 4, 5, up to 10 or more amino acid substitutions and / or deletions and / or insertions. In some embodiments, one or more amino acid substitutions are introduced into one or more of VP1, VP2, or VP3. In one embodiment, the modified capsid polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions relative to the wild-type polypeptide.

[0195] In another embodiment, the modified capsid polypeptide of the disclosure comprises a modified sequence, where such modifications can comprise both conservative and non-conservative substitutions, deletions, and / or additions, and typically comprise a peptide that shares at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, 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%, or at least 99% sequence identity to the corresponding wild-type capsid protein.

[0196] Methods of generating rAAV vectors Described herein are methods for generating a rAAV vector (e.g., a rAAV vector comprising a polynucleotide as described herein), comprising: contacting a cell in vitro with a polynucleotide described herein and a transfer plasmid, Rep / Cap, helper plasmid, or a combination thereof, to generate a recombinant adeno-associated viral (AAV) vector. In some embodiments, the cell is infected with the rAAV vector. In some embodiments, the cell is transduced with the rAAV vector. In some embodiments, the cell is an engineered stable cell line.

[0197] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungal cell), a mammalian cell (Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryonic kidney (HEK-293), mouse myeloma (NS0), Vero, or human retinal cell), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, a N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda (Sf9) Spodoptera frugiperda ) cell, a Trichoplusia ni (Tn5) Trichoplusia ni ) cell, a Drosophila melanogaster (Dm8 Drosophila melanogaster ) cell, an S2 cell, or a Manduca sexta (Mse Heliothis virescens ) cell), a yeast cell (e.g., a Saccharomyces cerevisiae (Sc1 Saccharomyces cerevisiae ) cell, a Cryptococcus (Cr1 Cryptococcus ) cell, or a Candida (Ca1 Candida ) cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., an Escherichia coli cell, a Streptococcus (St1 Streptococcus bacterium cell, a Streptomyces (St2 Streptomyces ) soil bacterium cell, or an archaeal cell). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.

[0198] In some embodiments, the cell is an A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, primary cell, or derivative thereof. In some embodiments, the cell is an A549 cell or derivative thereof. In some embodiments, the cell is a HEK-293 cell or derivative thereof. In some embodiments, the cell is a HEK-293T cell or derivative thereof. In some embodiments, the cell is a BHK cell or derivative thereof. In some embodiments, the cell is a CHO cell or derivative thereof. In some embodiments, the cell is a HeLa cell or derivative thereof. In some embodiments, the cell is a MRC5 cell or derivative thereof. In some embodiments, the cell is a Sf9 cell or derivative thereof. In some embodiments, the cell is a Cos-1 cell or derivative thereof. In some embodiments, the cell is a Cos-7 cell or derivative thereof. In some embodiments, the cell is a Vero cell or derivative thereof. In some embodiments, the cell is a BSC 1 cell or derivative thereof. In some embodiments, the cell is a BSC 40 cell or derivative thereof. In some embodiments, the cell is a BMT10 cell or derivative thereof. In some embodiments, the cell is a WI38 cell or derivative thereof. In some embodiments, the cell is a HeLa cell or derivative thereof. In some embodiments, the cell is a Saos cell or derivative thereof. In some embodiments, the cell is a C2C12 cell or derivative thereof. In some embodiments, the cell is an L cell or derivative thereof. In some embodiments, the cell is an HT1080 cell or derivative thereof. In some embodiments, the cell is a HepG2 cell or derivative thereof. In some embodiments, the cell is a Huh7 cell or derivative thereof. In some embodiments, the cell is a K562 cell or derivative thereof. In some embodiments, the cell is a primary cell.

[0199] In some embodiments, the recombinant viral vector is produced by a producer cell line method. Briefly, for example, a cell line (e.g., a HEK-293 cell line) is stably transfected with a plasmid containing a Rep gene, a Cap gene, and a promoter-payload sequence. The cell line is screened to select a lead clone for recombinant vector production, which is, in some embodiments, amplified to a production bioreactor and infected with a helper polynucleotide (e.g., a wild-type adenovirus) as a helper to initiate vector production. In some embodiments, the recombinant viral vector (e.g., rAAV) is subsequently harvested, inactivated (e.g., by heat) and / or adenovirus is removed, and the viral particles are purified. In some embodiments, the recombinant viral vector is purified and formulated.

[0200] In some embodiments, suitable media are used for the production of the recombinant vectors. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle’s Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle’s Minimum Essential Medium (EMEM), Ham’s F-10 Medium, Iscove’s Modified Dulbecco’s Medium (IMDM), Neural basal medium, Dulbecco’s Modified Eagle’s Medium (DMEM), custom formulations, particularly with respect to custom media formulations for use in the production of recombinant vectors.

[0201] In some embodiments, suitable production media of the present disclosure are supplemented with serum or serum-derived recombinant proteins at levels of 0.5-20% (v / v or w / v). In some embodiments, vectors are produced in serum-free conditions, also referred to as media free of animal-derived products. In some embodiments, commercial or custom media are designed to support the production of vectors, including but not limited to, supplementation with glucose, vitamins, amino acids, and / or growth factors to increase the titer and / or yield of vectors in the production culture.

[0202] Vector production cultures comprise a variety of conditions suitable for the particular host cell utilized (over a range of temperatures, for different lengths of time, etc.). Vector production cultures include adherent-dependent cultures, which are cultured in suitable adherent-dependent vessels, such as, for example, plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. In some embodiments, vector production cultures include suspension-adapted host cells such as HeLa, HEK-293, HEK-293T, HEK-293S, CHO, NS0, PER.C6, BHK, S2, and Sf9 cells, which are cultured in a variety of ways, including, for example, roller bottles, stirred tank bioreactors, single-use bioreactors such as Cytiva Xcellerex, Sartorius, and Wave, and disposable systems.

[0203] In some embodiments, viral particles of the present disclosure are harvested from vector production cultures by lysing the host cells of the production culture or by harvesting used culture medium from the production culture, provided that the cells are cultured under conditions that result in the release of viral particles from the intact cells into the culture medium. Suitable methods of lysing the cells include, for example, multiple freeze-thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0204] In additional embodiments, the viral particles are purified. As used herein, the term "purified" includes a preparation of viral particles that is free of at least some other components in which the viral particles naturally occur or in which they are present from the place of their initial preparation. Thus, for example, in some embodiments, isolated viral particles are prepared using purification techniques to enrich the isolated viral particles from a source mixture, such as a culture lysate or production culture supernatant. In some embodiments, the enrichment is measured in a variety of ways, such as, for example, by the proportion of DNAse resistant particles (DRPs) or genome copies (gc) present in solution, or by infectivity, or with respect to a second potentially interfering substance present in the source mixture, such as a contaminant and impurity, including product- and process-derived impurities, etc.

[0205] In some embodiments, the clarified vector production culture harvest is treated with a nuclease (e.g., a DNA nuclease or an RNA nuclease) to digest any high molecular weight DNA present in the production culture. In some embodiments, the nuclease digestion is performed under standard conditions.

[0206] In some embodiments, the viral particles are isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentration of viral particles; vector capture by phospho sphate chromatography; heat inactivation of helper virus; vector capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and vector capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. In some embodiments, these steps are used individually, in various combinations, or in different orders. In some embodiments, the method includes all of the steps in the order described below.

[0207] In some embodiments, the clarified vector production culture harvest is treated with a nuclease (e.g., a DNA nuclease or an RNA nuclease) to digest any high molecular weight DNA present in the production culture. In some embodiments, the nuclease digestion is performed under standard conditions.

[0208] Described herein are methods for generating recombinant vectors, wherein the method comprises providing a cell transfected with a helper polynucleotide. In some embodiments, the cell is transfected with a helper polynucleotide that provides helper functions to the AAV. In some embodiments, the helper polynucleotide provides adenovirus functions, including, for example, E1A, E1B, E2A, E4 ORF6, and / or VA RNA. In some embodiments, the sequences of the adenovirus genes providing these functions are obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 5, 7, 12, and 40, and also include any of the currently identified human types. In some embodiments, the adenovirus functions are provided from a defective adenovirus. In some embodiments, the method involves transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0209] Described herein are methods for generating recombinant vectors, wherein the method comprises providing a cell transfected with a helper polynucleotide under the control of a promoter. In some embodiments, the cell is a stable host cell comprising the desired components under the control of an inducible promoter. In some embodiments, the cell is a stable host cell comprising the desired components under the control of a constitutive promoter. In some embodiments, the cell is a stable host cell comprising selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell is generated that is derived from a HEK-293 cell (which contains El helper functions under the control of a constitutive promoter), but the host cell contains Rep protein and / or Cap protein under the control of an inducible promoter.

[0210] In some embodiments, the small genes, Rep sequences, Cap sequences, and helper functions required to produce rAAV of the disclosure are delivered to the packaging host cell in the form of any genetic element that transfers the sequences. In some embodiments, the selected genetic element is delivered by any suitable method.

[0211] In some embodiments, production of recombinant vectors, such as rAAV vectors, includes transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, bocavirus-AAV hybrids, and baculovirus-AAV hybrids.

[0212] In some embodiments, methods including transfection, stable cell line production, and infectious hybrid virus production systems are used for production of rAAV vectors. In some embodiments, the methods described herein include adenovirus-AAV hybrids, herpesvirus-AAV hybrids, bocavirus-AAV hybrids, and baculovirus-AAV hybrids. rAAV production cultures for production of rAAV viral particles all require: 1) a suitable host cell, including, for example, cell lines of human origin such as HeLa, A549, or HEK-293 cells or HEK-293T cells or HEK-293S cells, or cell lines of insect origin such as Sf9 (in the case of baculovirus production systems), or transgenic plant cells; 2) suitable helper virus functions, provided by wild-type or mutant adenovirus (such as temperature-sensitive adenovirus), herpesvirus, baculovirus, or plasmid constructs providing helper functions; 3) AAV Rep and Cap genes and gene products; 4) a desired genomic payload flanked by at least one AAV ITR sequence (such as a heterologous sequence encoding any desired sequence or fragment or variant thereof); and 5) suitable media and media components to support rAAV production. Suitable media for production of rAAV vectors include, but are not limited to, media produced by Hyclone Laboratories and JRH, including modified Eagle’s medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle’s Minimum Essential Medium (EMEM), Ham’s F-10 medium, Iscove’s Modified Dulbecco’s Medium (IMDM), Neuralbasal medium, Dulbecco’s Modified Eagle’s Medium (DMEM).

[0213] In some embodiments, recombinant AAV particles are produced by transfection of production cells with a plasmid containing the rAAV genome (a cis plasmid) comprising a payload flanked by one or more AAV ITRs and a separate construct expressing AAV Rep and Cap genes in trans. In some embodiments, adenovirus helper factors such as E1A, E1B, E2A, E4 ORF6, and VA RNA are provided by adenovirus infection or by transfection of a third plasmid providing adenovirus helper polynucleotides into the production cells. In some embodiments, the production cells are HEK-293 cells. In some embodiments, the helper polynucleotides provided will vary depending on the production cells used and whether the production cells already carry some of these helper polynucleotides.

[0214] In some embodiments, the rAAV vectors described herein are produced by a triple transfection method, such as the exemplary triple transfection method provided below. In some embodiments, a plasmid containing the Rep gene and Cap gene, and a helper adenovirus plasmid are transfected together (e.g., using extrusion poration, lipofection, optical transfection, calcium phosphate method, or polyethyleneimine (PEI)) into a cell line (e.g., HEK-293 cells), and the virus is collected and optionally purified.

[0215] In some embodiments, the rAAV vectors are produced by a producer cell line method, such as the exemplary producer cell line method provided below. Briefly, a cell line (e.g., HEK-293 cell line) is stably transfected with a plasmid containing the Rep gene, Cap gene, and promoter-payload sequence. The cell line is screened to select a lead clone for rAAV production, which is then expanded to a production bioreactor and infected with an adenovirus (e.g., wild-type adenovirus) as a helper to initiate rAAV production. The virus is subsequently harvested, can be inactivated (e.g., by heat) and / or adenovirus removed, and the rAAV vector is purified.

[0216] In some embodiments, provided are methods for producing any of the rAAV vectors as disclosed herein, comprising (a) culturing a host cell under conditions in which a rAAV vector is produced, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication and / or encapsidation protein; (ii) a rAAV provector comprising a nucleic acid encoding a therapeutic polypeptide and / or nucleic acid as described herein flanked by at least one AAV ITR, and (iii) AAV helper functions; and (b) recovering the rAAV vector produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, goat AAV, bovine AAV, or mouse AAV, among others. In some embodiments, the replication protein is an AAV2 replication protein.

[0217] Methods of treating cancer Described herein are methods of treating cancer in a subject in need thereof, comprising: administering to the subject an rAAV described herein. In some embodiments, the rAAV comprises a minigene comprising a splice modulator binding site. In some embodiments, the methods of treating cancer in a subject in need thereof further comprise administering a small molecule splice modulator.

[0218] Administration is not limited to any particular route, but can refer to any route accepted by the medical community as appropriate. In some embodiments, administration is intravenous (IV), intramuscular (IM), intraarterial, inhalation, intramedullary, intrathecal (IT), intracistema magna (ICM), intracerebroventricular (ICV), intraparenchymal, intranasal, subcutaneous (SQ), transdermal, intraventricular, intraperitoneal (IP), intragastric (IG), mucosal, buccal, enteral, intravitreal, and / or via a portal vein catheter; and / or a combination of any of the foregoing. In some embodiments, the rAAV vector compositions described herein are administered locally to a lesioned tissue (e.g., a tumor).

[0219] In some embodiments, the rAAV vector compositions provided by the present disclosure are administered in combination with a splicing modifier or modulator. In some embodiments, the rAAV vector compositions are provided sequentially with the splicing modifier. In some embodiments, the rAAV vector compositions are administered locally directly to a lesioned tissue and the splicing modifier is administered via a different route. In some embodiments, the rAAV vector compositions are administered locally directly to a lesioned tissue and the splicing modifier is administered orally. In some embodiments, the rAAV vector compositions are administered locally directly to a lesioned tissue and the splicing modifier is administered systemically. In some embodiments, both the rAAV vector compositions and the splicing modifier are administered locally to a lesioned tissue.

[0220] In some embodiments, the subject is a human. In some embodiments, the subject has a cancer. In some embodiments, the subject has a glioma, such as a grade III or grade IV glioma (glioblastoma). In some embodiments, the subject has a glioblastoma. In some embodiments, the subject has a primary tumor. In some embodiments, the subject has a metastasis. In some embodiments, the subject has a metastatic brain tumor. In some embodiments, the subject has a uveal melanoma.

[0221] In some embodiments, administration is local administration directly to a lesioned tissue (e.g., a tumor). In some embodiments, administration is administration to the central nervous system. In some embodiments, administration is administration to the brain. In some embodiments, administration is administration to the cerebral ventricles. In some embodiments, administration is intratumoral administration.

[0222] Any suitable route of administration or combination of routes can be used, including systemic administration (e.g., intravenous, intravascular, intra-arterial), local injection into the central nervous system (CNS; e.g., intra-tumoral injection, intracranial injection, intracerebral injection, intraventricular injection, intra-parenchymal injection, or injection into the cerebrospinal fluid (CSF) via the ventricular system, the cistema magna, or the intrathecal space), or local injection at other body sites (e.g., intraocular, intranasal, intramuscular, subcutaneous, intradermal, transdermal). In some embodiments, administration is by intra-tumoral injection, intracranial injection, intracerebral injection, intraventricular injection, or injection into the cerebrospinal fluid (CSF) via the ventricular system, the cistema magna, or the intrathecal space.

[0223] In some embodiments, intraventricular injection occurs in the right lateral ventricle, the left lateral ventricle, the third ventricle, the fourth ventricle, the interventricular foramen (also known as the Monro foramen), the cerebral aqueduct, the central canal, the median aperture, the right lateral aperture, the left lateral aperture, the perivascular space, or the subarachnoid space.

[0224] Administration can be by use of an osmotic pump, by electroporation, or by other means. In some embodiments, administration of the rAAV vectors described herein is performed prior to, after, or concurrently with surgical tumor removal or biopsy.

[0225] In some embodiments, administration is by convection-enhanced delivery (CED). CED uses direct infusion of a drug-containing liquid into tissue such that transport is dominated by convection. In some embodiments, the device is an osmotic pump. In some embodiments, the device is an infusion pump. In some embodiments, CED is performed with a ladder design catheter.

[0226] In some embodiments, magnetic resonance imaging (MRI)-guided CED is performed to deliver the rAAV vectors of the disclosure. In some embodiments, CED further comprises use of a tracer. In some embodiments, the tracer is an MRI contrast enhancer. In some embodiments, the MRI contrast enhancer is gadolinium and related chemical derivatives. In some embodiments, the MRI contrast enhancer is administered concurrently with the rAAV vector. In some embodiments, the MRI contrast enhancer is mixed directly with the rAAV vector prior to administration.

[0227] Pharmaceutical compositions In certain embodiments, described herein are pharmaceutical compositions comprising (a) a polynucleotide of the disclosure, a plasmid of the disclosure, or a rAAV comprising a polynucleotide of the disclosure, and (b) a pharmaceutically acceptable excipient. The pharmaceutical compositions disclosed herein are in a form suitable for administration to an individual in need thereof.

[0228] In some embodiments, the pharmaceutically acceptable excipient of the present disclosure is suitably selected from the group consisting of: injectable excipient liquids, such as sterile water for injection; and aqueous solutions such as saline.

[0229] An acceptable excipient is physiologically acceptable to the subject to which it is administered and retains the therapeutic properties of the compound with which it is administered / in which it is administered. Acceptable excipients and their formulations are generally described, for example, in Remington’s Pharmaceutical Sciences, supra. One exemplary excipient is physiological saline. The phrase “pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ or portion of the body or in the in vitro assay systems. Each excipient must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject to which it is administered. An acceptable excipient also does not modify the particular activity of the subject compound.

[0230] In another embodiment, the pharmaceutical compositions disclosed herein further comprise an acceptable additive to improve the stability of the compound in the composition and / or to control the release rate of the composition. The acceptable additive does not modify the particular activity of the subject compound. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, dextrose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, the acceptable additive is combined with an acceptable carrier and / or excipient such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and reduce the gelling of the solution. In some embodiments, the surfactant is added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0231] Also contemplated herein are suspensions, lyophilized and crystalline forms of the rAAV vectors, polynucleotides or compositions herein; methods of making suspensions, lyophilized and crystalline forms are known to those of skill in the art.

[0232] In some embodiments, the pharmaceutical compositions disclosed herein are sterile. In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by conventional, well- known sterilization techniques. For example, sterilization is readily accomplished by filtration through sterile filtration membranes. In some embodiments, the resulting solution is packaged for use or filtered and lyophilized under sterile conditions, the lyophilized article being combined with a sterile solution prior to administration.

[0233] In some embodiments, such as when the polypeptide is relatively unstable in a liquid composition, lyophilization is employed to stabilize the polypeptide for long-term storage.

[0234] In some embodiments, some excipients, such as, for example, polyols (including mannitol, glycerol, sorbitol, and derivatives thereof such as polysorbate 20); salts (including NaCl, MgCl2, and KC1), sugars (including glucose, sucrose, and trehalose); surfactants (including poloxamer 188), and amino acids (including alanine, glycine, and glutamic acid), are used as stabilizers for the lyophilized product. In some embodiments, polyols, surfactants, and sugars are also used to protect the polypeptide from freeze and dry-induced damage and to enhance stability during storage in the dry state. In some embodiments, sugars are effective both during the lyophilization process and during storage. Other classes of molecules, including monosaccharides and disaccharides, and polymers such as PVP, have also been reported as stabilizers for lyophilized products.

[0235] For injection, in some embodiments, the pharmaceutical compositions disclosed herein are in a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, lyophilized powders, spin-dried powders or spray-dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the compositions optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations of these.

[0236] In some embodiments, sustained release articles are prepared. Suitable examples of sustained release articles include a semipermeable matrix of a solid hydrophobic polymer containing a pharmaceutical composition herein, wherein the matrix is in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (see, for example, U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot™ (including injectable microspheres of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable the release of molecules for over 100 days, whereas certain hydrogels enable protein release for shorter periods of time.

[0237] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, fast-release, long-acting, or sustained-release as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or for slow release.

[0238] In some embodiments, the pharmaceutical composition is included in a container, a pack, or a dispenser together with instructions for administration. Examples

[0239] The following are examples of specific embodiments of carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) used in the examples, but some experimental errors and deviations should be accounted for.

[0240] Example 1. HEK-293T expression of inducible AAV-GFP This example describes exemplary regulatory minigenes for use in rAAV vectors of the present disclosure. Exemplary minigenes were tested in HEK-293T cells and used to control expression of a GFP reporter polypeptide.

[0241] Adherent human HEK-293T cells were transduced with inducible AAV-GFP and then fluorescently imaged for GFP expression before and after administration of an inducer molecule (e.g., LMI070). Data are found in Figure 3 .

[0242] Adherent human HEK-293T cells were transduced with inducible AAV-GFP at different doses, followed by administration of an inducer molecule (e.g., LMI070) and monitoring of GFP expression over several hours to determine initial expression kinetics of different AAV doses in vitro. Data are found in Figure 4 .

[0243] Example 2: Engineered rAAV vectors for interferon delivery This example describes exemplary engineered nucleic acids provided by the present disclosure that can be used to generate rAAV vectors capable of delivering interferon to a subject in need thereof.

[0244] As described herein, recombinant adeno-associated virus (AAV) vectors of the present disclosure can include nucleic acid elements, including but not limited to promoters, minigenes, polynucleotides comprising coding sequences for a payload of interest, one or more regulatory elements (e.g., WPRE, polyadenylation, antibiotic resistance genes, etc.), and / or ITRs. Exemplary nucleic acid element combinations are provided in Table 1, and each combination of nucleic acid elements was incorporated into a corresponding plasmid vector (Plasmids 1-30, also see Figures 5A-5DThe assay is used for (e.g., for interferon delivery). The CAG promoter is used for the nucleic acid to be tested and includes the CMV intermediate early enhancer (CMVie) sequence, chicken β-actin sequence, and rabbit β-globin splice acceptor. The CMVie sequence used is truncated and includes long (SEQ ID NO: 25), medium (SEQ ID NO: 26), or short (SEQ ID NO: 27) versions. Incorporation of small genes or the “Xon” system is also indicated (SEQ ID NO: 31 and SEQ ID NO: 51). A polynucleotide encoding human IFNβ is used, which is either CpG depleted (SEQ ID NO: 4) or a representative wild-type (SEQ ID NO: 2). A modified WPRE (SEQ ID NO: 33) or a representative wild-type WPRE (SEQ ID NO: 32) is used. Selected polyadenylates (such as SV40 shown in SEQ ID NO:19 and bGH shown in SEQ ID NO:34), antibiotic resistance genes (such as AmpR shown in SEQ ID NO:44 and KanR shown in SEQ ID NO:20 or SEQ ID NO:21), and ITR sequence lengths (such as truncated ITRs shown in SEQ ID NO:40-41 and full-length ITRs shown in SEQ ID NO:36-39) are also shown.

[0245] Table 1. Exemplary nucleic acid elements and generated plasmids

[0246] Materials and methods An exemplary workflow for generating engineered plasmids (by cloning) for producing the rAAV vector provided in this disclosure is shown in Figure 6 The document illustrates a multi-step cloning strategy for generating plasmids. Exemplary schemes for generating certain engineered rAAV vectors are provided below.

[0247] In order to generate plasmid #1 ( Figure 5A Using standard cloning methods, Kpnl and EcoRI Restriction endonucleases were used to subclone a newly synthesized 582 bp hIFNβ-CpG-deleted polynucleotide into the polynucleotide AAV-CAG-mCardinal to replace the mCardinal protein fluorescent marker ORF. This was done to generate plasmid #12 ( Figure 5L), two polynucleotide fragments were newly synthesized to cover both the bGH polyadenylation sequence (SEQ ID NO: 34) and the KanR-CpG-depleted up to the CMV i.e. enhancer sequence (SEQ ID NO: 35) and inserted into plasmid #1 using homologous recombination technique. Plasmid #14 ( Figure 5N ) was generated by inserting a 657 bp newly synthesized polynucleotide (SEQ ID NO: 54) into plasmid #12. BamHI / EcoRI Plasmid #18 ( Figure 5R ) and plasmid #16 ( Figure 5P ) were generated by replacing the hIFNp-CpG ORF from plasmid #12 or plasmid #14, respectively, with a newly synthesized fragment of eGFP. Plasmid #20 ( Figure 5T ) and plasmid #21 ( Figure 5U ) were obtained by replacing the hIFNp-CpG deleted ORF in plasmid #12 and plasmid #14, respectively, with a newly synthesized polynucleotide encoding mCardinal. A newly synthesized polynucleotide fragment (without ATG and without optional start codon) encompassing the spliced minigene (Xon) and the modified hIFNp-CpG deleted fragment was inserted into plasmid #12 and plasmid #14 to generate plasmid #13 ( Figure 5M ) and plasmid #15 ( Figure 5O ), respectively. A modified newly synthesized eGFP fragment (without ATG, without optional start codon) was inserted in plasmid #13 and plasmid #15 in place of the hIFNp-CpG deletion to generate plasmid #17 ( Figure 5Q ) and plasmid #19 ( Figure 5S ), respectively. A modified newly synthesized mCardinal fragment (without ATG, without optional start codon) was inserted in plasmid #13 and plasmid #15 in place of the hIFNp-CpG deletion to generate plasmid #22 ( Figure 5V ) and plasmid #23 ( Figure 5W ), respectively.

[0248] Example 2. Preparation of improved AAVs This example describes the preparation of improved AAVs with various combinations of regulatory elements for delivering a gene of interest to target cells.

[0249] Briefly, AAV stocks were generated using a standard triple transfection protocol in combination with pRepCap and adenoviral helper plasmids. The platform process uses a adherent HEK293T triple transfection protocol followed by a one-step purification by density gradient ultracentrifugation to remove empty AAV capsids. Rotary columns are then used to concentrate and formulate the samples in a one-step process. These research grade lots are then formulated into an AAV compatible excipient (1X dPBS / 0.005% Poloxamer). Each lot is tested for vector genome (vg) titer by RT qPCR with ITR primers within the ITRs of the AAV and the results are provided on the analysis certificate. AAV Preparation #5, AAV Preparation #6, AAV Preparation #8, and AAV Preparation #15 were produced using an established GMP-like large scale manufacturing suspension platform (using suspension adapted HEK293 cells). The AAV produced are listed in Table 2.

[0250] Table 2. List of recombinant AAV research stocks produced at research scale

[0251] *AAV preparations are arbitrarily numbered, but this nomenclature is consistently used across the experiments described in the examples herein.

[0252] **Plasmid numbers are consistent with Table 1, and this nomenclature is consistently used across the experiments described in the examples herein.

[0253] These results demonstrate that all AAVs were produced at high viral genome titers (>1e13 vg / mL), the suitability of the newly designed genomes for AAV packaging and transduction experiments, and robust and clinically relevant levels of virus production.

[0254] Example 3. Validation of various variants of therapeutic AAV candidates This example describes the preparation of improved AAVs for delivery of genes of interest to target cells with various combinations of regulatory elements, as well as expression and activity of the payloads.

[0255] The impact of variations in the payload and different regulatory elements on the productivity and function of various candidates was evaluated. In four constructs, the variables tested included: a CpG-depleted payload, two length variations of the CAG promoter, and variable polyadenylation and antibiotic resistance cassettes. AAV preparations were tested side-by-side in a series of assays to: 1) quantify vector genome titer using payload primers by digital PCR (dPCR); 2) quantify capsid titer using a commercial ELISA kit; and 3) measure viral potency using in vitro cell-based assays to quantify expression of the payload post-transduction in mammalian cells. Percent full capsids and volumetric yield were calculated using vg to capsid titer, and data is shown in Table 3 as relative ratios across production batches, with AAV Preparation #6 used as an internal reference for all values tested simultaneously. For viral potency, values obtained at an MOI of 5e5 vg / cell were used to calculate ratios across production batches for each construct.

[0256] Table 3. Production of various AAV candidates at research scale

[0257] In summary, these results demonstrate that the various elements utilized in combination within the AAV genomic plasmid do not significantly impact the production or activity of AAV.

[0258] Example 4. Large scale AAV batches for evaluation of clinical manufacturing suitability This example describes the preparation of improved AAV with various combinations of regulatory elements in large scale batches for delivery of genes of interest to target cells.

[0259] Candidates with therapeutic payloads were produced using a large scale suspension platform. AAV batches were produced in 2L to 4L scale harvests, fully purified, and evaluated against various quality standards. AAV batches were purified with a multi-step process to ensure high levels of purity in the final AAV material. Steps included tangential flow filtration (TFF), affinity capture chromatography, gradient ultracentrifugation or anion exchange chromatography, and final formulation in a centrifuge device or another TFF. AAV stock genome titers were obtained using a PCR-based protocol. These AAV preparations were used to evaluate various key quality attributes in side-by-side experiments.

[0260] Runs were performed using plasmid 1 (Table 4) or plasmid 24 (Table 5) as exemplary representatives of AAVs with therapeutic payloads described herein. In a series of assays, AAV preparations were tested side-by-side to: 1) quantify vector genome titer by digital PCR (dPCR) using primers located within the therapeutic payload; 2) quantify capsid titer using a commercial ELISA kit; and 3) measure viral potency using an in vitro cell-based assay to quantify expression of the payload post-transduction in mammalian cells. Percent AAV with intact capsids as well as volumetric yield were calculated using vg and capsid titers, and the data collected are shown in Tables 4 and 5, expressed as relative ratios across various production lots using AAV Preparation #5 (Table 4) or AAV Preparation #6 (tested in parallel, except for capsid; Table 5) as a control reference. In the experiments presented in Table 4, for viral potency, values obtained at an MOI of 5e5 vg / cell were used to calculate the ratios across production lots. In the experiments presented in Table 5, for viral potency, values obtained at an MOI of 3.9e5 vg / cell were used to calculate the ratios across production lots.

[0261] Table 4. Runs using plasmid #1

[0262] Table 5. Runs using plasmid #24

[0263] Purity was confirmed in several AAV preparations, as well as the presence of AAV capsid proteins in the expected ratios. Briefly, total vg (1e11) of each preparation was evaluated by gel electrophoresis in denaturing conditions followed by Coomassie blue staining. For each preparation, the three capsid proteins (VP1, VP2, and VP3) were identified in approximately 1:1:10 expected ratios, respectively, and no other proteins were detected, confirming high levels of purity and absence of detectable impurities or contaminants. Figure 7

[0264] These results demonstrate orthogonal validation of production performance for therapeutic AAV candidates (each produced at a different manufacturing supplier), highlighting the availability across facilities, and robust production processes across users and different processes. All subtle variability observed between different production service providers were within the expected range for small batch production.

[0265] ​In summary, these successful cloning experiments and virus production demonstrate that for each AAV construct, high viral genome titers (>1e13 vector genomes (vg) / mL) were obtained at high purity and homogeneity levels, as well as demonstrating the suitability of the newly designed genomes for AAV packaging and transduction experiments and robust and clinically relevant levels of virus production.

[0266] Example 5: Side-by-side production of improved AAV variants for validation of AAV vectors with therapeutic payloads This example describes a comparison of functionality and packaging of different AAV vectors with therapeutic payloads.

[0267] Briefly, suspension grown HEK293 cells (VPC2.0 cells, ThermoFisher Scientific) were transfected with individual pAAV plasmids alone as well as pRepCap and pAd helper plasmids to support packaging of AAV genomes. After production, cells were lysed and subjected to nuclease digestion (Benzonase) and the crude lysate was clarified by centrifugation to remove cell membranes and debris. Virus preparations were stored frozen until testing of vg titers using a digital dPCR protocol with primer and probe sets specific for the respective payload. Figures 8A-8C ) was performed.

[0268] In summary, these experiments demonstrate successful virus production for each AAV construct with titers obtained in the expected range (>1e10 vg / mL), as well as demonstrating the suitability of the newly designed genomes for AAV packaging and transduction experiments and clinically relevant levels of virus production.

[0269] Example 6: Demonstration of efficacy of AAV with therapeutic payloads This example describes the efficacy of payloads delivered with AAVs described herein.

[0270] Briefly, HEK293T cells were plated in 96-well plates and transduced with several variants of AAV-CAG-hIFNp at a multiplicity of infection ranging from about 3e4 to 1e6 vg / cell. 48 hours after transduction with AAV, culture supernatant was harvested for payload measurement. The amount of hIFNp was measured using commercial ELISA kits for human (h)IFNp Figures 9A-9D ) or mouse (m)IFNp Figure 9E ) at nanograms per milliliter (ng / mL; Figures 9A-9D ) or picograms per milliliter (pg / mL; Figure 9E ).

[0271] In particular, in a first set of experiments, all formulations were generated from plasmid #1, which carries the hIFNp payload, and packaged within the same AAV capsid serotype, but produced by four different suppliers (Table 1). Figure 9A Upon analysis of the results, it was observed that there was lower potency when produced at a particular supplier (AAV preparation #6). However, as shown previously, no other differences were detected. All other preparations generated from plasmid 1 (#5, #8, and #9) exhibited similar potency, as measured by payload expression.

[0272] In a set of experiments, plasmid #7 (corresponding to AAV preparation #1 in Figure 9A or AAV preparation #3 in Figure 9B ), plasmid #1 (corresponding to AAV preparation #9 in Figure 9D or AAV preparation #6 in Figure 9B ), plasmid #12 (corresponding to AAV preparation #10 in Figure 9D ), and plasmid #14 (corresponding to AAV preparation #11 in Figure 9B ) were all produced at one supplier and packaged in the same AAV capsid serotype, exhibiting no significant differences in potency across more than one variant of our AAV constructs and payloads. Figure 9B

[0273] As expected, AAV carrying payloads other than IFN did not produce measurable IFN expression. It was also demonstrated that AAV preparation #15 exhibited one of the highest levels of potency across constructs and suppliers (AAV preparation #15 was prepared with a process similar to those used for clinical batch production).

[0274] In a set of experiments, human (h) and mouse (m) cytokine payloads were compared, and it was demonstrated that mouse cytokines were able to be expressed in human cells Figure 9E ), thereby demonstrating that such payloads are useful as controls in various in vitro and in vivo models.

[0275] Taken together, these results demonstrate that the AAVs described herein can successfully deliver functional therapeutic payloads, such as hIFNp or mIFNp, into mammalian cells.

[0276] Example 7: Demonstration of payload activity following delivery in mammalian cells This example describes the activity of payloads delivered with the AAVs described herein.

[0277] ​An activity assay using a reporter cell line was used to measure therapeutic transgene activity. This method utilizes a reporter cell line of human pro-monocytic cells (U937) engineered to express firefly luciferase under the control of an IFN a / b-responsive promoter. In this model, when IFNa or IFNp binds to the IFN a / b receptor (IFNAR1) on the cell surface, the IFNa / b-regulated firefly luciferase reporter gene construct will be activated, producing a precise quantitation of luminescent signal.

[0278] Briefly, AAV preparations were incubated with reporter cells at an MOI of 5e5 vg / cell. AAV preparation #12 (expressing reporter payload mCardinal) and cell culture media (NC) were used as negative controls. Measurements were obtained after an 18-hour incubation period at 37°C and 5% CO2. A standard curve was generated using commercial recombinant hIFNp1 cytokine (Prospect CYT-234) and expressed in infectious units / mL. Luciferase detection and measurements were obtained using the reporter detection assay Figure 10 ). These results demonstrate that the various hIFNp payloads were successfully expressed in target cells and were biologically active. The data were also correlated to potency measurements via payload expression shown in Figures 9A-9E , where preparation #10 and preparation #13 showed the highest activity and expression Figure 9C ), and preparation #6 had the lowest activity and expression Figure 9A and Figure 9D ).

[0279] In summary, these data demonstrate the functionality of the vectors described herein, which successfully delivered biologically active hIFNp after transduction in human cells in an in vitro model.

[0280] Example 8: Demonstration of therapeutic activity of AAVs with therapeutic payloads in cancer cells This example describes the therapeutic activity of payloads delivered with the AAVs described herein to cancer cells (e.g., brain cancer cells, where the cancer metastasized from a primary cancer originating from a different location in the body).

[0281] Patient-derived xenograft organoid (PDXO) models were established from brain metastasis of secondary breast cancer and the resulting organoids were used to monitor the anti-tumor efficacy of AAVs with therapeutic payloads. Organoids were established by a qualified manufacturing supplier using a proprietary method. Prior to seeding into hydrogels, organoids were exposed to three doses of AAV preparation #6 (expressing hIFNp) as well as AAV negative control expressing GFP (AAV-GFP) in small volume media for 2-4 hours. Untreated organoids were exposed to media only. In addition to AAV dosing, replicate wells were included that received 1 mM staurosporine as a positive killing control, or AAV preparation buffer as a negative killing control. At day 6 post-treatment, replicate wells (n = 4) of treatment conditions were subjected to cell viability assessment using the CellTiter-Glo® reporter assay (Promega G7570), which measures total viable cells by the production of luminescent signal expressed as light units (lux) that is proportional to the amount of ATP present in the culture, and thus proportional to the amount of cells. In addition, organoids from technical replicate wells of each treatment condition were collected, fixed, washed in PBS, and stained with Hoechst and rhodamine phalloidin, respectively, to stain nuclei and eukaryotic cytoskeleton. Stained organoids were imaged at x4 magnification and subsequently processed in the 3D analysis platform Ominer® for high content imaging analysis to quantify compound-induced effects on tumor growth.

[0282] As Figure 11 As shown in FIG. 6, a dose-dependent decrease in organoid viability was observed at day 6 post-infection following infection with AAV-hIFNp but not AAV-GFP. A time-dependent decrease in cell viability was observed in all viral concentrations tested in the groups treated with AAV-hIFNp. It can be noted that no significant changes in high content imaging features were observed at day 6 post-treatment when treated with AAV-GFP, indicating that treatment with AAV-GFP was not therapeutic in this model at the tested doses. A dose-dependent increase and a time-dependent increase in the fraction of dead cells was observed in organoids treated with AAV-hIFNp, and a strong decrease in total organoid size and number of nuclei per organoid was also observed at all dose levels of AAV-hIFNp. At the highest viral dose tested, treatment with AAV-IFNp showed a small decrease in organoid and nuclei counts and total nuclei size compared to untreated and buffer-treated organoids.

[0283] These results demonstrate that the hIFNp payload is successfully expressed in target cancer cells (e.g., target brain cancer cells, where the cancer metastasizes from a primary cancer in another part of the subject’s body) and is biologically active and therapeutically potent for the treatment of cancer. Collectively, these results indicate that AAV-IFNp elicits an anti-tumor effect that is primarily cytotoxic, particularly at the highest tested dose level. The data demonstrate the functionality of AAV-hIFNp in eliciting an anti-neoplastic effect on human tumor cells in an ex vivo model of brain metastasis.

[0284] Example 9. Payload expression from prolonged AAV delivery in GBM cancer cells This example describes the therapeutic activity of payloads delivered to brain cancer cells with AAVs described herein.

[0285] In this example, immortalized glioblastoma (GBM) cells were exposed to AAVs described herein expressing the hIFNp payload or recombinant hIFNp cytokine (unvectorized). Following exposure, hIFNp levels were monitored in cell culture media for a period of 4 days.

[0286] Briefly, cells were plated in 48-well plates and exposed to defined MOIs of AAV Preparation #13 (hIFNp, 4e5 vg / cell) or AAV Preparation #12 (mCardinal, 4e5 vg / cell). Daily complete media changes were performed for a total of 4 days. Negative control wells were exposed to AAV expressing the control fluorescent protein mCardinal (Preparation #12), as well as media without active agent. hIFNp levels were measured from collected supernatants using a hIFNp high sensitivity ELISA kit (PBL Catalog #41415).

[0287] Secreted hIFNp levels were quantifiable as early as 10 hours post-transduction, with peak expression observed after 72 hours and maintained through 96 hours. AAV transduction alone did not stimulate the secretion of therapeutic levels of hIFNb from GBM cells in vitro, as evidenced by low levels of hIFNp from AAV-mCardinal transduced cells. In fact, supernatant from AAV-mCardinal treated cells was below the assay limit of quantification in 4 of 4 samples at 24 hours, 4 of 4 samples at 48 hours, 2 of 4 samples at 72 hours, and 3 of 4 samples at 96 hours post-treatment. Meanwhile, none of the 3 replicates described in this experiment with detectable levels (plotted in Figure 12 did not exceed 5 pg / mL.

[0288] Collectively, the results demonstrate that AAVs described herein can successfully transduce human GBM cells to deliver a sustained hIFNp payload.

[0289] Example 10. Production and testing of clinical grade therapeutic AAV to enable phase I / II clinical trial in brain cancer patients This example describes the production of clinical grade AAVs with various combinations of regulatory elements for delivery of genes of interest.

[0290] AAVs containing therapeutic payloads are produced in plasmid batches in compliance with current current good manufacturing practice (cGMP) regulations. The plasmid batches are used to produce the viral drug product. An exemplary cGMP-compliant manufacturing process is shown in Figure 13 .

[0291] To generate cGMP-compliant plasmid batches, master cell banks (MCBs) are generated for the plasmids described herein, such as, for example, plasmid #24 with long CMVie, short CBA, CpG-deleted hINF beta payload, mutant WPRE, bGH poly-adenylation, and CpG-deleted kanamycin resistance gene. In addition, E. coli strains are selected to produce plasmids of high quality, high uniformity, high stability, and mostly supercoiled conformation to enable optimal AAV production by transfection. The generated plasmids are used to produce various AAV batches of good laboratory practice (GLP) and good manufacturing practice (GMP) grade to support preclinical development, including but not limited to IND-enabling toxicology studies and clinical trials.

[0292] A series of highly specific assays are developed to test AAV GLP-compliant and GMP-compliant batches. These assays are used to assess safety, strength, potency, and purity standards of the AAV drug product, as well as any other suitable critical quality attributes.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) CAG promoter; and b) A polynucleotide encoding the payload of interest, wherein the polynucleotide comprises reduced CpG dinucleotides and / or increased CpG dinucleotide methylation compared to the parental equivalent.

2. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) CAG promoter; b) Small genes containing splicing regulator binding sites; c) Encoding a polynucleotide of interest, wherein the polynucleotide comprises reduced CpG dinucleotides and / or increased CpG dinucleotide methylation compared to the parental equivalent.

3. The rAAV vector according to claim 1 or 2, wherein the payload of interest is a therapeutic peptide.

4. The rAAV vector according to claim 3, wherein the therapeutic polypeptide is a cytokine or interleukin.

5. The rAAV vector according to claim 3 or 4, wherein the therapeutic polypeptide is a cytokine.

6. The rAAV vector according to claim 5, wherein the cytokine is colony-stimulating factor (CSF), transforming growth factor, tumor necrosis factor, interleukin, or interferon.

7. The rAAV vector according to claim 5, wherein the cytokine is interferon.

8. The rAAV vector according to any one of claims 1-6, wherein the methylation of the CpG dinucleotide is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to the parental equivalent.

9. The rAAV vector according to claim 8, wherein the CpG dinucleotide is fully methylated.

10. The rAAV vector according to claim 8 or 9, wherein the CpG dinucleotide is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to the parental equivalent.

11. The rAAV vector of claim 10, wherein the CpG dinucleotide is depleted.

12. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) CAG promoter; b) Polynucleotides encoding interferon; and c) A polynucleotide comprising WPRE, wherein the WPRE comprises SEQ ID NO:

33.

13. The rAAV vector according to any one of claims 1-12, wherein the CAG promoter comprises a cytomegalovirus (CMV) immediate early enhancer element, a promoter element, and a splice acceptor element.

14. The rAAV carrier of claim 13, wherein the early CMV enhancer element is derived from a wild-type CMV enhancer.

15. The rAAV carrier according to claim 13 or 14, wherein the CMV immediate early enhancer element is truncated relative to the wild-type CMV enhancer.

16. The rAAV vector according to any one of claims 13-15, wherein the CMV immediate early enhancer element comprises a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 25-27.

17. The rAAV vector according to any one of claims 13-16, wherein the CMV immediate early enhancer element comprises the polynucleotide sequences listed in SEQ ID NO: 25-27.

18. The rAAV vector according to any one of claims 13-17, wherein the promoter element is derived from the chicken β-actin gene.

19. The rAAV vector according to any one of claims 13-18, wherein the promoter element comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO: 28 or SEQ ID NO:

52.

20. The rAAV vector according to any one of claims 13-18, wherein the promoter element comprises the polynucleotide sequence listed in SEQ ID NO: 28 or SEQ ID NO:

52.

21. The rAAV vector according to any one of claims 13-20, wherein the splice acceptor is derived from the rabbit β-globin gene.

22. The rAAV vector according to any one of claims 13-21, wherein the splice acceptor comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO: 30 or SEQ ID NO:

53.

23. The rAAV vector according to any one of claims 13-21, wherein the splice acceptor comprises the polynucleotide sequence listed in SEQ ID NO:30 or SEQ ID NO:

53.

24. The rAAV vector according to any one of claims 6-23, wherein the interleukin is IL-1 α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19. IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 or IL-36.

25. The rAAV carrier according to any one of claims 6-23, wherein the CSF is CSF1 (M-CSF), CSF2 (GM-CSF), or CSF3 (G-CSF).

26. The rAAV vector according to any one of claims 6-23, wherein the TGF is TGF-β1, TGF-β2 or TGF-β3.

27. The rAAV vector according to any one of claims 6-23, wherein the TNF is TNF-α, TNF-β or LT-β.

28. The rAAV carrier according to any one of claims 6-23, wherein the interferon is IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ or a variant or derivative thereof.

29. The rAAV vector according to any one of claims 6-23, wherein the interferon is human IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ or variants or derivatives thereof.

30. The rAAV carrier of claim 29, wherein the interferon is human IFNβ.

31. The rAAV vector according to claim 29 or 30, wherein the human IFNβ comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

1.

32. The rAAV vector according to claim 29 or 30, wherein the human IFNβ comprises the amino acid sequence listed in SEQ ID NO:

1.

33. The rAAV vector according to claim 29 or 30, wherein the human IFNβ is encoded by a polynucleotide having at least 80% sequence identity with SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

34. The rAAV vector according to claim 29 or 30, wherein the human IFNβ is encoded by the polynucleotide listed in SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:

4.

35. The rAAV carrier according to claim 29, wherein the interferon is human IFNα.

36. The rAAV vector according to claim 29 or 35, wherein the human IFNα comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

5.

37. The rAAV vector according to claim 29 or 35, wherein the human IFNα comprises the amino acid sequence listed in SEQ ID NO:

5.

38. The rAAV vector according to claim 29 or 35, wherein the human IFNα is encoded by a polynucleotide having at least 80% sequence identity with SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

39. The rAAV vector according to claim 29 or 35, wherein the human IFNα is encoded by a polynucleotide listed in SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

40. The rAAV carrier of claim 29, wherein the interferon is human IFNγ.

41. The rAAV vector according to claim 29 or 40, wherein the human IFNγ comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

9.

42. The rAAV vector according to claim 29 or 40, wherein the human IFNγ comprises the amino acid sequence listed in SEQ ID NO:

9.

43. The rAAV vector according to claim 29 or 40, wherein the human IFNγ is encoded by a polynucleotide having at least 80% sequence identity with SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

44. The rAAV vector according to claim 29 or 40, wherein the human IFNγ is encoded by the polynucleotide sequence listed in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

45. The rAAV vector according to any one of claims 6-23, wherein the interferon is mouse IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ or a variant or derivative thereof.

46. ​​The rAAV vector according to any one of claims 6-45, wherein the interferon-encoding polynucleotide comprises reduced CpG dinucleotides and / or increased CpG dinucleotide methylation compared to the parental equivalent.

47. The rAAV vector of claim 46, wherein the methylation of the CpG dinucleotide is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parental equivalent.

48. The rAAV vector of claim 47, wherein the CpG dinucleotide is fully methylated.

49. The rAAV vector according to any one of claims 46-48, wherein the CpG dinucleotide is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parental equivalent.

50. The rAAV vector of claim 49, wherein the CpG dinucleotide is depleted.

51. The rAAV vector according to any one of claims 1-50, wherein the rAAV vector further comprises a first ITR sequence and a second ITR sequence.

52. The rAAV vector of claim 51, wherein the rAAV vector from 5' to 3' comprises: a) The first ITR sequence; b) The promoter described above; c) The polynucleotide encoding the payload of interest; and d) The second ITR sequence.

53. The rAAV vector of claim 51, wherein the rAAV vector from 5' to 3' comprises: a) The first ITR sequence; b) The promoter described above; c) The small gene mentioned; d) the polynucleotide encoding the payload of interest; and e) The second ITR sequence.

54. The rAAV vector according to any one of claims 51-53, wherein the first ITR sequence and / or the second ITR sequence are truncated compared to their corresponding wild-type ITR sequences.

55. The rAAV vector according to any one of claims 51-54, wherein the first ITR sequence and / or the second ITR sequence is truncated by at least about 5 nucleotides at the 5' end or the 3' end.

56. The rAAV vector according to any one of claims 51-55, wherein the first ITR sequence is truncated by 20 nucleotides at the 5' end.

57. The rAAV vector according to any one of claims 51-56, wherein the second ITR sequence is truncated by 20 nucleotides at the 3' end.

58. The rAAV vector according to any one of claims 51-57, wherein the first ITR sequence and / or the second ITR sequence comprises an ITR sequence derived from an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-P HP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV -HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16, or derivatives thereof.

59. The rAAV vector according to any one of claims 51-58, wherein the first ITR sequence and / or the second ITR sequence is an AAV2 ITR sequence or derived from an AAV2 ITR sequence.

60. The rAAV vector according to any one of claims 51-59, wherein the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO: 36-41.

61. The rAAV vector according to any one of claims 51-59, wherein the first ITR sequence and / or the second ITR sequence comprises the polynucleotide sequences listed in SEQ ID NO: 36-41.

62. The rAAV carrier according to any one of claims 1-61, wherein the rAAV carrier further comprises at least one adjustment element.

63. The rAAV vector according to claim 62, wherein the regulatory element is selected from the group consisting of: promoter, enhancer, terminator sequence, mRNA stability sequence, sequence allowing internal ribosome entry site (IRES) of bicistronic mRNA, intron, synthetic intron, sequence inhibiting viral recognition, sequence necessary for transduction into cells, and polyadenylate sequence.

64. The rAAV carrier according to claim 63, wherein the adjustment element is a promoter.

65. The rAAV vector according to claim 63 or 64, wherein the promoter is selected from the group consisting of: micropromoters, inducible promoters, constitutive promoters and their derivatives.

66. The rAAV vector according to any one of claims 63-65, wherein the promoter is selected from the group consisting of: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p19, p40, synaptic protein, GFAP, CaMKII, GRK1 and derivatives thereof.

67. The rAAV vector of claim 66, wherein the promoter is a CAG promoter.

68. The rAAV vector according to any one of claims 63-67, wherein the sequence of the internal ribosome entry site (IRES) that allows bicistronic mRNA is WPRE.

69. The rAAV vector of claim 68, wherein the WPRE is a wild-type WPRE.

70. The rAAV vector of claim 68, wherein the WPRE comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO:

32.

71. The rAAV vector of claim 68, wherein the WPRE comprises the polynucleotides listed in SEQ ID NO:

32.

72. The rAAV carrier of claim 68, wherein the WPRE is a modified WPRE.

73. The rAAV vector of claim 72, wherein the modified WPRE comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO:

33.

74. The rAAV vector of claim 72, wherein the modified WPRE comprises the amino acid sequence listed in SEQ ID NO:

33.

75. The rAAV vector according to any one of claims 63-74, wherein the polyadenylated sequence is selected from the group consisting of SV40, hGH, bGH and rbGlob.

76. The rAAV vector according to claim 75, wherein the polyadenylate sequence is an SV40 sequence.

77. The rAAV vector of claim 76, wherein the SV40 sequence comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO:

19.

78. The rAAV vector of claim 76, wherein the SV40 sequence comprises the polynucleotides listed in SEQ ID NO:

19.

79. The rAAV vector according to claim 75, wherein the polyadenylate sequence is a bGH sequence.

80. The rAAV vector of claim 79, wherein the bGH sequence comprises a polynucleotide having at least 80% sequence identity with SEQ ID NO:

34.

81. The rAAV vector of claim 79, wherein the bGH sequence comprises the polynucleotide sequence listed in SEQ ID NO:

34.

82. The rAAV vector according to any one of claims 1 or 3-81, wherein the rAAV vector further comprises a small gene located on the 5' side of the polynucleotide encoding the payload of interest.

83. The rAAV vector according to any one of claims 2-82, wherein the small gene encodes a splicing regulator binding site.

84. The rAAV vector of claim 83, wherein the splicing modulator binding site is located in an exon and / or an intron.

85. The rAAV vector according to claim 83 or 84, wherein the splice modulator binding site comprises one or more sequences required for spliceosome binding.

86. The rAAV vector according to any one of claims 83-85, wherein the splice modulator binding site comprises a donor site sequence, a branching site, and a receptor site.

87. The rAAV vector according to any one of claims 2-86, wherein the translation stop codon is located within the small gene coding frame.

88. The rAAV vector according to any one of claims 2-87, wherein the polynucleotide encoding the payload of interest further comprises a translation stop codon.

89. The rAAV vector according to any one of claims 2-88, wherein the polynucleotide encoding the payload of interest does not contain a start codon.

90. The rAAV vector according to any one of claims 2-89, wherein the polynucleotide encoding the payload of interest does not contain an in-frame open reading frame.

91. The rAAV vector according to any one of claims 2-90, wherein the small gene is regulated by a small molecule splicing modifier.

92. The rAAV vector according to claim 91, wherein the small molecule splicing modifier is sudemycin, LMI070, RG7916 or RG7800.

93. The rAAV vector according to claim 91, wherein the small molecule splicing modifier is selected from the group consisting of: 、 and 。 94. The rAAV vector according to any one of claims 2-93, wherein the small gene comprises exon 6, exon 7 and exon 8 of SMN2, and the splicing regulator binding site is recognized by LMI070.

95. The rAAV vector according to any one of claims 2-94, wherein the small gene contains at least about 80% sequence identity with SEQ ID NO:31 or SEQ ID NO:

51.

96. The rAAV vector according to any one of claims 2-94, wherein the small gene comprises the polynucleotide listed in SEQ ID NO: 31 or SEQ ID NO:

51.

97. The rAAV vector according to any one of claims 2-96, wherein the small gene is regulated by a disease state of the cell.

98. The rAAV vector of claim 97, wherein the disease state is cancer.

99. The rAAV vector according to claim 98, wherein the cancer is glioblastoma, metastatic brain tumor, or uveal melanoma.

100. The rAAV vector according to any one of claims 2-99, wherein the small gene is regulated by cell type or tissue type.

101. The rAAV vector according to any one of claims 2-100, wherein the polynucleotide of the small gene and the polynucleotide encoding the payload of interest are linked by a polynucleotide sequence encoding a cleavable peptide.

102. The rAAV vector according to claim 101, wherein the cleavable peptide is a substrate of a self-cleaving peptide, a drug-sensitive protease, or an endogenous endopeptide.

103. The rAAV carrier according to any one of claims 1-102, wherein the rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV- 2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV -HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16, or derivatives thereof.

104. The rAAV vector according to any one of claims 1-103 further comprises an antibiotic resistance gene.

105. The rAAV vector according to claim 104, wherein the antibiotic resistance gene confers resistance to aminoglycosides, β-lactams, macrolides, tetracyclines, or derivatives thereof.

106. The rAAV vector according to claim 104, wherein the antibiotic resistance gene confers resistance to kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, genomiconine, or derivatives thereof.

107. The rAAV vector of claim 104, wherein the antibiotic resistance gene confers resistance to kanamycin.

108. The rAAV vector according to any one of claims 104-107, wherein the antibiotic resistance gene comprises a nucleic acid sequence having at least about 80% sequence identity with SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 44 or SEQ ID NO:

55.

109. The rAAV vector according to any one of claims 104-107, wherein the antibiotic resistance gene comprises the nucleic acid listed in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 44 or SEQ ID NO:

55.

110. The rAAV vector according to any one of claims 104-109, wherein the antibiotic resistance gene comprises reduced CpG dinucleotide and / or increased CpG dinucleotide methylation compared to the parental equivalent.

111. The rAAV vector of claim 110, wherein the methylation of the CpG dinucleotide is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% compared to the parental equivalent.

112. The rAAV vector of claim 110, wherein the CpG dinucleotide is fully methylated.

113. The rAAV vector according to any one of claims 110-112, wherein the CpG dinucleotide is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% compared to the parental equivalent.

114. The rAAV vector according to any one of claims 110-112, wherein the CpG dinucleotide is depleted.

115. The rAAV vector according to any one of claims 1-114, wherein the rAAV vector further comprises a replication origin.

116. The rAAV vector according to claim 115, wherein the origin of replication is selected from the group consisting of: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, pBluescript, and combinations or derivatives thereof.

117. A method for treating cancer in a subject with a corresponding need, the method comprising: a) Applying the rAAV carrier according to any one of claims 1-116; and b) Apply small molecule splicing modifiers.

118. The method of claim 117, wherein the subject is a human.

119. The method of claim 117 or 118, wherein the administration is administered to the central nervous system.

120. The method according to any one of claims 117-119, wherein the application is administered to the brain.

121. The method according to any one of claims 117-120, wherein the application is administered to the ventricle.

122. The method according to any one of claims 117-121, wherein the application is performed via convection-enhanced delivery (CED).

123. The method according to any one of claims 117-121, wherein the administration is performed by intratumoral injection, intracranial injection, intracerebral injection, intraventricular injection, intraparenchymal injection, or injection into cerebrospinal fluid (CSF) via the ventricular system, cisterna magna, or intrathecal space.

124. The method according to any one of claims 117-123, wherein the small molecule splicing modifier is sudemycin, LMI070, RG7916 or RG7800.

125. The method according to any one of claims 117-123, wherein the small molecule splicing modifier is selected from the group consisting of: 、 and 。 126. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 40 or SEQ ID NO: 41; b) CAG promoters listed in any one of SEQ ID NO: 22-23, 48-50, and 64-65; c) Polynucleotides encoding CpG-depleted human interferon listed in SEQ ID NO: 4; d) The WPRE sequence listed in SEQ ID NO: 33; e) bGH polyadenylates listed in SEQ ID NO: 34; and f) The second ITR listed in SEQ ID NO: 40 or SEQ ID NO:

41.

127. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 40 or SEQ ID NO: 41; b) CAG promoters listed in any of SEQ ID NO: 22-23; c) Polynucleotides encoding CpG-depleted human interferon listed in SEQ ID NO: 4; d) The WPRE sequence listed in SEQ ID NO: 33; e) SV40 polyadenylates listed in SEQ ID NO: 19; and f) The second ITR listed in SEQ ID NO: 40 or SEQ ID NO:

41.

128. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) Contains the first ITR listed in any one of SEQ ID NO: 36-39; b) CAG promoters listed in any of SEQ ID NO: 48-50 and 64-65; c) Polynucleotides encoding CpG-depleted human interferon listed in SEQ ID NO: 4; d) The WPRE sequence listed in SEQ ID NO: 33; e) bGH polyadenylates listed in SEQ ID NO: 34; and f) The second ITR sequence listed in any of SEQ ID NO: 36-39.

129. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 40 or SEQ ID NO: 41; b) CAG promoters listed in any of SEQ ID NO: 22-23; c) Polynucleotides encoding human interferons listed in SEQ ID NO: 2 or SEQ ID NO: 3; d) The WPRE sequence listed in SEQ ID NO: 33; e) SV40 polyadenylates listed in SEQ ID NO: 19; and f) The second ITR listed in SEQ ID NO: 40 or SEQ ID NO:

41.

130. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 40 or SEQ ID NO: 41; b) CAG promoters listed in any of SEQ ID NO: 22-23; c) The small genes listed in SEQ ID NO: 31 or SEQ ID NO: 51; d) The polynucleotide encoding the CpG-depleted human interferon listed in SEQ ID NO: 4; e) The WPRE sequence listed in SEQ ID NO: 33; f) SV40 polyadenylates listed in SEQ ID NO: 19; and g) The second ITR listed in SEQ ID NO: 36-39.

131. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 40 or SEQ ID NO: 41; b) CAG promoters listed in any of SEQ ID NO: 48-50 and 64-65; c) The small genes listed in SEQ ID NO: 31; d) The polynucleotide encoding the CpG-depleted human interferon listed in SEQ ID NO: 4; e) The WPRE sequence listed in SEQ ID NO: 33; f) bGH polyadenylates listed in SEQ ID NO: 34; and g) The second ITR listed in SEQ ID NO: 40 or SEQ ID NO:

41.

132. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) The first ITR listed in SEQ ID NO: 36-39; b) CAG promoters listed in any of SEQ ID NO: 48-50 and 64-65; c) The small genes listed in SEQ ID NO: 31 or SEQ ID NO: 51; d) The polynucleotide encoding the CpG-depleted human interferon listed in SEQ ID NO: 4; e) The WPRE sequence listed in SEQ ID NO: 33; f) bGH polyadenylates listed in SEQ ID NO: 34; and g) The second ITR listed in SEQ ID NO: 36-39.

133. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) First ITR sequence; b) CAG promoter; c) The coding sequence of CpG-depleted human interferon; d) WPRE sequence; e) bGH polyadenylation sequence; and f) Second ITR sequence.

134. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) First ITR sequence; b) CAG promoter; c) The coding sequence of CpG-depleted human interferon; d) WPRE sequence; e) SV40 polyadenylation sequence; and f) Second ITR sequence.

135. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) First ITR sequence; b) CAG promoter; c) The coding sequence of human interferon; d) WPRE sequence; e) SV40 polyadenylation sequence; and f) Second ITR sequence.

136. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) First ITR sequence; b) CAG promoter; c) The small genes listed in SEQ ID NO: 31 or SEQ ID NO: 51; d) The coding sequence of CpG-depleted human interferon; e) WPRE sequence; f) SV40 polyadenylation sequence; and g) Second ITR sequence.

137. A recombinant adeno-associated virus (rAAV) vector comprising, from 5' to 3': a) First ITR sequence; b) CAG promoter; c) The small genes listed in SEQ ID NO: 31 or SEQ ID NO: 51; d) The coding sequence of CpG-depleted human interferon; e) WPRE sequence; f) bGH polyadenylation sequence; and g) Second ITR sequence.

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