Adeno-associated virus vector composition and method of use

Abstract

Nucleic acids and recombinant viral vectors containing said nucleic acids are described herein, wherein the said nucleic acids encode a target payload (e.g., interferon).

Description

Technical Field

[0001] This application claims the benefit and priority of U.S. Provisional Patent Application Nos. 63 / 496,916 and 63 / 502,546, filed on April 18, 2023 and May 16, 2023, respectively, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

Summary of the Invention

[0002] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors for use in delivering a payload of interest (e.g., a therapeutic payload) to a desired target are disclosed herein, and the recombinant adeno-associated virus (rAAV) vectors exhibit improved efficacy and / or reduced immunogenicity.

[0003] In certain embodiments, rAAV vectors comprising a coding sequence for a payload of interest (e.g., a therapeutic payload) are further described herein, and CpG dinucleotides within the coding sequence are decreased, depleted, and / or methylated to result in certain beneficial therapeutic properties. In some embodiments, as described herein, sequences comprising one or more modified CpG dinucleotides (e.g., decrease of CpG dinucleotides, depletion of CpG dinucleotides, and / or methylation of CpG dinucleotides) encode a therapeutic polypeptide. In some embodiments, as described herein, sequences comprising one or more modified CpG dinucleotides encode an interferon (e.g., any interferon known in the art, such as those described herein).

[0004] In certain embodiments, methods for treating a disease or disorder of a subject requiring treatment are also described herein, the methods comprising the step of administering a modified rAAV vector as described herein. In some embodiments, the disclosure provides a method for treating a cancer of a subject, comprising the step of administering a modified rAAV vector as described herein. In some embodiments, the cancer is a brain tumor. 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 grade IV glioma (glioblastoma). In some embodiments, the cancer is a glioblastoma. In some embodiments, the cancer is a high-grade glioma, a metastatic brain tumor, or a uveal melanoma. In some embodiments, the administration of the rAAV vector containing cytokines (e.g., any cytokines that deliver the rAAV vector as described herein) is local to the tumor of the subject having cancer. In some embodiments, the administration of the rAAV vector containing cytokines to the subject having cancer is systemic (e.g., intravenous). Furthermore, the rAAV vector compositions and methods described herein offer long-term cytokine generation (e.g., by vectorization, robust expression, etc.), high specificity with respect to payload delivery (e.g., by local administration), and minimal risk of systemic toxicity (e.g., by low-dose administration of self-limiting AAV genomes).

[0005] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a CAG promoter and b) a polynucleotide encoding a payload of interest, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

[0006] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a CAG promoter, b) a minigene including a splice modulator binding site, and c) a polynucleotide encoding a payload of interest, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

[0007] In some embodiments, the payload of interest is a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide is a cytokine or 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 the CpG nucleotide 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 dinucleotide is completely methylated. In some embodiments, 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 its parental equivalent. In some embodiments, the CpG dinucleotide is depleted.

[0008] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a CAG promoter, b) a polynucleotide encoding an interferon, and c) a polynucleotide comprising a WPRE including sequence number 33.

[0009] In some embodiments, the CAG promoter includes a cytomegalovirus (CMV) initial enhancer element, a promoter element, and a splice acceptor element. In some embodiments, the CMV initial enhancer element is derived from a wild-type CMV enhancer. In some embodiments, the CMV initial enhancer element is cleaved with respect to the wild-type CMV enhancer. In some embodiments, the CMV initial enhancer element includes 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. In some embodiments, the CMV initial enhancer element includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 26. In some embodiments, the CMV initial enhancer element includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 26. In some embodiments, the CMV initial enhancer element includes the polynucleotide sequence described in SEQ ID NO: 26. In some embodiments, the CMV early enhancer element includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 27. In some embodiments, the CMV very early enhancer element includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 27. In some embodiments, the CMV very early enhancer element includes the polynucleotide sequence described in SEQ ID NO: 27. In some embodiments, the promoter element is derived from the chicken β-actin gene. In some embodiments, the promoter element includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 28. In some embodiments, the promoter element includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 52. In some embodiments, the promoter element includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 28. In some embodiments, the promoter element includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 52.In some embodiments, the promoter element includes the polynucleotide sequence described in SEQ ID NO: 28. In some embodiments, the promoter element includes the polynucleotide sequence described in SEQ ID NO: 52. In some embodiments, the splice acceptor is derived from the rabbit beta-globin gene. In some embodiments, the splice acceptor includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 30. In some embodiments, the splice acceptor includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 52. In some embodiments, the splice acceptor includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 30. In some embodiments, the splice acceptor includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 52. In some embodiments, the splice acceptor includes the polynucleotide sequence described in SEQ ID NO: 30. In some embodiments, the splice acceptor includes the polynucleotide sequence described in SEQ ID NO: 53. In some embodiments, the interleukin is IL-1α, 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. In some embodiments, the CSF is CSF1 (M-CSF), CSF2 (GM-CSF), or CSF3 (G-CSF). In some embodiments, TGF is TGF-β1, TGF-β2, or TGF-β3. In some embodiments, TNF is TNF-α, TNF-β, or LT-β. In some embodiments, interferon is IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ, or a variant or derivative thereof.In some embodiments, the interferon is human IFNα, human IFNβ, human IFNγ, human IFNε, human IFNκ, human IFNω, human IFNλ, or a variant or derivative thereof. In some embodiments, the interferon is human IFNβ. In some embodiments, human IFNβ comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, human IFNβ comprises amino acids having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, human IFNβ comprises the amino acid sequence described in SEQ ID NO: 1. In some embodiments, human IFNβ 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, human IFNβ 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, human IFNβ is encoded by a polynucleotide described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the interferon is human IFNα. In some embodiments, human IFNα includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5. In some embodiments, human IFNα includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5. In some embodiments, human IFNα includes the amino acid sequence described in SEQ ID NO: 5. In some embodiments, human IFNα 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, human IFNα 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, human IFNα is encoded by a polynucleotide described in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the interferon is human IFNγ. In some embodiments, human IFNγ includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9.In some embodiments, human IFNγ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9. In some embodiments, human IFNγ comprises the amino acid sequence described in SEQ ID NO: 9. In some embodiments, human IFNγ 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, human IFNγ 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, human IFNγ is encoded by a polynucleotide sequence described in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the interferon is mouse IFNα, mouse IFNβ, mouse IFNγ, mouse IFNε, mouse IFNκ, mouse IFNω, mouse IFNλ, or a variant or derivative thereof. In some embodiments, the interferon is mouse IFNβ. In some embodiments, mouse IFNβ comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, mouse IFNβ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, mouse IFNβ comprises the amino acid sequence described in SEQ ID NO: 13. In some embodiments, mouse IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, mouse IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, mouse IFNβ is encoded by the polynucleotide described in SEQ ID NO: 14. In some embodiments, mouse IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, mouse IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, mouse IFNβ is encoded by the polynucleotide described in SEQ ID NO: 42.In some embodiments, the interferon is canine IFNα, canine IFNβ, canine IFNγ, canine IFNε, canine IFNκ, canine IFNω, canine IFNλ, or a variant or derivative thereof. In some embodiments, the interferon is canine IFNβ. In some embodiments, canine IFNβ includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15. In some embodiments, canine IFNβ includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 15. In some embodiments, canine IFNβ includes the amino acid sequence described in SEQ ID NO: 15. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 16. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, canine IFNβ is encoded by a polynucleotide described in SEQ ID NO: 16. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, canine IFNβ is encoded by the polynucleotide described in SEQ ID NO: 43. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 60. In some embodiments, canine IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 60. In some embodiments, canine IFNβ is encoded by the polynucleotide described in SEQ ID NO: 60. In some embodiments, the interferon is rat IFNα, rat IFNβ, rat IFNγ, rat IFNε, rat IFNκ, rat IFNω, rat IFNλ, or a variant or derivative thereof. In some embodiments, the interferon is rat IFNβ.In some embodiments, rat IFNβ includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 61. In some embodiments, rat IFNβ includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 61. In some embodiments, rat IFNβ includes the amino acid sequence described in SEQ ID NO: 61. In some embodiments, rat IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 62. How many? In some embodiments, rat IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 62. In some embodiments, rat IFNβ is encoded by the polynucleotide described in SEQ ID NO: 62. In some embodiments, rat IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 63. In some embodiments, rat IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 63. In some embodiments, rat IFNβ is encoded by the polynucleotide described in SEQ ID NO: 63. In some embodiments, the interferon is guinea pig IFNα, guinea pig IFNβ, guinea pig IFNγ, guinea pig IFNε, guinea pig IFNκ, guinea pig IFNω, guinea pig IFNλ, or a variant or derivative thereof. In some embodiments, the interferon is guinea pig IFNβ. In some embodiments, guinea pig IFNβ contains an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 71. In some embodiments, guinea pig IFNβ comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 71. In some embodiments, guinea pig IFNβ comprises the amino acid sequence described in SEQ ID NO: 71. In some embodiments, guinea pig IFNβ is encoded by a polynucleotide having at least 80% sequence identity to SEQ ID NO: 72. In some embodiments, guinea pig IFNβ is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 72. In some embodiments, guinea pig IFNβ is encoded by the polynucleotide described in SEQ ID NO: 72. In some embodiments, the polynucleotide encoding the interferon comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to the parental equivalent.In some embodiments, the methylation of the CpG nucleotide 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 95% compared to the parental equivalent. In some embodiments, the CpG dinucleotide is completely methylated. In some embodiments, the CpG dinucleotide 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 99% compared to the parental equivalent. In some embodiments, the CpG dinucleotide is 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 the 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 the payload of interest, and e) a second ITR sequence. In some embodiments, the first ITR sequence and / or the second ITR sequence are cleaved relative to their corresponding wild-type ITR sequences. In some embodiments, the first ITR sequence and / or the second ITR sequence are cleaved at the 5' or 3' end by at least about 5 nucleotides. In some embodiments, the first ITR sequence is cleaved at the 5' end by 20 nucleotides. In some embodiments, the second ITR sequence is cleaved at the 3' end by 20 nucleotides.In some embodiments, the first ITR sequence and / or the second ITR sequence are 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, A The ITR sequence comprises an AAV serotype selected from the group consisting of AV-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. In some embodiments, the first ITR sequence and / or the second ITR sequence is either an AAV ITR sequence or derived from an AAV ITR sequence. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 80% sequence identity with SEQ ID NOs. 36-41. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 90% sequence identity with SEQ ID NOs. 36-41. In some embodiments, the first ITR sequence and / or the second ITR sequence comprises the polynucleotide sequence described in SEQ ID NOs. 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 promoters, enhancers, terminator sequences, mRNA stability sequences, sequences enabling internal ribosome entry sites (IRESs) for bicistronic mRNA, introns, synthetic introns, sequences inhibiting virus recognition, sequences required for transduction into cells, and poly(A) sequences. In some embodiments, the regulatory element is a promoter. In some embodiments, the promoter is selected from the group consisting of minipromoters, inducible promoters, constitutive promoters, and their derivatives. 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 their derivatives. In some embodiments, the promoter is a CAG promoter. In some embodiments, the sequence enabling internal ribosome entry sites (IRESs) for 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 with SEQ ID NO: 32. In some embodiments, the WPRE comprises a polynucleotide having at least 90% sequence identity with SEQ ID NO: 32. In some embodiments, the WPRE comprises the polynucleotide described 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 with SEQ ID NO: 33. In some embodiments, the modified WPRE comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 33. In some embodiments, the modified WPRE comprises the amino acid sequence described in SEQ ID NO: 33. In some embodiments, the polyA sequence is selected from the group consisting of SV40, hGH, bGH, and rbGlob. In some embodiments, the polyA sequence is the SV40 sequence.In some embodiments, the SV40 sequence includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 19. In some embodiments, the SV40 sequence includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 19. In some embodiments, the SV40 sequence includes the polynucleotide described in SEQ ID NO: 19. In some embodiments, the polyA sequence is a bGH sequence. In some embodiments, the bGH sequence includes a polynucleotide having at least 80% sequence identity with SEQ ID NO: 34. In some embodiments, the bGH sequence includes a polynucleotide having at least 90% sequence identity with SEQ ID NO: 34. In some embodiments, the bGH sequence includes the polynucleotide sequence described in SEQ ID NO: 34. In some embodiments, the rAAV vector further includes a minigene at the 5' end of 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 intron. In some embodiments, the splice modulator binding site includes one or more sequences required for spliceosome binding. In some embodiments, the splice modulator binding site includes a donor site sequence, a branching site, and an acceptor site. In some embodiments, the minigene encodes an in-frame translation termination codon. In some embodiments, the polynucleotide encoding the payload of interest further includes a translation termination codon. In some embodiments, the polynucleotide encoding the payload of interest does not include a start codon. In some embodiments, the polynucleotide encoding the payload of interest does not include 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 sudemycin, LMI070, RG7916, or RG7800. In some embodiments, the small molecule splicing modifier is...

[0010]

change

[0011] In certain embodiments, a method for treating a target cancer requiring treatment is described herein, the method comprising the steps of a) administering an rAAV vector as described herein, and b) administering a small molecule splicing modifier. In some embodiments, the subject is human. In some embodiments, the administration is to the central nervous system. In some embodiments, the administration is to the brain. In some embodiments, the administration is to the 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, intraventricular, intraparenchymal, or injection into cerebrospinal fluid (CSF) via the ventricular system, cisterna magna, or intrathecal space. In some embodiments, the small molecule splicing modifier is sudemycin, LMI070, RG7916, or RG7800. In some embodiments, the small molecule splicing modifier is

[0012] [ka] It is selected from the group consisting of the following.

[0013] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NO: 40 or 41, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a polynucleotide encoding CpG-depleted human interferon as described in SEQ ID NO: 4, d) a WPRE sequence described in SEQ ID NO: 33, e) bGH polyA described in SEQ ID NO: 34, and f) a second ITR sequence described in SEQ ID NO: 40 or 41.

[0014] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NO: 40 or 41, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a polynucleotide encoding a CpG-depleted human interferon as described in SEQ ID NO: 4, d) a WPRE sequence described in SEQ ID NO: 33, e) SV40 polyA as described in SEQ ID NO: 19, and f) a second ITR described in SEQ ID NO: 40 or 41.

[0015] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence including one of SEQ ID NOs: 36-39; b) a CAG promoter as described in one of SEQ ID NOs: 22-23, 48-50, and 64-65; c) a polynucleotide encoding CpG-depleted human interferon as described in SEQ ID NO: 4; d) a WPRE sequence as described in SEQ ID NO: 33; e) bGH polyA as described in SEQ ID NO: 34; and f) a second ITR sequence as described in one of SEQ ID NOs: 36-39.

[0016] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NO: 40 or 41, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a polynucleotide encoding human interferon as described in SEQ ID NO: 2 or SEQ ID NO: 3, d) a WPRE sequence described in SEQ ID NO: 33, e) SV40 polyA described in SEQ ID NO: 19, and f) a second ITR described in SEQ ID NO: 40 or 41.

[0017] In certain embodiments, a recombinant adeno-associated virus (rAAV) vector is disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NO: 40 or 41, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a minigene described in SEQ ID NO: 31 or 51, d) a polynucleotide encoding CpG-depleted human interferon as described in SEQ ID NO: 4, e) a WPRE sequence described in SEQ ID NO: 33, f) SV40 polyA described in SEQ ID NO: 19, and g) a second ITR described in SEQ ID NO: 40 or 41.

[0018] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NO: 40 or 41, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a minigene described in SEQ ID NO: 31, d) a polynucleotide encoding CpG-depleted human interferon as described in SEQ ID NO: 4, e) a WPRE sequence described in SEQ ID NO: 33, f) a bGH polyA described in SEQ ID NO: 34, and g) a second ITR described in SEQ ID NO: 40 or 41.

[0019] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence described in SEQ ID NOs: 36-39, b) a CAG promoter described in any one of SEQ ID NOs: 22-23, 48-50, and 64-65, c) a minigene described in SEQ ID NOs: 31 or 51, d) a polynucleotide encoding CpG-depleted human interferon as described in SEQ ID NOs: 4, e) a WPRE sequence described in SEQ ID NOs: 33, f) a bGH polyA described in SEQ ID NOs: 34, and g) a second ITR described in SEQ ID NOs: 36-39.

[0020] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for CpG-depleted human interferon, d) a WPRE sequence, e) an SV40 polyA sequence, and f) a second ITR sequence.

[0021] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for CpG-depleted human interferon, d) a WPRE sequence, e) a bGH polyA sequence, and f) a second ITR sequence.

[0022] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for human interferon, d) a WPRE sequence, e) an SV40 polyA sequence, and f) a second ITR sequence.

[0023] In certain embodiments, a recombinant adeno-associated virus (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 51; d) a coding sequence for CpG-depleted human interferon; e) a WPRE sequence; f) an SV40 polyA sequence; and g) a second ITR sequence is disclosed herein.

[0024] In certain embodiments, a recombinant adeno-associated virus (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 51; d) a coding sequence for CpG-depleted human interferon; e) a WPRE sequence; f) a bGH polyA sequence; and g) a second ITR sequence is disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present disclosure will be more fully understood by reference to the accompanying drawings.

[0026] [Figure 1A] An exemplary workflow depicting the mechanism of rAAV described herein is depicted. [Figure 1B] An exemplary inducible AAV expression system plasmid map depicting various components used in the AAV expression system through certain embodiments is depicted. [Figure 2] An exemplary constitutive AAV expression system plasmid map depicting various components used in the AAV expression system through certain embodiments is depicted. [Figure 3] Data obtained from in vitro verification of inducible GFP fluorescence is depicted. [Figure 4] Data depicting the in vitro kinetics of inducible GFP fluorescence with increasing viral dose is depicted. [Figure 5A]Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5A shows plasmid #1, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted human interferon-beta (hIFNβ) gene sequence, the polyadenylation signal sequence (SV40 pA) derived from SV40, and the ampicillin resistance (AmpR) gene. [Figure 5B] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5B shows plasmid #2, an exemplary plasmid containing the CAG promoter sequence, a minigene (Xon), a CpG-depleted hIFNβ gene sequence lacking the ATG codon, SV40 pA, and the AmpR gene. [Figure 5C] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5C shows plasmid #3, an exemplary plasmid containing the CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking the ATG codon, the bovine growth hormone polyadenylation signal (bGH pA), and the kanamycin resistance (KanR) gene. [Figure 5D] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5D shows an exemplary plasmid, plasmid #4, containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the KanR gene. [Figure 5E] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5E shows plasmid #5, an exemplary plasmid containing the CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking the ATG codon, bGH pA, and the KanR gene. [Figure 5F] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5F shows plasmid #6, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the KanR gene. [Figure 5G]Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5G shows an exemplary plasmid, plasmid #7, containing the CAG promoter, wild-type hIFNβ gene sequence, SV40 pA, and AmpR gene. [Figure 5H] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5H shows plasmid #8, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the KanR gene. [Figure 5I] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5I shows an exemplary plasmid, plasmid #9, containing the CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking the ATG codon, bGH pA, and the KanR gene. [Figure 5J] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5J shows plasmid #10, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the KanR gene. [Figure 5K] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5K shows exemplary plasmid #11 containing the CAG promoter sequence, Xon, CpG depleted, and the hIFNβ gene sequence lacking the ATG codon, bGH pA, and KanR gene. [Figure 5L] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5L shows plasmid #12, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the CpG-depleted KanR gene. [Figure 5M] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5M shows plasmid #13, an exemplary plasmid containing the CAG promoter sequence, Xon, CpG depleted hIFNβ gene sequence lacking the ATG codon, bGH pA, and CpG depleted KanR gene. [Figure 5N] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5N shows plasmid #14, an exemplary plasmid containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the CpG-depleted KanR gene. [Figure 5O] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 50 shows exemplary plasmid #15, which contains a CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking an ATG codon, a bGH pA, and a CpG-depleted KanR gene. [Figure 5P] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5P shows an exemplary plasmid, plasmid #16, containing the CAG promoter sequence, enhanced fluorescent protein (eGFP) gene sequence, bGH pA, and KanR gene. [Figure 5Q] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5Q shows an exemplary plasmid, plasmid #17, containing the CAG promoter sequence, Xon, eGFP gene sequence lacking the ATG codon, bGH pA, and KanR gene. [Figure 5R] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5R shows an exemplary plasmid, plasmid #18, containing the CAG promoter sequence, eGFP gene sequence, bGH pA, and the KanR gene depleted of CpG. [Figure 5S] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5S shows exemplary plasmid #19 containing the eGFP gene sequence lacking the CAG promoter sequence, Xon, and ATG codon, as well as the bGH pA and CpG-depleted KanR gene. [Figure 5T]Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5T shows an exemplary plasmid, plasmid #20, containing the CAG promoter sequence, mCardinal gene sequence, bGH pA, and CpG-depleted KanR gene. [Figure 5U] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5U shows plasmid #21, an exemplary plasmid containing the CAG promoter sequence, mCardinal gene sequence, bGH pA, and CpG-depleted KanR gene. [Figure 5V] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5V shows plasmid #22, an exemplary plasmid containing the CAG promoter sequence, Xon, mCardinal gene sequence lacking the ATG codon, bGH pA, and CpG-depleted KanR gene. [Figure 5W] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5W shows plasmid #23, an exemplary plasmid containing the CAG promoter sequence, Xon, mCardinal gene sequence lacking the ATG codon, bGH pA, and CpG-depleted KanR gene. [Figure 5X] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5X shows an exemplary plasmid, plasmid #24, containing the CAG promoter sequence, the CpG-depleted hIFNβ gene sequence, the bGH pA, and the CpG-depleted KanR gene. [Figure 5Y] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5Y shows plasmid #25, an exemplary plasmid containing the CAG promoter sequence, mCardinal gene sequence, bGH pA, and CpG-depleted KanR gene. [Figure 5Z]Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5Z shows an exemplary plasmid, plasmid #26, containing the CAG promoter sequence, mouse interferon beta (mIFNβ) gene sequence, bGH pA, and the CpG-depleted KanR gene. [Figure 5AA] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5AA shows an exemplary plasmid, plasmid #27, containing the CAG promoter sequence, rat interferon beta (rIFNβ) gene sequence, bGH pA, and the CpG-depleted KanR gene. [Figure 5AB] Exemplary plasmid vector maps are drawn for plasmids 1–30. Figure 5AB shows plasmid #28, an exemplary plasmid containing the CAG promoter sequence, the canine interferon beta (cIFNβ) gene sequence, bGH pA, and the KanR gene depleted of CpG. [Figure 5AC] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5AC shows plasmid #29, an exemplary plasmid containing a CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking an ATG codon, a bGH pA, and a CpG-depleted KanR gene. [Figure 5AD] Exemplary plasmid vector maps for plasmids 1–30 are drawn. Figure 5AD shows plasmid #30, an exemplary plasmid containing a CAG promoter sequence, Xon, a CpG-depleted hIFNβ gene sequence lacking an ATG codon, a bGH pA, and a CpG-depleted KanR gene. [Figure 6] This section illustrates an exemplary workflow for producing the rAAV vector plasmid used to produce the rAAV vectors described herein. [Figure 7]The following are SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel) results, in which 1 e11 vg of each AAV preparation, subjected to electrophoresis for protein separation on a 10% polyacrylamide gel, was loaded into a well and its purity was evaluated by Coomassie blue staining. [Figure 8A] Data from in vitro evaluations of payload production efficiency are shown. Figures 8A-8B show the relative production efficiency of various expressed AAV preparations as a ratio of each vg titer (vg / mL) divided by the vg titer of the reference stock (AAV produced using plasmid #1 (Figure 8A) or plasmid #12 (Figure 8B)). The plasmid used to produce the corresponding AAV is identified on the X axis. [Figure 8B] Data from in vitro evaluations of payload production efficiency are shown. Figures 8A-8B show the relative production efficiency of various expressed AAV preparations as a ratio of each vg titer (vg / mL) divided by the vg titer of the reference stock (AAV produced using plasmid #1 (Figure 8A) or plasmid #12 (Figure 8B)). The plasmid used to produce the corresponding AAV is identified on the X axis. [Figure 8C] Data from in vitro evaluations of payload production efficiency are shown. Figure 8C shows the vg titers obtained from each of five 1-liter (L) shaking flasks of suspended cells using plasmid #1. [Figure 9A] Data from in vitro evaluation of payload expression are shown. Figure 9A shows the measured payload expression for AAV preparations #6, #5, #8, and #9, each produced by different vendors and processes. All preparations were produced using plasmid #1, which carried a human IFNβ (hIFNβ) payload and was packaged within the same AAV capsid serotype. [Figure 9B]The data from in vitro evaluation of payload expression are shown. Figure 9B shows the measured payload expression for AAV preparations #1, #9, #10, and #11 (produced from plasmids #7, #1, #12, and #14, respectively) of various AAV genomes, produced by a single vendor using the same process and packaged within the same AAV capsid serotype. [Figure 9C] Data from in vitro evaluation of payload expression are shown. Figure 9C shows the measured payload expression for AAV preparations #6, #9, #10, #2, #13, and #16 (produced from plasmids #1, #1, #12, #7, #24, and #25, respectively) that express hIFNβ (with the exception of AAV preparation #16, which contained the reporter payload (mCardinal) and was used as a negative control). All other preparations, except for AAV preparation #6, were prepared using the same process from the same vendor. [Figure 9D] Data from in vitro evaluation of payload expression are shown. Figure 9D shows payload expression for AAV preparations #3, #6, #14, and #15 (produced from plasmids #7, #1, #24, and #24, respectively). Both AAV preparations #14 and #15 utilized plasmid #24 but were produced by two different vendors. [Figure 9E] Data from in vitro evaluation of payload expression are shown. Figure 9E shows the measured payload expression for AAV preparations #6 and #17 (produced using plasmids #1 and #26 expressing human IFNβ and mouse IFNβ, respectively). In all plots, data represent the mean ± SD of technically redundant measurements. Abbreviations: MOI, degree of multiple infection; prep, preparation. [Figure 10]This shows in vitro measurement results of cytokine payload activity for six exemplary AAVs and media (NC) expressing hIFNβ (AAV preparations #6, #9, #10, #2, and #13), as well as one AAV expressing a reporter payload (mCardinal) (AAV preparation #12) used as a negative control. Values ​​represent the mean ± SD for eight technical overlaps. [Figure 11] This report presents an in vitro evaluation of the reduction in cancer cell viability upon exposure to the AAV expression system expressing human IFNβ (AAV-hIFNβ) described herein. As shown, relative cell viability is expressed as a percentage at day 6 after exposure to low, medium, or high doses of AAV-hIFNβ or AAV-GFP. Staurosporine (STS) was used as the positive killing target, and data are expressed as mean ± SD using technical replication of n=4. [Figure 12] This shows the levels of hIFNβ secreted from in vitro transduced GBM cells, as measured by ELISA. The values ​​represent the mean ± SD of four technical replications. [Figure 13] This specification provides exemplary processes and assays for the production of the AAV constructs described herein. [Modes for carrying out the invention]

[0027] Cancer remains the second leading cause of death in the United States, after heart disease. In 2023, the U.S. is projected to see a total of 1.9 million new cancer cases (approximately 5,370 per day) and 609,820 cancer-related deaths (approximately 1,670 per day). The treatment repertoire for many cancers (e.g., glioblastoma, metastatic brain tumors, and uveal melanoma) remains largely limited to invasive and / or cytotoxic approaches, including resection, radiation, chemotherapy, and combinations thereof. Even common immuno-based oncological drugs, such as cytokine therapy, exhibit negative characteristics, including toxicity from systemic administration, short half-lives, and lack of specificity. Furthermore, there remain challenges in methods for effectively delivering therapeutic agents to cancer tissue.

[0028] There is a need for improved treatments for cancer (e.g., glioblastoma, metastatic brain tumors, uveal melanoma), including cytokine therapies with improved characteristics such as low toxicity, long-term and stable sustained expression, improved delivery (e.g., local delivery to tumors), and low doses. Compositions and methods relating to rAAV delivery of target payloads, such as cytokines including interferon, are provided herein.

[0029] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to the extent of the subject matter. In general, the nomenclature and techniques used in relation to immunology, oncology, cell and tissue culture, molecular biology, and protein expression and oligonucleotide or polynucleotide chemistry, and hybridization described herein are well known and commonly used in the art. Units of measurement, unless otherwise defined, follow the International System of Units (SI), NIST SP330, 2019 edition.

[0030] Where used herein, all numbers or numerical ranges include the entire integer within or encompassing such a range, and any value or integer within that range, unless the context clearly indicates otherwise. Thus, for example, a reference to the range 90–100% includes 91%, 92%, 93%, 94%, 95%, 95%, 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. In another example, references to the range of 1 to 5,000 times include 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, etc., as well as 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, etc., and 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, etc.

[0031] The terms used herein are intended solely to describe specific embodiments and are not intended to limit any embodiments. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context otherwise expressly indicates otherwise. Furthermore, the terms “comprises” and / or “comprising,” where used herein, specify the presence of the described features, integers, processes, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. Where used herein, the terms “and / or” include any and all combinations of one or more of the enumerated items relating to the subject.

[0032] As used herein, “adeno-associated virus vector” or “AAV vector” refers to, but is 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-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AA This refers to vectors derived from adeno-associated virus serotypes including V-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 may have one or more AAV wild-type genes, e.g., Rep genes and / or Cap genes, that are entirely or partially deleted, but retain functional adjacent inverted terminal repeat (ITR) sequences. Functional ITR sequences facilitate rescue, replication, and packaging of AAV virions. Therefore, an AAV vector is defined herein to contain at least those sequences on the same side for at least replication and viral packaging (e.g., functional ITRs). The ITRs do not have to be wild-type polynucleotide sequences and, in some embodiments, can be modified, for example, by nucleotide insertions, deletions, or substitutions, as long as the sequences provide functional rescue, replication, and packaging.

[0033] The term “adeno-associated virus inverted terminal repeat” or “AAV ITR” refers to a region adjacent to each end of the AAV genome that functions together on the same side as an origin for DNA replication and as a viral packaging signal. As used herein, “AAV ITR” does not necessarily include wild-type polynucleotide sequences that are modified, for example, by nucleotide insertions, deletions, or substitutions. Furthermore, AAV ITRs include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, A AV-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 The AAV vectors are derived from any multiple serotypes, including 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. Furthermore, the 5' and 3' ITRs adjacent to the selected polynucleotide sequences in the AAV vector do not have to be identical and do not have to be derived from the same AAV serotype or isolate, as long as they function as intended to enable, for example, the desired therapeutic or genome editing effect. Furthermore, 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).

[0034] Unless otherwise specified or the context makes clear, when used herein, the term “about” with respect to a number or range of numbers is understood to mean the specified number and ±10% of that number, or 10% below the listed lower limit and 10% above the listed upper limit for any range of values.

[0035] As used herein, “CpG dinucleotide,” “CpG site,” or “CpG” refers to a region of nucleic acid (e.g., DNA or RNA) where a cytosine nucleotide occurs adjacent to a guanine nucleotide in a linear nucleic acid sequence of nucleotides along its length, e.g., cytosine and guanine separated by only one phosphate group, or cytosine at the 5' end of a guanine nucleotide.

[0036] The terms “recipient,” “individual,” “subject,” “host,” and “patient” are used interchangeably herein and, in some embodiments, refer to any mammalian subject, particularly humans, to whom diagnosis, treatment, or therapy is desired. “Mammal” for treatment purposes refers to any animal classified as a mammal, including humans, domesticated and livestock, as well as laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is human. None of these terms require the supervision of a medical professional.

[0037] "Identity percentage," "identity %," or "sequence identity" refers to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in their alignment. For example, "The nucleotide sequence is X% identical to sequence number Y" refers to the identity percentage of the nucleotide sequence to sequence number Y, stating that X% of the residues in the nucleotide sequence are identical to the corresponding residues in the sequence disclosed in sequence number Y. A sequence that is said to be X% identical to a reference sequence may contain more nucleotide or amino acid residues than those identified in the reference sequence, but it must contain the sequence corresponding to the reference sequence. In most cases, the sequence in question contains the sequence corresponding to all identified reference sequences. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, GAPBLAST, BLASTP, BLASTN, or GCG.

[0038] "Polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a chain of nucleotides of any length, including DNA or RNA. In some embodiments, nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or analogs thereof. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs (e.g., increased CpG dinucleotides as described herein). Where present, modifications to the nucleotide structure are conferred before or after the assembly of the chain. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, polynucleotides are further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “cap” substitutions of one or more naturally occurring nucleotides with analogs, internucleotide modifications, e.g., those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates) and those with charged bonds (e.g., phosphorothioates, phosphorodithioates), those containing pendant moieties, e.g., proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine), those containing intercalators (e.g., acridine, psoralens), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides), those containing alkylating agents, those having modified bonds (e.g., alpha-anomeric nucleic acids), and unmodified forms of polynucleotides. In some embodiments, any of the hydroxyl groups normally present in the sugar are, for example, substituted with a phosphonate group, a phosphate group, protected with a standard protecting group, activated to prepare further linkage to further nucleotides, or conjugated to a solid support. In some embodiments, the 5' and 3' terminal OH groups are phosphorylated or substituted with amine or organic capping groups of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.In some embodiments, polynucleotides also include analogous forms of ribose or deoxyribose sugars, such as 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, 2'-azidol-ribose, carbocic sugar analogs, α- or β-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and base-deficient nucleoside analogs such as methylriboside. In some embodiments, one or more phosphodiester bonds are substituted with morphological linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is substituted with P(O)S ("thioate"), P(S)S ("dithioate"), (O)NRi ("amidate"), P(O)R, P(O)OR', or CH2 ("formacetal"), where R or R' is independently a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralgyl, optionally containing H or an ether (-O-) bond. Not all bonds in the polynucleotide need to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA. In some embodiments, the nucleic acids or polynucleotides disclosed herein have a reduced number of CpG dinucleotides compared to their parental equivalents. In some embodiments, the nucleic acids or polynucleotides disclosed herein are depleted of CpG dinucleotides compared to their parental equivalents (i.e., all CpG dinucleotides are modified so that they are no longer CpG dinucleotides, or all CpG dinucleotides are deleted). In some embodiments, the nucleic acids or polynucleotides disclosed herein are increased in methylated CpG dinucleotides.

[0039] The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable in mammals, particularly humans or animal patients.

[0040] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector derived from AAV and containing one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) adjacent to at least one AAV ITR. In some embodiments, such an rAAV vector, when introduced into a host cell that has (or expresses) appropriate helper polynucleotides or viruses and expresses AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins), replicates and packages into viral particles. When an rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in other vectors such as plasmids used for cloning or transfection), this rAAV vector is called a "provector" and is "rescued" by replication and capsid formation in the presence of AAV packaging function and appropriate helper function.

[0041] As used herein, the term “treatment” (also “treat” or “treating”) means any administration of a substance or composition (e.g., an AAV vector as described herein) that partially or completely alleviates, improves, helps survive, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or illness (e.g., cancer). Such treatment may be for subjects who do not show signs of the disease, disorder, and / or illness in question, or for subjects who show only initial signs of the disease, disorder, and / or illness. Alternatively, such treatment may be for subjects who show one or more established signs of the disease, disorder, and / or illness in question. In some embodiments, the treatment may be for subjects who have been diagnosed with the disease, disorder, and / or illness in question. In some embodiments, the treatment may be for subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, and / or illness in question.

[0042] As used herein, “operably linked,” “operably linked,” “operatably linked,” or their grammatical equivalents refer to the arrangement of genetic elements, such as promoters, enhancers, polyadenylated sequences, etc., where the action of a first genetic element (e.g., migration or activation) has some effect on a second genetic element. The effect on the second genetic element may, but does not have to be, the same type of action as that of the first genetic element. For example, two genetic elements are operationally linked if the migration of the first element causes the activation of the second element. For example, a regulatory element, which may include a promoter and / or enhancer, is operationally linked to a coding region if the regulatory element assists in the initiation of transcription of a coding sequence. Intervening residues may exist between the regulatory element and the coding region as long as this functional relationship is maintained.

[0043] As used herein, the terms “peptide,” “polypeptide,” or “protein” refer to a chain of amino acids. As used herein, the term “protein” further refers to a larger molecule containing one or more chains of amino acids, and in some embodiments, a protein fragment or domain or a full-length protein. Furthermore, as used herein, the term “protein” refers to either a linear chain of amino acids or a chain of amino acids that has been processed and folded into a functional protein. Protein structure can be divided into four distinct levels: (1) primary structure – referring to the sequence of amino acids in a polypeptide chain; (2) secondary structure – referring to regular local substructures on the polypeptide backbone, such as α-helices and β-sheets; (3) tertiary structure – referring to the three-dimensional structure in the case of monomeric and polymeric protein molecules; and (4) quaternary structure – referring to a three-dimensional structure that includes the aggregation of two or more individual polypeptide chains acting as a single functional unit. The use of peptide or polypeptide herein does not imply that a chain of amino acids is not a protein (i.e., a chain of amino acids having a secondary, tertiary, or quaternary structure).

[0044] As used herein, the term “wild type” refers to a naturally occurring form of organism, strain, gene, nucleic acid, vector, or vector component that is distinguishable from a mutant or variant form.

[0045] The recombinant adeno-associated virus (rAAV) vector disclosed herein Cytokines are regulators of innate and adaptive immunity that enable signal transduction within the immune system. Due to their ability to recognize and destroy cancer cells in the immune system, there is considerable interest in using cytokines for cancer treatment. However, current cytokine therapies exhibit negative characteristics, including toxicity from systemic administration, short half-lives, and a lack of specificity. Improvements in cytokine therapy for treating cancers (e.g., glioblastoma, metastatic brain tumors, uveal melanoma) are needed.

[0046] This specification provides compositions and methods for the delivery of a target cytokine payload via rAAV, enabling targeted, low-dose, and direct administration to tumors and the tumor microenvironment. Local tumor delivery and low-dose administration can result in reduced local and systemic toxicity, for example, by bypassing neutralizing antibodies generated during systemic administration of rAAV. Furthermore, the rAAVs described herein have longer half-lives, enabling long-term and sustained stable expression, and are also self-limiting, ceasing with tumor death via payload activity. The compositions and methods described herein can further provide reduction of nonspecific inflammation by being optimized immunologically and for vector integrity.

[0047] An exemplary workflow of the mechanism of rAAV described herein is found in Figure 1. The rAAV described herein enables local delivery to the tumor (1), which then leads to the expression of engineered cytokines and direct tumor cell lysis in tumor cells. As a result, inflammatory cytokines are released, triggering an innate immune response (3), activating 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 and local and systemic toxicity is reduced.

[0048] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are described herein, comprising, from 5' to 3', a) a promoter, b) a polynucleotide encoding a payload of interest, and c) a polynucleotide containing a regulatory element. In some embodiments, the regulatory element is an inducible regulatory element (e.g., an inducible system such as a splice modulator binding site and / or promoter). In some embodiments, the regulatory element is a constitutively active regulatory element.

[0049] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for interferon, d) a WPRE sequence, e) an SV40 polyA sequence, and f) a second ITR sequence.

[0050] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are further disclosed herein, comprising, from 5' to 3', a) a promoter and b) a polynucleotide encoding a payload of interest, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

[0051] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for CpG-depleted interferon, d) a WPRE sequence, e) an SV40 polyA sequence, and f) a second ITR sequence.

[0052] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a coding sequence for CpG-depleted interferon, d) a WPRE sequence, e) a bGH polyA sequence, and f) a second ITR sequence.

[0053] In certain embodiments, recombinant adeno-associated virus (rAAV) vectors are further disclosed herein, comprising, from 5' to 3', a) a promoter, b) a minigene including a splice modulator binding site, and c) a polynucleotide encoding a payload of interest, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

[0054] In certain embodiments, a recombinant adeno-associated virus (rAAV) vector is further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a minigene described in Sequence ID No. 31, d) a coding sequence for CpG-depleted human interferon, e) a WPRE sequence, f) an SV40 polyA sequence, and g) a second ITR sequence.

[0055] In certain embodiments, a recombinant adeno-associated virus (rAAV) vector is further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a minigene described in Sequence ID No. 51, d) a coding sequence for CpG-depleted interferon, e) a WPRE sequence, f) an SV40 polyA sequence, and g) a second ITR sequence.

[0056] In certain embodiments, rAAV vectors are further disclosed herein, comprising, from 5' to 3', a) a promoter, b) a regulatory element including a splice modulator binding site, and c) a polynucleotide encoding the payload of interest, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

[0057] In certain embodiments, an rAAV vector is further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a regulatory element described in Sequence ID No. 31, d) a coding sequence for CpG-depleted interferon, e) a WPRE sequence, f) a bGH poly-A sequence, and g) a second ITR sequence.

[0058] In certain embodiments, an rAAV vector is further disclosed herein, comprising, from 5' to 3', a) a first ITR sequence, b) a CAG promoter, c) a regulatory element described in Sequence ID No. 51, d) a coding sequence for CpG-depleted interferon, e) a WPRE sequence, f) a bGH poly-A sequence, and g) a second ITR sequence.

[0059] CpG dinucleotide modification In certain embodiments, recombinant adeno-associated virus (rAAV) vectors comprising a reduction in CpG dinucleotides compared to a parental equivalent are described herein. In some embodiments, the rAAV vector comprises increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the rAAV vector comprises depletion of CpG dinucleotides compared to a parental equivalent. In some embodiments, the rAAV vector comprises a reduction in CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, CpG dinucleotides are depleted or reduced if one or more nucleotides of the CpG dinucleotide are replaced with one or more different nucleotides, resulting in the sequence no longer being a CpG dinucleotide sequence. In some embodiments, if CpG dinucleotides of an rAAV containing a coding sequence (e.g., for a regulatory element, a gene (e.g., a transgene), an antibiotic resistance gene, etc.) are replaced with one or more different nucleotides, the one or more nucleotides are selected so as to preserve the function of the coding sequence. In other words, if the coding sequence is a regulator such as a promoter or enhancer, the function of the promoter or enhancer is preserved, or if the coding sequence codes for a polypeptide, the amino acid sequence of the polypeptide and / or the polypeptide function are preserved.

[0060] In some embodiments, the rAAV vector includes a reduction in CpG dinucleotides compared to its 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 its 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 dinucleotide is reduced in the range of approximately 10% to approximately 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 dinucleotide is reduced in the range of approximately 15% to approximately 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 dinucleotide is reduced in the range of approximately 20% to approximately 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 dinucleotide is reduced in the range of approximately 25% to approximately 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 dinucleotide is reduced in the range of approximately 30% to approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, CpG dinucleotides decrease in the range of approximately 40%–45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 50%–55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 60%–65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 90%–95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by at least approximately 50%. In some embodiments, CpG dinucleotides are reduced by at least approximately 75%.

[0061] In some embodiments, the rAAV vector involves CpG dinucleotide depletion compared to its parental equivalent. In some embodiments, CpG dinucleotides are reduced by the deletion of specific CpG dinucleotides. In some embodiments, CpG dinucleotides are reduced by substituting one or more nucleotides within a CpG dinucleotide to create a sequence that is not a CpG dinucleotide. In some embodiments, CpG dinucleotides are depleted by the deletion of all CpG dinucleotides. In some embodiments, CpG dinucleotides are depleted by substituting one or more nucleotides within each CpG dinucleotide to create a sequence that is not a CpG dinucleotide.

[0062] In some embodiments, the rAAV vector includes increased CpG dinucleotide methylation compared to the parental equivalent. In some embodiments, the CpG dinucleotide methylation 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 dinucleotide methylation is increased 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 methylation of CpG dinucleotides increases 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 methylation of CpG dinucleotides increases 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 methylation of CpG dinucleotides increases 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 methylation of the CpG dinucleotide increases in the range of approximately 30% to approximately 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 increases in the range of approximately 40% to approximately 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 increases in the range of approximately 50% to approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 60% to approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 90%–95%, 96%, 97%, 98%, 99%, or 100%.

[0063] In some embodiments, the CpG dinucleotide is modified throughout the entire length of the provided rAAV vector (e.g., by reduction, depletion, and / or increased methylation). In some embodiments, the CpG dinucleotide is modified only in one or more significant portions or elements (e.g., regulatory elements, polynucleotides containing coding sequences, etc.) of the provided rAAV vector. This disclosure provides, among other things, the insight that when the CpG dinucleotide is modified within the coding sequence of the payload of interest, a particular polynucleotide encoding the payload of interest (e.g., a polynucleotide encoding one or more interferons such as IFNβ) exhibits superior therapeutic outcomes (e.g., increased efficacy, reduced toxicity, etc.). Thus, in one non-limiting example, the rAAV vector provided by this disclosure comprises a polynucleotide encoding an interferon, wherein the coding sequence of the interferon contains a modified CpG dinucleotide (e.g., reduction, depletion, and / or increased methylation) compared to its parental equivalent, but other elements in the adjacent nucleic acid do not have the modified CpG dinucleotide. In another non-limiting example, the rAAV vector provided by this disclosure comprises a polynucleotide encoding an interferon, wherein the interferon encoding sequence comprises a modified CpG dinucleotide (e.g., reduced, depleted, and / or increased methylation) compared to its parental equivalent, and one or more other polynucleotides in the rAAV vector comprises a modified CpG dinucleotide (e.g., a promoter, antibiotic resistance gene, minigene, and / or polynucleotide element as described herein).

[0064] Mini gene regulation In some embodiments, the regulatory element of this disclosure is an inductive regulatory element (e.g., an inductive system such as a splice modulator binding site and / or promoter). In some embodiments, the inductive regulatory element is a minigene. In some embodiments, the regulatory element is a constitutive regulatory element.

[0065] In some embodiments, recombinant adeno-associated virus (rAAV) vectors comprising a minigene containing a splice modulator binding site are described herein. In some embodiments, the minigene is located at the 5' end of a polynucleotide encoding the 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, pseudoexons, fragments thereof, or combinations thereof.

[0066] In some embodiments, a minigene comprises a minimal gene fragment containing at least one exon and regulatory region, or a splice modulator site, necessary for controlling the expression of a downstream polynucleotide sequence. In some embodiments, a minigene comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8) exons, introns, pseudoexons, fragments thereof, or combinations thereof. In many embodiments, the splice modulator site of a minigene controls the splicing outcome of the RNA transcript encoded by the minigene. In some embodiments, the splice modulator site induces the RNA transcript to a specific splicing outcome based on factors present in the context of a particular cell or tissue (e.g., splicing outcome 1 when the minigene is in a stem cell, splicing outcome 2 when the minigene is in a retinal cell, splicing outcome 3 when the minigene is in a kidney cell, etc.). In some embodiments, the splice modulator site induces the RNA transcript to a specific splicing outcome by binding to a splice modulator, for example, in one or more nucleic acid sequences. In some embodiments, the splice modulator is a polypeptide, nucleic acid, or small molecule. In some embodiments, a minigene is used to control the expression of a downstream polynucleotide sequence by manipulating the minigene to allow (or activate) only the expression of the downstream polynucleotide sequence in the presence of a splice modulator and / or in the context of a particular cell type / tissue type.

[0067] In some embodiments, the minigene encodes an in-frame translation stop codon. In some embodiments, the in-frame translation stop codon is removed by alternative splicing of the minigene transcript. In some embodiments, alternative splicing of the minigene transcript modifies the transcript so that the stop codon is deleted or invalidated, an initialization or start codon is introduced, the open reading frame is restored, or a deleted portion of the protein is provided. In some embodiments, alternative splicing of the minigene transcript enables the transcription and translation of a downstream polynucleotide, for example, a polynucleotide encoding a payload of interest as described herein.

[0068] In some embodiments, the polynucleotide downstream of the minigene (e.g., a polynucleotide encoding the payload of interest as described herein) includes a translation termination codon. In some embodiments, the polynucleotide downstream of the minigene does not include a start codon. In some embodiments, the polynucleotide downstream of the minigene does not include an open reading frame.

[0069] In some embodiments, the minigene and the polynucleotide encoding the payload of interest are linked by a cleavable peptide. The cleavable peptide is a self-cleaving peptide, a drug-sensitive protease, or a substrate for an endogenous endoprotease.

[0070] In some embodiments, the minigene includes the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the minigene includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 31.

[0071] In some embodiments, the minigene includes the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the minigene includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 51.

[0072] In some embodiments, the minigene is regulated by a small molecule splicing modifier. In some embodiments, the small molecule splicing modifier is sudemycin (FR901464, prazienolide B), LMI070, RG7916, or RG7800, and their derivatives.

[0073] In some embodiments, the small molecule splicing modifier has the following structure:

[0074] [ka] This is LMI070 (CAS number: 1562338-42-4), which has [specific features / features].

[0075] In some embodiments, the small molecule splice modulator has the following structure:

[0076] [ka] This is RG7916 (Roche / PTC / SMAF,7-(4,7-diazaspiro[2.5]octan-7-yl)-2-(2,8-dimethylimidazo[l,2-b]pyridazin-6-yl)-4H-pyrido[l,2-a]pyrimidine-4-one) (CAS number: 1825352-65-5).

[0077] In some embodiments, the small molecule splice modulator has the following structure:

[0078] [ka] This is the RG7800 (Roche) (CAS number: 1449598-06-4) which has [specific features / features].

[0079] In some embodiments, the small molecule splice modulator is an analogue of RG7916 or RG7800.

[0080] In some embodiments, the small molecule splice modulator is (5',Z)-5-(((lR,4R)-4-((2JE',4JE)-5-((3R,55')-7,7-dimethyl-l,6-dioxaspiro[2.5]octan-5-yl)-3-methylpenta-2,4-dien-l-yl)cyclohexyl)amino)-5-oxopenta-3-en-2-ylmethylcarbame Sudemycin is 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-methylpenta-2,4-dien-l-yl)cyclohexyl)amino)-5-oxopenta-3-en-2-yldimethylcarbamate.

[0081] In some embodiments, the small molecule splice modulator is a prazienolide compound. An example of a prazienolide compound is (8E,12E,14E)-7-((4-cycloheptylpiperazine-l-yl)carbonyl)oxy-3,6,16,21-tetrahydroxy-6,10,12,16,20-pentamethyl-l8,l9-epoxytricosa-8,l2,l4-triene-ll-olido, also known as E7107, which is a semi-synthetic derivative of the natural product prazienolide D.

[0082] In some embodiments, the minigene is regulated by an intracellular disease state. In some embodiments, the disease state is cancer. In some embodiments, the cancer is glioblastoma.

[0083] In some embodiments, the minigenes are regulated by cell type or tissue type.

[0084] Target payload In certain embodiments, polynucleotides encoding the payload of interest are described herein. In some embodiments, the payload of interest is a therapeutic agent, such as a therapeutic polypeptide.

[0085] In some embodiments, a therapeutic agent is any agent that, when administered to an organism, elicits a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, a suitable population is a population of model organisms. In some embodiments, a suitable population is defined by various criteria such as a specific age group, sex, genetic background, or pre-existing clinical condition. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or illness. In some embodiments, a therapeutic agent is an agent that has been approved or requires approval 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 for administration to humans. In some embodiments, a therapeutic agent is a therapeutic polypeptide or therapeutic polynucleotide. In some embodiments, a therapeutic polypeptide is a cytokine (e.g., interferon).

[0086] In some embodiments, the therapeutic polypeptide is a cytokine. In some embodiments, the cytokine is colony-stimulating factor (CSF), transforming growth factor (e.g., transforming growth factor-β), tumor necrosis factor (e.g., tumor necrosis α), interleukin, or interferon. In some embodiments, the cytokine is interferon.

[0087] Some of the key components are interleukin-1α (IL-1α), interleukin-1β (IL-1β), 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).

[0088] In some embodiments, the colony-stimulating factor (CSF) is CSF1 (M-CSF), CSF2 (GM-CSF), or CSF3 (G-CSF).

[0089] In some embodiments, the transforming growth factor (TGF) is TGF-β1, TGF-β2, or TGF-β3.

[0090] In some embodiments, tumor necrosis factor (TNF) is TNF-α, TNF-β, or LT-β.

[0091] In some embodiments, interferon (IFN) is type I IFN, type II IFN, or type III IFN. In some embodiments, IFN is IFN-alpha (IFNα), IFN-beta (IFNβ), IFN-gamma (IFNγ), IFN-epsilon (IFNε), IFN-kappa (IFNκ), IFN-omega (IFNω), IFN-lambda (IFNλ), IFN-chi (IFNχ), IFN-xai (IFNξ), IFN-tau (IFNτ), IFN-delta (IFNδ), IFN-nu (IFNν), IFN-zeta (IFNζ), IFN-alpha (alfa), and their derivatives. In some embodiments, interferon is IFNα, IFNβ, IFNγ, or a combination thereof. In some embodiments, the interferon is IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ, IFNχ, IFNξ, IFNτ, IFNδ, IFNν, IFNζ, IFN-alpha (alfa), or a variant or derivative thereof. In some embodiments, the IFN 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, or a variant or derivative thereof. In some embodiments, the IFN is IFN-β1, IFN-β2, IFN-β3, or a variant or derivative thereof.

[0092] In some embodiments, IFN is from mice, rats, horses, ruminants (e.g., sheep, cattle, goats), primates (e.g., chimpanzees, baboons, gorillas, orangutans, monkeys), dogs, cats, pigs, donkeys, rabbits, fish, flies, humans, non-primate placental mammals, or non-rodent placental mammals. In some embodiments, IFN is human IFNβ. In some embodiments, IFN is human IFNα, human IFNβ, human IFNγ, human IFNω, human IFNε, human IFNκ, human IFNτ, human IFNζ, human IFN-alpha, or variants or derivatives thereof. In some embodiments, IFN is mouse IFNα, mouse IFNβ, mouse IFNγ, or variants or derivatives thereof. In some embodiments, IFN is mouse IFNα, mouse IFNβ, mouse IFNγ, mouse IFNε, mouse IFNκ, mouse IFNω, mouse IFNλ, mouse IFNζ, or a variant or derivative thereof. In some embodiments, IFN is mouse IFNβ. In some embodiments, IFN is canine IFNα, canine IFNβ, canine IFNγ, canine IFNε, canine IFNκ, canine IFNω, canine IFNλ, or a variant or derivative thereof. In some embodiments, IFN is canine IFNβ, or a variant or derivative thereof. In some embodiments, IFN is rat IFNα, rat IFNβ, rat IFNγ, rat IFNε, rat IFNκ, rat IFNω, rat IFNλ, or a variant or derivative thereof. In some embodiments, IFN is rat IFNβ, or a variant or derivative thereof. In some embodiments, IFN is guinea pig IFNα, guinea pig IFNβ, guinea pig IFNγ, guinea pig IFNε, guinea pig IFNκ, guinea pig IFNω, guinea pig IFNλ, or a variant or derivative thereof. In some embodiments, IFN is guinea pig IFNβ, or a variant or derivative thereof. In some embodiments, IFN is IFNδ of non-primate and non-rodent placental mammals, or a variant or derivative thereof.In some embodiments, IFN is IFNε, IFNκ, or variants or derivatives thereof from placental mammals.

[0093] In some embodiments, IFN is a subtype of mouse IFNα. In some embodiments, IFNα is IFNα1, IFNα2, IFNα3, IFNα4, IFNα5, IFNα6, IFNα7, IFNα8, IFNα10, IFNα13, IFNα14, IFNα16, IFNα17, IFNα21, and their derivatives.

[0094] In some embodiments, the therapeutic polypeptide comprises one or more interferons. In some embodiments, the therapeutic polypeptide comprises IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, and IFNα-IFNβ-IFNγ. In some embodiments, the therapeutic polypeptide is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, or IFNα-IFNβ-IFNγ, where IFN is mouse. In some embodiments, the therapeutic polypeptide is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, or IFNα-IFNβ-IFNγ, where IFN is human. In some embodiments, the therapeutic polypeptide is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, or IFNα-IFNβ-IFNγ, where IFN is from a dog. In some embodiments, the therapeutic polypeptide is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, or IFNα-IFNβ-IFNγ, where IFN is from a rat. In some embodiments, the therapeutic polypeptide is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, or IFNα-IFNβ-IFNγ, where IFN is from a guinea pig.

[0095] In some embodiments, human interferon beta (hIFNβ) has a sequence (SEQ ID NO: 1) represented by UniProt / SwissProt database entry number P01574. In some embodiments, hIFNβ is encoded by one of the nucleic acid sequences SEQ ID NOs: 2-4. In some embodiments, hIFNβ 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 one of the nucleic acid sequences SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 75% sequence identity to one of the nucleic acid sequences SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 80% sequence identity to one of the nucleic acid sequences SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 90% sequence identity to one of the nucleic acid sequences SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-4. In some embodiments, hIFNβ is encoded by a nucleic acid sequence having any one of the nucleic acid sequences of SEQ ID NOs: 2-4.

[0096] In some embodiments, the interferon is human IFNβ. In some embodiments, the human 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: 1. In some embodiments, the human IFNβ comprises the amino acid sequence described in SEQ ID NO: 1. In some embodiments, the human IFNβ is encoded by a polynucleotide 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: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the human IFNβ is encoded by a polynucleotide sequence described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0097] In some embodiments, human interferon alpha 1 (hIFNα1) has a sequence (SEQ ID NO: 5) represented by UniProt / SwissProt database entry number P01562. In some embodiments, hIFNα is encoded by one of the nucleic acid sequences SEQ ID NOs: 6-8. In some embodiments, hIFNα 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 one of the nucleic acid sequences SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 75% sequence identity to one of the nucleic acid sequences SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 80% sequence identity to one of the nucleic acid sequences SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 6-8. In some embodiments, hIFNα is encoded by a nucleic acid sequence having any one of the nucleic acid sequences of SEQ ID NOs: 6-8.

[0098] In some embodiments, the interferon is human IFNα. In some embodiments, human IFNα 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, human IFNα comprises the amino acid sequence described in SEQ ID NO: 5. In some embodiments, human IFNα is encoded by a polynucleotide 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: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0099] In some embodiments, 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, human IFNα includes 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: 7. In some embodiments, 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, human IFNα is encoded by the polynucleotide sequence described in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, human IFNα is encoded by the polynucleotide sequence described in SEQ ID NO: 6. In some embodiments, human IFNα is encoded by the polynucleotide sequence described in SEQ ID NO: 7. In some embodiments, human IFNα is encoded by the polynucleotide sequence described in SEQ ID NO: 8.

[0100] In some embodiments, human interferon gamma (hIFNγ) has a sequence (SEQ ID NO: 9) represented by UniProt / SwissProt database entry number P01579. In some embodiments, hIFNγ is encoded by the nucleic acid sequences SEQ ID NOs: 10-12. In some embodiments, hIFNγ 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 sequences SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 75% sequence identity to any one of the nucleic acid sequences SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 80% sequence identity to any one of the nucleic acid sequences SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 10-12. In some embodiments, hIFNγ is encoded by a nucleic acid sequence having any one of the nucleic acid sequences of SEQ ID NOs: 10-12.

[0101] In some embodiments, the interferon is human IFNγ. In some embodiments, the human IFNγ 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 IFNγ comprises the amino acid sequence described in SEQ ID NO: 9. In some embodiments, the human IFNγ is encoded by a polynucleotide 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: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the human IFNγ 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, human IFNγ 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 with SEQ ID NO: 11. In some embodiments, human IFNγ 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 with SEQ ID NO: 12. In some embodiments, human IFNγ is encoded by the polynucleotide sequence described in SEQ ID NO: 10. In some embodiments, human IFNγ is encoded by the polynucleotide sequence described in SEQ ID NO: 11. In some embodiments, human IFNγ is encoded by the polynucleotide sequence described in SEQ ID NO: 12.

[0102] In some embodiments, the interferon is mouse interferon beta (mIFNβ). In some embodiments, mouse interferon beta (hIFNβ) has a sequence (SEQ ID NO: 13) represented by UniProt / SwissProt database entry number P01575. In some embodiments, mIFNβ is encoded by the nucleic acid sequence SEQ ID NO: 14 or 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 to the nucleic acid sequence SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14 or 42. In some embodiments, mIFNγ is encoded by a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 14 or 42.

[0103] In some embodiments, the interferon is mouse IFNβ. In some embodiments, the mouse 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: 13. In some embodiments, the mouse IFNβ comprises the amino acid sequence described in SEQ ID NO: 13. In some embodiments, the mouse 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: 14. In some embodiments, the mouse 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: 42. In some embodiments, the mouse IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 14. In some embodiments, human IFNγ is encoded by the polynucleotide sequence described in SEQ ID NO: 42.

[0104] In some embodiments, the interferon is canine interferon beta (cIFNβ). In some embodiments, canine interferon beta (cIFNβ) has a sequence (sequence number 15) represented by UniProt / UniProtKB database entry number B6E116. In some embodiments, the interferon is canine interferon beta (cIFNβ) and is encoded by the nucleic acid sequence of sequence number 16, 43, or 60. In some embodiments, cIFNβ 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 sequence number 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of sequence number 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNβ is encoded by a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, 43, or 60. In some embodiments, cIFNγ is encoded by a nucleic acid sequence having the nucleic acid sequence of sequence number 16, 43, or 60.

[0105] In some embodiments, the interferon is canine IFNβ. In some embodiments, the canine 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: 15. In some embodiments, the canine IFNβ comprises the amino acid sequence described in SEQ ID NO: 15. In some embodiments, the canine 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: 16. In some embodiments, the canine 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: 43. In some embodiments, canine 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 with respect to SEQ ID NO: 60. In some embodiments, canine IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 16. In some embodiments, canine IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 43. In some embodiments, canine IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 60.

[0106] In some embodiments, the interferon is rat IFNβ. In some embodiments, the rat IFNβ contains 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 with SEQ ID NO: 61. In some embodiments, the rat IFNβ contains the amino acid sequence described in SEQ ID NO: 61.

[0107] In some embodiments, 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, 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, rat IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 62. In some embodiments, rat IFNβ is encoded by the polynucleotide sequence described in SEQ ID NO: 63.

[0108] 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 with SEQ ID NO: 71. In some embodiments, the guinea pig IFNβ comprises the amino acid sequence described in SEQ ID NO: 71.

[0109] In some embodiments, 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 with respect to SEQ ID NO: 72.

[0110] In some embodiments, the polynucleotide encoding interferon is codon-optimized. In some embodiments, the polynucleotide encoding interferon is optimized for interferon expression. By using codon optimization, it is possible to increase the translation rate or produce recombinant RNA transcripts with desired properties, such as superior expression efficiency, compared to transcripts produced using non-optimized sequences. In certain embodiments, the polynucleotide encoding interferon is codon-optimized for expression in mammalian and human cells.

[0111] In some embodiments, recombinant adeno-associated virus (rAAV) vectors are disclosed herein that include a polynucleotide encoding a payload of interest (e.g., a cytokine such as any interferon described herein), wherein the encoding sequence of the payload of interest includes a modified CpG dinucleotide (e.g., reduced, depleted, and / or increased methylation) compared to its parental equivalent. In one non-limiting example, the rAAV vector includes a polynucleotide encoding an interferon (e.g., IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ, IFNχ, IFNξ, IFNτ, IFNδ, IFNν, IFNζ, IFN-alpha (alfa), or a variant or derivative thereof), wherein the encoding sequence of the interferon includes a modified CpG dinucleotide (e.g., reduced, depleted, and / or increased methylation) compared to its parental equivalent. In some embodiments, the polynucleotide encoding the interferon includes a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent. In some embodiments, the CpG dinucleotides are fully methylated. In some embodiments, the CpG dinucleotides are depleted.

[0112] In some embodiments, the polynucleotide encoding the payload of interest includes a reduction in 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 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 dinucleotide is reduced in the range of approximately 10% to approximately 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 dinucleotide is reduced in the range of approximately 15% to approximately 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 dinucleotide is reduced in the range of approximately 20% to approximately 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 dinucleotide is reduced in the range of approximately 25% to approximately 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 dinucleotide is reduced in the range of approximately 30% to approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, CpG dinucleotides decrease in the range of approximately 40%–45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 50%–55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 60%–65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by approximately 90%–95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides are reduced by at least approximately 50%. In some embodiments, CpG dinucleotides are reduced by at least approximately 75%.

[0113] In some embodiments, the polynucleotide encoding the desired payload includes a depletion of CpG dinucleotides compared to its parental equivalent.

[0114] In some embodiments, CpG dinucleotides are reduced by the deletion of a specific CpG dinucleotide. In some embodiments, CpG dinucleotides are reduced by substituting one or more nucleotides within a CpG dinucleotide to create a sequence that is not a CpG dinucleotide. In some embodiments, CpG dinucleotides are depleted by the deletion of all CpG dinucleotides. In some embodiments, CpG dinucleotides are depleted by substituting one or more nucleotides within each CpG dinucleotide to create a sequence that is not a CpG dinucleotide.

[0115] In some embodiments, the polynucleotide encoding the payload of interest includes increased methylation of the CpG dinucleotide compared to its parental equivalent. In some embodiments, 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 its parental equivalent. In some embodiments, the methylation of CpG dinucleotides increases 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 methylation of the CpG dinucleotide increases 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 methylation of the CpG dinucleotide increases 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 methylation of CpG dinucleotides increases in the range of approximately 25% to approximately 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 increases in the range of approximately 30% to approximately 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 increases in the range of approximately 40% to approximately 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 increases in the range of approximately 50% to approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 60% to approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 90%–95%, 96%, 97%, 98%, 99%, or 100%.

[0116] In some embodiments, polynucleotides encoding interferon (including, for example, IFNα, IFNβ, IFNγ, IFNε, IFNκ, IFNω, IFNλ, IFNχ, IFNξ, IFNτ, IFNδ, IFNν, IFNζ, IFN-alpha, variants or derivatives thereof from mouse, human, dog, cat, ruminant, rat, guinea pig, primate, pig, or ferret) include a reduction in CpG dinucleotides compared to their parental equivalents. 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 99% compared to their parental equivalents. In some embodiments, the CpG dinucleotide is reduced in the range of approximately 5% to approximately 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 dinucleotide is reduced in the range of approximately 10% to approximately 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 dinucleotide is reduced in the range of approximately 15% to approximately 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 dinucleotide is reduced in the range of approximately 20% to approximately 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 dinucleotide is reduced in the range of approximately 25% to approximately 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 dinucleotide is reduced in the range of approximately 30% to approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 50%–55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 60%–65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, CpG dinucleotides decrease in the range of approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotide is reduced by about 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotide is reduced by about 90%–95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the CpG dinucleotide is reduced by at least about 50%. In some embodiments, the CpG dinucleotide is reduced by at least about 75%.

[0117] In some embodiments, the polynucleotide encoding the interferon includes a depletion of CpG dinucleotides compared to its parental equivalent.

[0118] In some embodiments, CpG dinucleotides are reduced by the deletion of a specific CpG dinucleotide. In some embodiments, CpG dinucleotides are reduced by substituting one or more nucleotides within a CpG dinucleotide to create a sequence that is not a CpG dinucleotide. In some embodiments, CpG dinucleotides are depleted by the deletion of all CpG dinucleotides. In some embodiments, CpG dinucleotides are depleted by substituting one or more nucleotides within each CpG dinucleotide to create a sequence that is not a CpG dinucleotide.

[0119] In some embodiments, the polynucleotide encoding the interferon includes increased methylation of the CpG dinucleotide compared to its parental equivalent. In some embodiments, 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 its parental equivalent. In some embodiments, the methylation of CpG dinucleotides increases 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 methylation of the CpG dinucleotide increases 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 methylation of the CpG dinucleotide increases 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 methylation of CpG dinucleotides increases in the range of approximately 25% to approximately 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 increases in the range of approximately 30% to approximately 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 increases in the range of approximately 40% to approximately 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 increases in the range of approximately 50% to approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 60% to approximately 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 70%–75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 80%–85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the methylation of CpG dinucleotides increases in the range of approximately 90%–95%, 96%, 97%, 98%, 99%, or 100%.

[0120] In some embodiments, the rAAV vector described herein comprises one or more polynucleotides encoding one or more interferons. In some embodiments, one or more polynucleotides encode polypeptides separated by self-cleaving peptides. 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 the first self-cleaving peptide. Optionally, a linker sequence (e.g., Gly-Ser-Gly) is located 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 located upstream (3') of the polynucleotide encoding the second linker peptide.

[0121] Suitable self-cleaving peptides include 2A self-cleaving peptides such as P2A self-cleaving peptides, T2A self-cleaving peptides, F2A self-cleaving peptides, or E2A self-cleaving peptides. In some embodiments, the self-cleaving peptide is a P2A self-cleaving peptide having the sequence of Sequence ID No. 17.

[0122] 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 P2A 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 Sequence ID No. 18.

[0123] Vector and virus packaging In some embodiments, the polynucleotides of this disclosure (e.g., polynucleotides encoding the payload of interest) are delivered by a vector. In some embodiments, the polynucleotides are delivered by plasmids (e.g., circular DNA molecules capable of autonomously replicating within cells), cosmids (e.g., pWE or sCOs vectors), artificial chromosomes, human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), minicircles, doggybone, nanoplasmids, P1-derived artificial chromosomes (PAC), phagemids, phage derivatives, bacmids, or viruses. In some embodiments, the vectors are 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 The following are selected from the list consisting of 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.

[0124] In some embodiments, the plasmid comprises a polynucleotide and an antibiotic resistance gene (e.g., any antibiotic resistance gene 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., promoter, enhancer, WPRE, and / or poly-A, etc.) are operably ligated to one or more coding polynucleotides in the plasmid (e.g., polynucleotides encoding the payload of interest).

[0125] In some embodiments, the polynucleotides of the Disclosure (e.g., polynucleotides encoding a payload of interest) are delivered by a virus. In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anerovirus, vacciniavirus, or 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 anerovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a poliovirus. In some embodiments, the virus is a vacciniavirus. In some embodiments, the virus is a retrovirus.

[0126] 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- The herpesvirus is 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 herpesvirus is HSV1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8.

[0127] In some embodiments, AAV is AAV1 or a derivative thereof. In some embodiments, AAV is AAV2 or a derivative thereof. In some embodiments, AAV is AAV3 or a derivative thereof. In some embodiments, AAV is AAV4 or a derivative thereof. In some embodiments, AAV is AAV5 or a derivative thereof. In some embodiments, AAV is AAV6 or a derivative thereof. In some embodiments, AAV is AAV7 or a derivative thereof. In some embodiments, AAV is AAV8 or a derivative thereof. In some embodiments, AAV is AAV9 or a derivative thereof. In some embodiments, AAV is AAV10 or a derivative thereof. In some embodiments, AAV is AAV11 or a derivative thereof. In some embodiments, AAV is AAV12 or a derivative thereof. In some embodiments, AAV is AAV13 or a derivative thereof. In some embodiments, AAV is AAV14 or a derivative thereof. In some embodiments, AAV is AAV15 or a derivative thereof. In some embodiments, AAV is AAV16 or a derivative thereof. In some embodiments, AAV is AAV-rh8 or a derivative thereof. In some embodiments, AAV is AAV-rh10 or a derivative thereof. In some embodiments, AAV is AAV-rh20 or a derivative thereof. In some embodiments, AAV is AAV-rh39 or a derivative thereof. In some embodiments, AAV is AAV-rh74 or a derivative thereof. In some embodiments, AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, AAV is AAV-hu37 or a derivative thereof. In some embodiments, AAV is AAV-Anc80 or a derivative thereof. In some embodiments, AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, AAV is AAV-7m8 or a derivative thereof. In some embodiments, AAV is AAV-PHP-B or a derivative thereof. In some embodiments, AAV is AAV-PHP-EB or a derivative thereof.In some embodiments, AAV is AAV-2.5 or a derivative thereof. In some embodiments, AAV is AAV-2tYF or a derivative thereof. In some embodiments, AAV is AAV-3B or a derivative thereof. In some embodiments, AAV is AAV-LK03 or a derivative thereof. In some embodiments, AAV is AAV-HSC1 or a derivative thereof. In some embodiments, AAV is AAV-HSC2 or a derivative thereof. In some embodiments, AAV is AAV-HSC3 or a derivative thereof. In some embodiments, AAV is AAV-HSC4 or a derivative thereof. In some embodiments, AAV is AAV-HSC5 or a derivative thereof. In some embodiments, AAV is AAV-HSC6 or a derivative thereof. In some embodiments, AAV is AAV-HSC7 or a derivative thereof. In some embodiments, AAV is AAV-HSC8 or a derivative thereof. In some embodiments, AAV is AAV-HSC9 or a derivative thereof. In some embodiments, AAV is AAV-HSC10 or a derivative thereof. In some embodiments, AAV is AAV-HSC11 or a derivative thereof. In some embodiments, AAV is AAV-HSC12 or a derivative thereof. In some embodiments, AAV is AAV-HSC13 or a derivative thereof. In some embodiments, AAV is AAV-HSC14 or a derivative thereof. In some embodiments, AAV is AAV-HSC15 or a derivative thereof. In some embodiments, AAV is AAV-TT or a derivative thereof. In some embodiments, AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, AAV is AAV-Myo or a derivative thereof. In some embodiments, AAV is AAV-NP40 or a derivative thereof. In some embodiments, AAV is AAV-NP59 or a derivative thereof. In some embodiments, AAV is AAV-NP22 or a derivative thereof. In some embodiments, AAV is AAV-NP66 or a derivative thereof. In some embodiments, AAV is AAV-HSC16 or a derivative thereof.

[0128] 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.

[0129] Adjustment element In some embodiments, the recombinant adeno-associated virus (rAAV) vectors of this disclosure include one or more regulatory elements. In some embodiments, one or more regulatory elements are operably ligated to a polynucleotide containing 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 elements facilitate specific functions (e.g., RNA splicing, translation initiation, translation termination, etc.) during and / or after transcription of an mRNA transcript. In some embodiments, the regulatory elements facilitate specific functions when present on a DNA template. In some embodiments, the regulatory elements facilitate specific functions when present on an RNA template. In some embodiments, the regulatory elements are selected from the group consisting of a promoter, enhancer, terminator sequence, mRNA stability sequence, sequence enabling internal ribosome entry sites (IRES) for bicistronic mRNA, sequence inhibiting viral recognition (e.g., by Toll-like or RIG-like receptors such as TLR7, TLR8, TLR9, MDA5, RIG1, and / or DAI), sequence required for transduction into cells, introns, synthetic introns, exons, synthetic exons, and polyA signaling elements. In some embodiments, the rAAV vector includes a promoter, enhancer, intron, microRNA, linker, splicing elements, sequence enabling internal ribosome entry sites (IRES) for bicistronic mRNA, polyA signaling sequence, or a combination thereof. In some embodiments, the sequence enabling internal ribosome entry sites (IRES) for bicistronic mRNA is a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In some embodiments, rAAV includes a promoter. In some embodiments, the rAAV vector includes an enhancer. In some embodiments, the rAAV vector includes introns. In some embodiments, the rAAV vector includes synthetic introns. In some embodiments, the rAAV vector includes microRNA. In some embodiments, the rAAV vector includes a linker. In some embodiments, the rAAV vector includes a splicing element.In some embodiments, the rAAV vector includes a poly-A signal sequence.

[0130] In some embodiments, the polyA signal sequence is derived from SV40. In some embodiments, the polyA signal sequence includes the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the polyA signal sequence is derived from bovine growth hormone (bGH). Other suitable polyA signals include synthetic polyA signals, polyA derived from human growth hormone (hGH), rabbit beta-globin (RGB), or modified RGB (mRGB).

[0131] In some embodiments, the polyA sequence is selected from SV40, hGH, bGH, rbGlob, and their derivatives and variants. In some embodiments, the polyA sequence is an SV40 sequence or a derivative or variant thereof. In some embodiments, the SV40 sequence includes the polynucleotide described in SEQ ID NO: 19. In some embodiments, the polyA sequence is a bGH sequence or a derivative or variant thereof. In some embodiments, the bGH sequence includes the polynucleotide sequence described in SEQ ID NO: 34. In some embodiments, the polyA sequence is a bGH sequence or a derivative or variant thereof. In some embodiments, the polyA sequence is an rbGlob sequence or a derivative or variant thereof.

[0132] In some embodiments, the rAAV vector includes a promoter. In some embodiments, the promoter is selected from the group consisting of mini-promoters, inducible promoters, constitutive promoters, and derivatives thereof. In some embodiments, the constitutive promoter includes 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 the CMV promoter or a derivative thereof. In some embodiments, the promoter is the CBA promoter or a derivative thereof. In some embodiments, the promoter is the EF1a promoter or a derivative thereof. In some embodiments, the promoter is the CAG promoter or a derivative thereof. In some embodiments, the promoter is the PGK promoter or a derivative thereof. In some embodiments, the promoter is the TRE promoter or a derivative thereof. In some embodiments, the promoter is the U6 promoter or a derivative thereof. In some embodiments, the promoter is the UAS promoter or a derivative thereof. In some embodiments, the promoter is the T7 promoter or a derivative thereof. In some embodiments, the promoter is the Sp6 promoter or a derivative thereof. In some embodiments, the promoter is the lac promoter or a derivative thereof. In some embodiments, the promoter is the araBad promoter or a derivative thereof. In some embodiments, the promoter is the trp promoter or a derivative thereof. In some embodiments, the promoter is the Ptac promoter or a derivative thereof. In some embodiments, the promoter is the p5 promoter or a derivative thereof. In some embodiments, the promoter is the p19 promoter or a derivative thereof. In some embodiments, the promoter is the 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.

[0133] In some embodiments, the promoter is a CAG promoter. The CAG promoter is a hybrid of a cytomegalovirus (CMV) early enhancer element and a chicken beta-actin (CBA) promoter, comprising (1) a CMV early enhancer element, (2) a CBA promoter element containing the first exon and first intron of the CBA gene, and (3) a splice acceptor element of the rabbit beta-globin gene. In some embodiments, the CMV early enhancer element is derived from a wild-type CMV enhancer. In some embodiments, the CMV early enhancer element is cleaved with respect 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 respect to SEQ ID NO: 48. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 49. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 50. In some embodiments, the CAG promoter includes the polynucleotide described in SEQ ID NO: 48. In some embodiments, the CAG promoter includes the polynucleotide described in SEQ ID NO: 49. In some embodiments, the CAG promoter includes the polynucleotide described in SEQ ID NO: 50. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 22.In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 23. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 24. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 64. In some embodiments, the CAG promoter includes 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 respect to SEQ ID NO: 65.

[0134] In some embodiments, the CMV initial 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 respect to SEQ ID NO: 25.

[0135] In some embodiments, the CMV initial 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 respect to SEQ ID NO: 26. In some embodiments, the CMV initial enhancer element comprises the polynucleotide sequence described in SEQ ID NO: 26.

[0136] In some embodiments, the CMV initial 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 respect to SEQ ID NO: 27. In some embodiments, the CMV initial enhancer element comprises the polynucleotide sequence described in SEQ ID NO: 27.

[0137] 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 with SEQ ID NO: 28. In some embodiments, the promoter element comprises the polynucleotide sequence described 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 with SEQ ID NO: 52. In some embodiments, the promoter element comprises the polynucleotide sequence described in SEQ ID NO: 52.

[0138] In some embodiments, the splice acceptor is derived from the rabbit beta-globin gene. In some embodiments, the splice acceptor refers to an intron containing the splice acceptor polynucleotide sequence. In some embodiments, the splice acceptor contains 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: 30. In some embodiments, the splice acceptor contains the polynucleotide sequence described in SEQ ID NO: 30. In some embodiments, the splice acceptor contains 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: 53. In some embodiments, the splice acceptor contains the polynucleotide sequence described in SEQ ID NO: 53.

[0139] In some embodiments, the polynucleotide sequence enabling the internal ribosome entry site (IRES) of 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 sequence comprises the nucleic acid sequence described 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 the amino acid sequence described in SEQ ID NO: 33.

[0140] Inverted terminal repeat sequence Inverted terminal repeat (ITR) sequences typically contain 145 bases each (i.e., both 5'ITR sequences and 3'ITR sequences each contain 145 bases).

[0141] In some embodiments, the recombinant adeno-associated virus (rAAV) vector described herein 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 the 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 including a splice modulator site, d) a polynucleotide encoding the payload of interest, and e) a second ITR sequence.

[0142] In some embodiments, the first ITR sequence and / or the second ITR sequence are cleaved compared to their corresponding wild-type ITR sequence or parental equivalent. In some embodiments, the first ITR sequence and / or the second ITR sequence are cleaved at the 5' or 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 cleaved at the 5' end by about 5 nucleotides. In some embodiments, the first ITR sequence is cleaved at the 5' end by about 10 nucleotides. In some embodiments, the first ITR sequence is cleaved at the 5' end by about 15 nucleotides. In some embodiments, the first ITR sequence is cleaved at the 5' end by about 20 nucleotides. In some embodiments, the first ITR sequence is cleaved at its 5' end by approximately 25 nucleotides. In some embodiments, the first ITR sequence is cleaved at its 5' end by approximately 30 nucleotides. In some embodiments, the first ITR sequence is cleaved at its 5' end by 15 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 5 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 10 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 15 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 20 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 25 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by approximately 30 nucleotides. In some embodiments, the second ITR sequence is cleaved at its 3' end by 15 nucleotides. In some embodiments, the first ITR sequence is modified to promote the formation of a self-complementary AAV genome (scAAV). In some embodiments, the second ITR sequence is modified to promote the formation of a self-complementary AAV genome.

[0143] In some embodiments, the first ITR sequence and / or the second ITR sequence are 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, It contains an ITR derived from an AAV serotype selected from the group consisting of 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 their derivatives.

[0144] In some embodiments, the first ITR sequence and / or the second ITR sequence is an AAV ITR sequence or derived from an AAV ITR sequence. In some embodiments, the first ITR sequence is an AAV2 ITR sequence or derived from an AAV2 ITR sequence. In some embodiments, the second ITR sequence is an AAV2 ITR sequence or derived from an AAV2 ITR sequence.

[0145] In some embodiments, the first ITR sequence and / or the second ITR sequence include a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with respect 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 include a nucleic acid sequence having the sequence identity described 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 includes the nucleic acid sequence described in SEQ ID NO: 36. In some embodiments, the first ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 37. In some embodiments, the first ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 38. In some embodiments, the first ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 39. In some embodiments, the first ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 40. In some embodiments, the first ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 41. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 36. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 37. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 38. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 39. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 40. In some embodiments, the second ITR sequence includes the nucleic acid sequence described in SEQ ID NO: 41.

[0146] Additional nucleic acid elements In some embodiments, the recombinant adeno-associated virus (rAAV) vector described herein further comprises 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.

[0147] In some embodiments, the rAAV vector further includes an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes resistance to aminoglycosides, β-lactams, macrolides, tetracyclines, or their derivatives. In some embodiments, the rAAV vector includes an antibiotic resistance gene to aminoglycosides or their derivatives. In some embodiments, the rAAV vector includes an antibiotic resistance gene to β-lactams or their derivatives. In some embodiments, the rAAV vector includes an antibiotic resistance gene to macrolides or their derivatives. In some embodiments, the rAAV vector includes an antibiotic resistance gene that confers resistance to tetracyclines or their derivatives.

[0148] In some embodiments, the antibiotic resistance gene confers resistance to kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeosin, or derivatives thereof.

[0149] In some embodiments, the ampicillin resistance gene includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 44.

[0150] In some embodiments, the ampicillin resistance gene includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 55.

[0151] In some embodiments, the kanamycin resistance gene includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 20 or 21.

[0152] In some embodiments, the antibiotic resistance gene is operably ligated to the promoter. In some embodiments, the promoter includes the polynucleotide sequence described in SEQ ID NO: 45, SEQ ID NO: 46, or SEQ ID NO: 47. In some embodiments, the promoter includes the polynucleotide sequence described in SEQ ID NO: 45. In some embodiments, the promoter includes the polynucleotide sequence described in SEQ ID NO: 46. In some embodiments, the promoter includes the polynucleotide sequence described in SEQ ID NO: 47. In some embodiments, the promoter includes 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.

[0153] In some embodiments, the antibiotic resistance gene (e.g., any antibiotic resistance gene described herein, e.g., kanamycin resistance gene) includes a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to the parental equivalent.

[0154] In some embodiments, methylation of the CpG dinucleotide in an antibiotic resistance gene (e.g., any antibiotic resistance gene 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 the parental equivalent. In some embodiments, the CpG dinucleotide is completely methylated.

[0155] In some embodiments, the CpG dinucleotide in an antibiotic resistance gene (e.g., any antibiotic resistance gene described herein, e.g., a kanamycin resistance gene) 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 99% compared to its parental equivalent. In some embodiments, the CpG dinucleotide in the antibiotic resistance gene is depleted.

[0156] In some embodiments, the rAAV vector further includes an origin of replication. In some embodiments, the origin of replication is derived from plasmids 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.

[0157] In some embodiments, the rAAV vector comprises a woodchuck hepatitis virus post-transcriptional regulator (WPRE) or a derivative thereof. In some embodiments, the WPRE is a mut6 variant of WPRE.

[0158] The rAAV vectors described herein, in some embodiments, include, but are not limited to, reporter sequences for co-expression such as 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 vector includes a selection marker.

[0159] In some embodiments, the reporter sequence is an 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.

[0160] 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 sequence number 58 or 59.

[0161] In some embodiments, the reporter sequence is fLuc. In some embodiments, fLuc comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 66-68.

[0162] In some embodiments, the reporter sequence is nLuc. In some embodiments, nLuc comprises a polynucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with SEQ ID NOs. 69-70.

[0163] Recombinant viral vectors In some embodiments, the recombinant adeno-associated virus (rAAV) vector comprises any polynucleotide or any plasmid described herein. In some embodiments, the viral vector is derived from an anerovirus. In some embodiments, the viral vector is derived from double-stranded DNA viruses, including parvoviruses (e.g., AAV and bocavirus), retroviruses (e.g., retroviridae family viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), aneroviruses, arenaviruses, coronaviruses, single-stranded negative-sense RNA viruses, e.g., orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (measles and Sendai), single-stranded positive-sense RNA viruses, e.g., picornaviruses and alphaviruses (e.g., poliovirus), as well as 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, Togavirus, Flavivirus, Reovirus, Papovavirus, Hepadnavirus, Human Papillomavirus, Human Formyvirus, and Hepatitis viruses. Examples of retroviruses are Avian leukemia / sarcoma, Avian type C virus, Mammalian type C, B, and D viruses, Onchoretrovirus, HTLV-BLV group, Lentivirus, Alpharetrovirus, Gammaretrovirus, and Supumavirus. Other examples include Mouse leukemia virus, Mouse sarcoma virus, Mouse mammary tumor virus, Bovine leukemia virus, Feline leukemia virus, Feline sarcoma virus, Avian leukemia virus, Human T-cell leukemia virus, Baboon endogenous virus, Gibbon leukemia virus, Pfizer monkey virus, Monkey immunodeficiency virus, Monkey sarcoma virus, Rous sarcoma virus, and Lentivirus.

[0164] Various recombinant viral vectors are intended herein. In some embodiments, the recombinant viral vector is a recombinant herpesvirus, such as herpes simplex virus (HSV), e.g., HSV-1, HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV) 6 (HHV-6), HHV-7, or HHV-8. In some embodiments, HSV is HSV-1. In some embodiments, HSV is HSV-2. In some embodiments, HSV is VZV. In some embodiments, HSV is EBV. In some embodiments, HSV is CMV. In some embodiments, HSV is HHV-6. In some embodiments, HSV is HHV-7. In some embodiments, HSV is HHV-8.

[0165] In some embodiments, the recombinant viral vector is AAV, for example, 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 These include 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.

[0166] In some embodiments, recombinant AAV is AAV1 or a derivative thereof. In some embodiments, recombinant AAV is AAV2 or a derivative thereof. In some embodiments, recombinant AAV is AAV3 or a derivative thereof. In some embodiments, recombinant AAV is AAV4 or a derivative thereof. In some embodiments, recombinant AAV is AAV5 or a derivative thereof. In some embodiments, recombinant AAV is AAV6 or a derivative thereof. In some embodiments, recombinant AAV is AAV7 or a derivative thereof. In some embodiments, recombinant AAV is AAV8 or a derivative thereof. In some embodiments, recombinant AAV is AAV9 or a derivative thereof. In some embodiments, recombinant AAV is AAV10 or a derivative thereof. In some embodiments, recombinant AAV is AAV11 or a derivative thereof. In some embodiments, recombinant AAV is AAV12 or a derivative thereof. In some embodiments, recombinant AAV is AAV13 or a derivative thereof. In some embodiments, recombinant AAV is AAV14 or a derivative thereof. In some embodiments, recombinant AAV is AAV15 or a derivative thereof. In some embodiments, recombinant AAV is AAV16 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh10 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, recombinant AAV is AAV-7m8 or a derivative thereof.In some embodiments, recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC4 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Myo or a derivative thereof.In some embodiments, recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC16 or a derivative thereof.

[0167] Recombinant AAV vectors Recombinant adeno-associated virus vectors (rAAVs) can be used to facilitate gene delivery into cells, such as target cells, for the introduction of genes (e.g., polynucleotides encoding a payload of interest). This disclosure provides rAAV vectors comprising any nucleic acids described herein. In particular, rAAV vectors comprising nucleic acids encoding one or more payloads of interest are described herein. In some embodiments, 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 bicistronic or tricistronic nucleic acids encoding multiple interferon polypeptides.

[0168] In some embodiments, the polynucleotides described herein encode one or more target payloads (e.g., therapeutic polypeptides such as cytokines) delivered to cells or tissues, as well as regulatory elements that control the expression of said target payloads. In some embodiments, the polynucleotides described herein encode interferons delivered to cells or tissues, as well as regulatory elements that control the expression of said interferons. Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylated sequences, introns, synthetic introns, mRNA stability sequences (e.g., Woodchuck hepatitis virus posttranscriptional regulators; WPREs), sequences that enable internal ribosome entry sites (IRESs) for bicistronic mRNA, sequences required for episome maintenance (e.g., ITRs), sequences that evade 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 DAIs), and / or sequences required for transduction into cells.

[0169] In some embodiments, the rAAV vector described herein comprises a CAG promoter operably ligated to one or more polynucleotides encoding one or more interferon polypeptides. In some embodiments, the CAG promoter comprises a first segment containing a cytomegalovirus (CMV) enhancer sequence, a second segment containing a chicken beta-actin (CBA) gene promoter element, a third segment containing a spacer sequence, and a fourth segment containing a rabbit beta-globin splice acceptor. In some embodiments, the order of the segments from 5-prime to 3-prime is first, second, third, and fourth.

[0170] In some embodiments, the CAG promoter includes one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 75% sequence identity to one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 80% sequence identity to one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 90% sequence identity to one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 95% sequence identity to one nucleic acid sequence from sequence numbers 22 to 23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs. 22-23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs. 22-23. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs. 22-23. In some embodiments, the CAG promoter includes 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 consecutive nucleotides to any one of SEQ ID NOs. 22-23.In some embodiments, the CAG promoter includes at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to any one of the sequence numbers 22-23, approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or approximately 240 consecutive nucleotides. In some embodiments, the CAG promoter includes any one of the sequence numbers 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with any one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 75% sequence identity with any one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 80% sequence identity with any one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 90% sequence identity with any one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 95% sequence identity with any one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 97% sequence identity with respect to one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 98% sequence identity with respect to one of the nucleic acid sequences 48-50 and 64-65. In some embodiments, the CAG promoter includes a nucleic acid sequence having at least 99% sequence identity with respect to one of the nucleic acid sequences 48-50 and 64-65.In some embodiments, the CAG promoter contains approximately 20, approximately 25, approximately 40, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 175, approximately 180, approximately 200, approximately 220, approximately 240, approximately 260, approximately 280, approximately 300, approximately 320, approximately 340, approximately 360, approximately 380, approximately 400, or approximately 240 consecutive nucleotides of any one of the sequence numbers 48-50 and 64-65. In some embodiments, the CAG promoter includes at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity for approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or approximately 240 consecutive nucleotides of any one of sequence numbers 48-50 and 64-65.

[0171] In some embodiments, the CMV enhancer is derived from human CMV. In some embodiments, the CMV enhancer includes various repeating sequence elements. In some embodiments, the CMV enhancer includes one nucleic acid sequence of any one of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to one of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 75% sequence identity to one of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 90% sequence identity to one of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 95% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 98% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having at least 99% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer includes a nucleic acid sequence having any one of the nucleic acid sequences of SEQ ID NOs. 25-27. In some embodiments, the CMV enhancer comprises approximately 20, approximately 25, approximately 40, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 175, approximately 180, approximately 200, approximately 220, approximately 240, approximately 260, approximately 280, approximately 300, approximately 320, approximately 340, approximately 360, approximately 380, approximately 400, or approximately 240 consecutive nucleotides of any one of SEQ ID NOs.In some embodiments, the CMV enhancer contains at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity for approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or approximately 240 consecutive nucleotides of any one of sequence numbers 25-27.

[0172] In some embodiments, the CAG promoter includes a chicken beta-actin (CBA) gene promoter element. In some embodiments, the CBA gene promoter element includes a CBA gene promoter sequence, the CBA gene exon 1, and the CBA gene intron 1. In some embodiments, the CBA promoter includes the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter includes 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 includes 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 includes 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 includes 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 includes a nucleic acid sequence having at least 95% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 97% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 98% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter includes a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the CBA promoter contains approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or 240 consecutive nucleotides of SEQ ID NO: 28.In some embodiments, the CBA promoter includes at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to about 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, or about 240 consecutive nucleotides of SEQ ID NO: 28. In some embodiments, the CBA promoter includes 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 includes a nucleic acid sequence having at least 75% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 80% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 90% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 95% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 97% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 98% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter includes a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the CBA promoter contains approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 520, 300, 320, 340, 360, 380, 400, or 240 consecutive nucleotides of SEQ ID NO: 52.In some embodiments, the CBA promoter includes at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity for approximately 20, 25, 40, 50, 75, 100, 125, 150, 175, 180, 200, 220, 240, 260, 520, 300, 320, 340, 360, 380, 400, or approximately 240 consecutive nucleotides of SEQ ID NO: 52.

[0173] In some embodiments, the CAG promoter includes a spacer sequence immediately after the 3' end of the CBA promoter element. In some embodiments, the spacer sequence includes an intron element. In some embodiments, the spacer sequence includes an exon 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 long. 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 long. In some embodiments, the spacer sequence is approximately 20 to approximately 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides long. In some embodiments, the spacer sequence is approximately 50 to approximately 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, or 250 nucleotides long. In some embodiments, the spacer sequence is approximately 80 to approximately 100, 120, 140, 160, 180, 200, or 250 nucleotides long. In some embodiments, the spacer sequence is at least 10 nucleotides long. In some embodiments, the spacer sequence is at least 12 nucleotides long. In some embodiments, the spacer sequence is at least 20 nucleotides long. In some embodiments, the spacer sequence is at least 50 nucleotides long. In some embodiments, the spacer sequence is at least 100 nucleotides long. In some embodiments, the spacer sequence is at least 250 nucleotides long. In some embodiments, the spacer sequence is 5 to 20 nucleotides long. In some embodiments, the spacer is 10 to 20 nucleotides long. In some embodiments, the spacer sequence is 10 to 20 nucleotides long. In some embodiments, the spacer sequence is 10 to 15 nucleotides long. In some embodiments, the spacer sequence is 250 to 350 nucleotides long.In some embodiments, the spacer sequence includes the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 75% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 80% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 90% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 95% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 97% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 98% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the spacer sequence includes 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 long. In some embodiments, the spacer sequence is 500 to 1000 nucleotides long. In some embodiments, the spacer sequence is 600 to 1000 nucleotides long. In some embodiments, the spacer sequence is 700 to 1000 nucleotides long. In some embodiments, the spacer sequence is 800 to 1000 nucleotides long. In some embodiments, the spacer sequence is 900 to 1000 nucleotides long.

[0174] In some embodiments, the CAG promoter includes a rabbit beta-globin splice acceptor. In some embodiments, the rabbit beta-globin splice acceptor includes the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the rabbit beta-globin splice acceptor includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the CAG promoter includes the rabbit beta-globin splice acceptor. In some embodiments, the rabbit beta-globin splice acceptor includes the nucleic acid sequence of SEQ ID NO: 53.In some embodiments, the rabbit beta-globin splice acceptor includes 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 includes 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 includes 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 includes 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 includes 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 includes a nucleic acid sequence having at least 97% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor includes a nucleic acid sequence having at least 98% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor includes a nucleic acid sequence having at least 99% sequence identity with respect to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the rabbit beta-globin splice acceptor includes a nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 53.

[0175] Useful rAAV vectors include those having one or more naturally occurring AAV genes, either entirely or partially deleted, but retaining a functional adjacent ITR sequence. In some embodiments, the AAV ITR is any serotype suitable for a particular application. In some embodiments, the nucleic acid does not contain an ITR. In some embodiments, the AAV ITR is any suitable AAV serotype, including any currently known or hereafter discovered serotype, or modified, evolved, selected, or chimeric capsid serotypes. In some embodiments, the ITR is of a different length or configuration.

[0176] The AAV capsid protein constitutes the non-nucleic acid portion outside the virion and is encoded by the AAV Cap gene. In some embodiments, the Cap gene encodes VP1, VP2, VP3, MAAP, AAP, or a combination thereof.

[0177] In some embodiments, Rep, Cap, or other polynucleotides necessary for producing the rAAV of this disclosure are provided herein. In some embodiments, Rep, Cap, or other polynucleotides are delivered to a packaging host cell using any suitable genetic element (e.g., a 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 into the packaging host cell by two vectors (a first vector containing a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2) and a second vector containing a second nucleic acid encoding a single capsid protein (e.g., VP3)). In some embodiments, three vectors, each containing nucleic acids encoding a different capsid protein, are delivered to the packaging host cell.

[0178] In some embodiments, a single nucleic acid encoding multiple replication proteins (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 into the packaging host cell by two vectors (a first vector containing a first nucleic acid encoding 1 to 3 replication proteins, and a second vector containing a second nucleic acid encoding 1 to 3 replication proteins). In some embodiments, four vectors, each containing a nucleic acid encoding a different replication protein, are delivered to the packaging host cell.

[0179] In some embodiments, a single nucleic acid encoding multiple adenovirus co-proteins (e.g., E1A, E1B, E4, E2A, and VA RNA) is delivered to a packaging host cell in a single vector. In some embodiments, the nucleic acid encoding the adenovirus co-protein is delivered into the packaging host cell by two vectors. In some embodiments, two or more vectors, each containing nucleic acids encoding a different capsid protein, are delivered to the packaging host cell. The selected genetic elements are delivered by any preferred method, including those described herein. Methods used to construct any embodiment of this disclosure include genetic engineering, recombinant engineering, and synthetic techniques.

[0180] AAV, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV AV9,AAV10,AAV11,AAV12,AAV13,AAV14,AAV15,AAV16,AAV-rh8,AAV-r h10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc8 0、AAV-Anc80L65、AAV-7m8、AAV-PHP-B、AAV-PHP-EB、AAV-2.5、AAV-2tY F、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-HSC1 1、AAV-HSC12、AAV-HSC13、AAV-HSC14、AAV-HSC15、AAV-TT、AAV-DJ / 8、A AV-Myo、AAV-NP40、AAV-NP59、AAV-NP22、AAV-NP66、またはAAV-HSC16である。

[0181] In some embodiments, recombinant AAV is AAV1 or a derivative thereof. In some embodiments, recombinant AAV is AAV2 or a derivative thereof. In some embodiments, recombinant AAV is AAV3 or a derivative thereof. In some embodiments, recombinant AAV is AAV4 or a derivative thereof. In some embodiments, recombinant AAV is AAV5 or a derivative thereof. In some embodiments, recombinant AAV is AAV6 or a derivative thereof. In some embodiments, recombinant AAV is AAV7 or a derivative thereof. In some embodiments, recombinant AAV is AAV8 or a derivative thereof. In some embodiments, recombinant AAV is AAV9 or a derivative thereof. In some embodiments, recombinant AAV is AAV10 or a derivative thereof. In some embodiments, recombinant AAV is AAV11 or a derivative thereof. In some embodiments, recombinant AAV is AAV12 or a derivative thereof. In some embodiments, recombinant AAV is AAV13 or a derivative thereof. In some embodiments, recombinant AAV is AAV14 or a derivative thereof. In some embodiments, recombinant AAV is AAV15 or a derivative thereof. In some embodiments, recombinant AAV is AAV16 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh10 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, recombinant AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, recombinant AAV is AAV-7m8 or a derivative thereof.In some embodiments, recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC4 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, recombinant AAV is AAV-Myo or a derivative thereof.In some embodiments, recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, recombinant AAV is AAV-HSC16 or a derivative thereof.

[0182] In some embodiments, the vector is a pseudotyped rAAV vector. The pseudotyped vector includes an AAV genome vector of a given serotype that has been pseudotyped with a Cap gene product derived from a serotype other than the given serotype (e.g., particularly AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9). For example, a typical pseudotyped vector is an AAV serotype 2 genome vector that has been pseudotyped with a Cap gene product derived from AAV serotype 9.

[0183] In some embodiments, AAVs have intravirion mutations used to transduce specific cell types more efficiently than unmutated capsid virions. In some embodiments, preferred AAV variants have ligand insertions to facilitate AAV targeting to specific cell types. In some embodiments, construction and characterization of AAV capsid variants, including insertion variants, alanine screening variants, and epitope tagged variants, are used.

[0184] In some embodiments, artificial AAV capsids are used. Such artificial capsids are generated by any suitable technique that combines a selected AAV sequence (e.g., a fragment of the VP1 capsid protein) with a heterologous sequence derived from a non-AAV virus or non-viral source, obtained from a discontinuous portion of the same serotype, or obtained from a separately selected AAV serotype. Artificial AAV serotypes are, but are not limited to, pseudotyped AAV, chimeric AAV capsids, recombinant AAV capsids, engineered AAV capsids, or "humanized" AAV capsids.

[0185] Other rAAV virions used in the compositions and methods of this disclosure include, but are not limited to, capsid hybrids produced by molecular breeding of the virus, as well as those capsid hybrids produced by exon shuffling, directed evolution, rational design, error-prone PCR, via computational algorithms, or via the use of artificial intelligence or machine learning.

[0186] In some embodiments, the capsid is modified to improve therapeutic effects. In some embodiments, the capsid is modified for minimizing immunogenicity and / or immunocloaking, better stability and / or particle durability, efficient degradation, and / or precise delivery of heterogeneous coding sequences or functional fragments or variants to the nucleus. In some embodiments, the modification or mutation is a deletion, insertion, substitution, or any combination thereof of amino acids in the capsid polypeptide. In some embodiments, the capsid polypeptide contains 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 contains 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.

[0187] In another embodiment, the modified capsid polypeptides of the present disclosure comprise a modified sequence, such modification may include both conserved and non-conserved 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 with respect to the corresponding wild-type capsid protein.

[0188] Method for generating rAAV vectors Methods for generating rAAV vectors (e.g., rAAV vectors comprising polynucleotides as described herein) are disclosed herein, the methods comprising the step of in vitro contacting cells with the polynucleotides as described herein and a transfer plasmid, Rep / Cap, helper plasmid, or a combination thereof to generate a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the cells are infected with the rAAV vector. In some embodiments, the cells are transduced with the rAAV vector. In some embodiments, the cells are engineered stable cell lines.

[0189] In some embodiments, the cells are eukaryotic cells (e.g., plant cells, animal cells, protist cells, or fungal cells), mammalian cells (Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, human fetal kidney (HEK-293), mouse myeloma (NS0), Vero, or human retinal cells), immortalized cells (e.g., HeLa cells, COS cells, HEK-293T cells, MDCK cells, 3T3 cells, PC12 cells, Huh7 cells, HepG2 cells, K562 cells, N2a cells, or SY5Y cells), insect cells (e.g.) These include fall armyworm cells, golden looper cells, Drosophila melanogaster cells, S2 cells, or tobacco budworm cells), yeast cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells, or Candida cells), plant cells (e.g., parenchyma cells, placoceral cells, or plastile cells), fungal cells (e.g., Saccharomyces cerevisiae cells, Cryptococcus cells, or Candida cells), or prokaryotic cells (e.g., Escherichia coli cells, Streptococcus bacterial cells, Streptomyces soil bacterial cells, or archaeal cells). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells are insect cells. In some embodiments, the cells are yeast cells. In some embodiments, the cells are plant cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are prokaryotic cells.

[0190] In some embodiments, the cells are 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 cells, HT1080, HepG2, Huh7, K562, primary cells, or derivatives thereof. In some embodiments, the cells are A549 cells or derivatives thereof. In some embodiments, the cells are HEK-293 cells or derivatives thereof. In some embodiments, the cells are HEK-293T cells or derivatives thereof. In some embodiments, the cells are BHK cells or derivatives thereof. In some embodiments, the cells are CHO cells or derivatives thereof. In some embodiments, the cells are HeLa cells or derivatives thereof. In some embodiments, the cells are MRC5 cells or derivatives thereof. In some embodiments, the cells are Sf9 cells or derivatives thereof. In some embodiments, the cells are Cos-1 cells or derivatives thereof. In some embodiments, the cells are Cos-7 cells or derivatives thereof. In some embodiments, the cells are Vero cells or derivatives thereof. In some embodiments, the cells are BSC-1 cells or derivatives thereof. In some embodiments, the cells are BSC-40 cells or derivatives thereof. In some embodiments, the cells are BMT-10 cells or derivatives thereof. In some embodiments, the cells are WI38 cells or derivatives thereof. In some embodiments, the cells are HeLa cells or derivatives thereof. In some embodiments, the cells are Saos cells or derivatives thereof. In some embodiments, the cells are C2C12 cells or derivatives thereof. In some embodiments, the cells are L cells or derivatives thereof. In some embodiments, the cells are HT1080 cells or derivatives thereof. In some embodiments, the cells are HepG2 cells or derivatives thereof. In some embodiments, the cells are Huh7 cells or derivatives thereof. In some embodiments, the cells are K562 cells or derivatives thereof. In some embodiments, the cells are primary cells.

[0191] In some embodiments, recombinant viral vectors are produced by a producer cell line. Briefly, for example, 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 recombinant vector production and, in some embodiments, is expanded into a production bioreactor and infected with a helper polynucleotide (e.g., wild-type adenovirus) as a helper to initiate vector production. In some embodiments, the recombinant viral vector (e.g., rAAV) is then recovered, the adenovirus is inactivated and / or removed (e.g., by heat), and the viral particles are purified. In some embodiments, the recombinant viral vector is purified and formulated.

[0192] In some embodiments, media suitable for the production of recombinant vectors are used. These media include, but are not limited to, modified Eagle medium (MEM), Roswell Park Memorial Institute medium (RPMI) 1640, Eagle minimal essential medium (EMEM), Ham F10 medium, Iskov modified Dulbecco medium (IMDM), Neuralbasal medium, Dulbecco modified Eagle medium (DMEM), and custom formulations, particularly those used for the production of recombinant vectors, manufactured by Hyclone Laboratories and JRH.

[0193] In some embodiments, suitable production culture media of this disclosure are supplemented with serum or serum-derived recombinant protein at levels of 0.5–20 (v / v or w / v). In some embodiments, the vector is produced under serum-free conditions, also known as a medium without animal-derived products. In some embodiments, commercially available or custom media are designed to support vector production and include, but are not limited to, supplementation with glucose, vitamins, amino acids, and other growth factors to increase the titer and / or yield of the vector in production cultures.

[0194] Vector-producing cultures involve various conditions (such as a wide temperature range and varying durations) suitable for the specific host cells being used. Vector-producing cultures include adhesion-dependent cultures cultured in suitable adhesion-dependent culture vessels such as plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. In some embodiments, vector-producing cultures include suspension-adapted host cells such as HeLa, HEK-293, HEK-293T, HEK-293S, CHO, NS0, PER.C6, BHK, S2, and Sf9 cells, cultured in various methods including, for example, spinner flasks, agitated tank bioreactors, single-use bioreactors such as Cytiva Xcellerex, Sartorius, and Wave, as well as disposable systems.

[0195] In some embodiments, the viral particles of the present disclosure are recovered by lysing host cells of a vector-producing culture, or by collecting the culture supernatant from a producing culture, under conditions that allow the cells to release the viral particles into the culture medium in an intact state. Preferred methods for lysing cells include, for example, multiple freeze / thaw cycles, sonication, microfluidic treatment, and treatment with chemicals such as detergents and / or proteases.

[0196] In further embodiments, the virus particles are purified. As used herein, the term “purified” includes preparations of virus particles from which at least some of the other components present in the naturally occurring state of the virus particles or in the initially prepared state have been removed. Thus, for example, in some embodiments, isolated virus particles are prepared using purification techniques to concentrate from a source mixture, such as a culture lysate or a productive culture supernatant. In some embodiments, concentration is evaluated in various ways, measured by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in the solution, or by infectivity, or in relation to second potentially interfering substances present in the source mixture, such as contaminants and impurities, including product-derived and process-derived impurities.

[0197] In some embodiments, the recovered product of the vector-producing culture is clarified to remove host cell debris. In some embodiments, the recovered product of the culture is clarified by filtration through a series of depth filters, including, for example, a Grade DOHC Millipore Millistak+HC pod filter, a Grade A1HC Millipore Millistak+HC pod filter, and an Opticap XL 10 Millipore Express SHC hydrophilic membrane filter, which is a 0.2 μm filter. Clarification can also be achieved by various other standard techniques, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or larger.

[0198] In some embodiments, the recovered vector-producing culture is further treated with a nuclease (e.g., DNA or RNA nuclease) to digest any high molecular weight DNA present in the product culture. In some embodiments, nuclease digestion is carried out under standard conditions.

[0199] In some embodiments, 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 concentrating viral particles, vector capture by apatite chromatography, thermal inactivation of helper viruses, 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 steps in the order described below.

[0200] Methods for generating recombinant vectors are described herein, the methods comprising the step of providing cells transfected with helper polynucleotides. In some embodiments, cells are transfected with helper polynucleotides that provide helper functions to AAV. In some embodiments, the helper polynucleotides provide adenoviral functions including, for example, E1A, E1B, E2A, E4ORF6, and / or VA RNA. The sequences of adenoviral genes that provide these functions are obtained in some embodiments from any known adenovirus serotype such as serotypes 2, 3, 4, 5, 7, 12, and 40, and further include any human type currently identified. In some embodiments, the adenoviral functions are provided by self-attenuating adenoviruses. In some embodiments, the method comprises transfecting cells with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0201] Methods for generating recombinant vectors are described herein, the methods comprising the step of providing cells transfected with helper polynucleotides under the control of a promoter. In some embodiments, the cells are stable host cells containing the required(s) components under the control of an inductive promoter. In some embodiments, the cells are stable host cells containing the required(s) components under the control of a constitutive promoter. In some embodiments, the cells are stable host cells containing selected components under the control of a constitutive promoter and other selected components under the control of one or more inductive promoters. For example, stable host cells are generated that are derived from HEK-293 cells (containing E1 helper function under the control of a constitutive promoter) but containing Rep and / or Cap proteins under the control of an inductive promoter.

[0202] In some embodiments, the minigene, Rep sequence, Cap sequence, and helper function required to produce the rAAV of this 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 preferred method.

[0203] The production of recombinant vectors such as rAAV vectors, in some embodiments, includes transfection, production of stable cell lines, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, bocavirus-AAV hybrids, and baculovirus-AAV hybrids.

[0204] Methods comprising transfection, production of stable cell lines, and infectious hybrid virus production systems are used in some embodiments to produce rAAV vectors. In some embodiments, the methods described herein include adenovirus-AAV hybrids, herpesvirus-AAV hybrids, bocavirus-AAV hybrids, and baculovirus-AAV hybrids. All rAAV-producing cultures for the production of rAAV virus particles require: 1) suitable host cells, including, for example, human cell lines such as HeLa, A549, or HEK-293 cells, or HEK-293T cells, or HEK-293S cells, or insect cell lines such as Sf9 in the case of baculovirus-producing systems, or transgenic plant cells; 2) suitable helper virus functions provided by wild-type or mutant adenoviruses (e.g., temperature-sensitive adenoviruses), herpesviruses, baculoviruses, or plasmid constructs that provide 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 heterologous sequences encoding any desired sequence or fragment or variant thereof); and 5) suitable media and media components to support rAAV production. Suitable media for the production of rAAV vectors include, but are not limited to, modified Eagle medium (MEM), Roswell Park Memorial Institute medium (RPMI) 1640, Eagle minimal essential medium (EMEM), Ham F10 medium, Iskov modified Dulbecco medium (IMDM), Neuralbasal medium, and Dulbecco modified Eagle medium (DMEM), as well as media produced by Hyclone Laboratories and JRH.

[0205] In some embodiments, recombinant AAV particles are produced by transfecting producer cells with a plasmid containing an rAAV genome (cis-plasmid) that includes a payload adjacent to one or more AAV ITRs, and a separate construct that trans-expresses the AAV Rep and Cap genes. In some embodiments, adenovirus helper factors such as E1A, E1B, E2A, E4ORF6, and VA RNA are provided by either adenovirus infection or by transfecting producer cells with a third plasmid that provides adenovirus helper polynucleotides. In some embodiments, the producer cells are HEK-293 cells. In some embodiments, the helper polynucleotides provided vary depending on the producer cells used and whether those producer cells already carry some of these helper polynucleotides.

[0206] 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 the Cap gene is transfected with a helper adenovirus plasmid into a cell line (e.g., HEK-293 cells) (e.g., using squeeze poration, lipofection, optical transfection, calcium phosphate method, or polyethyleneimine (PEI)), the virus is recovered, and optionally purified.

[0207] In some embodiments, the rAAV vector is 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, the Cap gene, and the promoter-payload sequence. The cell line is screened to select a lead clone for rAAV production and then expanded into a production bioreactor and infected with an adenovirus (e.g., wild-type adenovirus) as a helper to initiate AAV production. The virus is then recovered, and the adenovirus may be inactivated (e.g., by heat) and / or removed, and the rAAV is purified.

[0208] In some embodiments, a method is provided to produce any rAAV vector disclosed herein, comprising: (a) culturing host cells under conditions that produce rAAV, wherein the host cells comprise (i) one or more AAV packaging genes, each of which encodes an AAV byproduct and / or capsidized protein; (ii) an rAAV provector comprising a nucleic acid encoding a therapeutic polypeptide and / or nucleic acid as described herein, and adjacent to at least one AAV ITR; and (iii) an AAV helper function; and (b) recovering the rAAV vector produced by the host cells. In some embodiments, the above at least one AAV ITR 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 - Selected from the group consisting of 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. In some embodiments, the replicating protein is the AAV2 replicating protein.

[0209] Methods of treating cancer Methods for treating target cancers requiring treatment are disclosed herein, the methods comprising the step of administering the rAAV described herein. In some embodiments, the rAAV comprises a minigene containing a splice modulator binding site. In some embodiments, the method for treating target cancers requiring treatment further comprises the step of administering a small molecule splice modulator.

[0210] Administration is not limited to any specific route, but rather may refer to any route recognized as appropriate in the medical community. In some embodiments, administration is intravenous (IV), intramuscular (IM), intra-arterial, inhalation, intramedullary, intrathecal (IT), intracisional (ICM), intraventricular (ICV), intraparenchymal, transnasal, subcutaneous (SQ), transcutaneous, intraventricular, intraperitoneal (IP), intragastric (IG), mucosal, oral, enteral, intravitreous, and / or via portal vein catheter, and / or any combination thereof. In some embodiments, the rAAV vector compositions described herein are administered locally to diseased tissue (e.g., tumor).

[0211] In some embodiments, the rAAV vector composition provided by this disclosure is administered in combination with a splicing modifier or modulator. In some embodiments, the rAAV vector composition is provided sequentially with a splicing modifier. In some embodiments, the rAAV vector composition is administered topically directly to diseased tissue, and the splicing modifier is administered via a different route. In some embodiments, the rAAV vector composition is administered topically directly to diseased tissue, and the splicing modifier is administered orally. In some embodiments, the rAAV vector composition is administered topically directly to diseased tissue, and the splicing modifier is administered systemically. In some embodiments, both the rAAV vector composition and the splicing modifier are administered topically directly to diseased tissue.

[0212] In some embodiments, the subject is human. In some embodiments, the subject has 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 glioblastoma. In some embodiments, the subject has primary cancer. In some embodiments, the subject has metastases. In some embodiments, the subject has a brain tumor. In some embodiments, the subject has uveal melanoma.

[0213] In some embodiments, administration is a direct, local administration to diseased tissue (e.g., a tumor). In some embodiments, administration is directed to the central nervous system. In some embodiments, administration is directed to the brain. In some embodiments, administration is directed to the ventricles of the brain. In some embodiments, administration is intratumorally.

[0214] Any preferred route of administration or a combination of different routes of administration may be used, including systemic administration (e.g., intravenous, intravascular, intraarterial), local injection into the central nervous system (CNS) (e.g., intratumoral injection, intracranial injection, intracerebral injection, intraventricular injection, intraparenchymal injection, or injection into cerebrospinal fluid (CSF) via the ventricular system, cisterna magna, spinal cavity), or local injection into other body sites (e.g., intraocular, nasal, intramuscular, subcutaneous, intradermal injection, transdermal administration). In some embodiments, administration is by intratumoral injection, intracranial injection, intracerebral injection, intraventricular, intraparenchymal, or injection into cerebrospinal fluid (CSF) via the ventricular system or intrathecal space.

[0215] In some embodiments, intraventricular injections are performed in the right lateral ventricle, left lateral ventricle, third ventricle, fourth ventricle, interventricular foramen (also called foramen of Monro), cerebral aqueduct, central canal, median orifice, right lateral orifice, left lateral orifice, perivascular space, or subarachnoid space.

[0216] Administration may be carried out using an osmotic pump, electroporation, or other means. In some embodiments, the administration of the rAAV vector described herein is performed before, after, or concurrently with surgical tumor removal or biopsy.

[0217] In some embodiments, administration is by convection-enhanced delivery (CED). CED uses the direct injection of a drug-containing solution into tissue so that transport is governed 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 using a step-designed cannula.

[0218] In some embodiments, magnetic resonance imaging (MRI)-guided CED is performed to deliver the rAAV vector of this disclosure. In some embodiments, the CED further includes the use of a tracing agent. In some embodiments, the tracing agent 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 and the rAAV vector are administered simultaneously. In some embodiments, the MRI contrast enhancer is mixed directly with the rAAV vector before administration.

[0219] Pharmaceutical composition In certain embodiments, pharmaceutical compositions comprising (a) a polynucleotide of the Disclosure, a plasmid of the Disclosure, or an rAAV comprising a polynucleotide of the Disclosure, and (b) a pharmaceutically acceptable excipient are described herein. The pharmaceutical compositions described herein are in a form suitable for administration to an individual requiring it.

[0220] In some embodiments, the pharmaceutically acceptable excipients of this disclosure are appropriately selected from injectable excipient solutions such as sterile water for injection and aqueous solutions such as physiological saline.

[0221] Acceptable excipients are physiologically acceptable to the recipient and maintain the therapeutic properties of the compound administered with them. Acceptable excipients and their formulations are generally as described, for example, in Remington's Pharmaceutical Sciences. One exemplary excipient is saline. As used herein, the term "pharmaceutically acceptable excipient" means a pharmaceutically acceptable substance, composition, or carrier, including liquid or solid fillers, diluents, excipients, solvents, or encapsulants that are involved in the transport of the compound of interest from one organ or part of the body to another, or in an in vitro assay system. Each excipient is acceptable in the sense that it is compatible with the other components of the formulation and is not harmful to the recipient. Furthermore, acceptable excipients should not alter the specific activity of the compound of interest.

[0222] In another embodiment, the pharmaceutical compositions disclosed herein further include acceptable additives for improving the stability of compounds in the composition and / or controlling the release rate of the composition. Acceptable additives do not alter the specific activity of the compound of interest. Exemplary acceptable additives include, but are not limited to, sugars such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose, and mixtures thereof. In some embodiments, acceptable additives are combined with acceptable carriers and / or excipients such as dextrose. Alternatively, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 for improving peptide stability and inhibiting gelation of the solution. In some embodiments, surfactants are added to the composition in an amount of 0.01% to 5% of the solution. By adding such acceptable additives, the storage stability and half-life of the formulation are improved.

[0223] The suspensions, lyophilized products, and crystalline forms of rAAV vectors, polynucleotides, or compositions described herein are also considered herein, and methods for preparing suspensions, lyophilized products, and crystalline forms are known to those skilled in the art.

[0224] 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 achieved by filtration through a sterile filtration membrane. In some embodiments, the resulting solution is packaged for use or filtered and lyophilized under sterile conditions, and the lyophilized preparation is combined with the sterile solution before administration.

[0225] In some embodiments, freeze-drying is used to stabilize polypeptides for long-term storage, such as when polypeptides are relatively unstable in liquid compositions.

[0226] In some embodiments, certain excipients, such as polyols (including mannitol, glycerol, sorbitol, and its derivatives like polysorbate 20), salts (including NaCl, MgCl2, and KCl), sugars (including glucose, sucrose, and trehalose), surfactants (including poloxamer 188), and amino acids (including alanine, glycine, and glutamic acid), act as stabilizers for lyophilized products. In some embodiments, polyols, surfactants, and sugars are also used to protect polypeptides from damage due to freezing and drying and to improve stability during storage in a dry state. Sugars are effective in some embodiments both during the lyophilization process and during storage. Other molecular classes, including monosaccharides and disaccharides, as well as polymers such as polyvinylpyrrolidone (PVP), have also been reported as stabilizers for lyophilized products.

[0227] For injection, in some embodiments, the pharmaceutical compositions disclosed herein are provided in powder form suitable for reconstitution with the appropriate solutions described above. Examples of these include, but are not limited to, lyophilized powders, rotary-dried powders, spray-dried powders, amorphous powders, granules, precipitates, or fine particles. For injection, the compositions optionally include stabilizers, pH adjusters, surfactants, bioavailability adjusters, and combinations thereof.

[0228] In some embodiments, sustained-release formulations are prepared. Preferred examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the pharmaceutical compositions herein, where these matrices take the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (e.g., see U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., Lupron Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and luprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, while certain hydrogels release proteins in a shorter period.

[0229] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, immediate-release, long-acting, or sustained-release, as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled-release or sustained-release.

[0230] In some embodiments, the pharmaceutical composition is contained in a container, pack, or dispenser along with instructions for administration. [Examples]

[0231] The following are examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. While various efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperatures, etc.), some experimental error and deviation should naturally be acceptable.

[0232] Example 1. HEK-293T expression of inducible AAP-GFP This example describes an exemplary regulatory minigene for use in the rAAV vector of this disclosure. The exemplary minigene was tested in HEK-293T cells and used to control the expression of a GFP reporter polypeptide.

[0233] Adherent human HEK-293T cells were transduced with inducible AAV-GFP, and fluorescence imaging was performed on GFP expression before and after administration of an inducer molecule (e.g., LMI070). The data are shown in Figure 3.

[0234] Adherent human HEK-293T cells were transduced with inducible AAV-GFP at various doses, followed by administration of an inducer molecule (e.g., LMI070). GFP expression was monitored for several hours to determine the initial expression dynamics at different AAV doses in vitro. The data are shown in Figure 4.

[0235] Example 2: Manipulated rAAV vector for interferon delivery This embodiment describes exemplary engineered nucleic acids useful for producing rAAV vectors, which are provided by this disclosure and capable of delivering interferon to the target of interest.

[0236] As described herein, the recombinant adeno-associated virus (rAAV) vectors of this disclosure may include, but are not limited to, a promoter, a minigene, a polynucleotide containing a coding sequence for the payload of interest, one or more regulatory elements (e.g., WPRE, polyA, antibiotic resistance gene, etc.), and / or ITR. Exemplary combinations of nucleic acid elements are provided in Table 1, and each combination of nucleic acid elements was incorporated into its respective plasmid vector (plasmids 1–30, see also Figures 5A–5D) and subjected to assays (e.g., for interferon delivery). The nucleic acids were tested using a CAG promoter and included the CMV earliest enhancer element (CMVie) sequence, chicken beta-actin sequence, and rabbit beta-globin splice acceptor. The CMVie sequence used was cleaved and contained either a long (SEQ ID NO: 25), medium (SEQ ID NO: 26), or short (SEQ ID NO: 27) version. Incorporation of minigenes or "Xon" systems is further shown (SEQ ID NOs: 31 and 51). Polynucleotides encoding human IFNβ, either CpG-depleted (SEQ ID NO: 4) or representative wild-type (SEQ ID NO: 2), were used. WPREs, either modified (SEQ ID NO: 33) or representative wild-type (SEQ ID NO: 32), were also used. Selected poly(A) (SV40 shown in SEQ ID NO: 19, and bGH shown in SEQ ID NO: 34), antibiotic resistance genes (AmpR shown in SEQ ID NO: 44, and KanR shown in SEQ ID NO: 20 or 21), and ITR sequence lengths (cleaved ITRs shown in SEQ ID NOs: 40-41 and full-length ITRs shown in SEQ ID NOs: 36-39) are also shown.

[0237] [Table 1-1]

[0238] [Table 1-2]

[0239] material and method Figure 6 shows an exemplary workflow for producing an engineered plasmid (by cloning) used to produce an rAAV vector, which is provided in this disclosure and illustrates a multi-step cloning strategy for plasmid production. An exemplary protocol for producing a specific engineered rAAV vector is provided below.

[0240] To generate plasmid #1 (Figure 5A), a 582 bp novel synthetic hIFNβ-CpGdel polynucleotide was subcloned to an AAV-CAG-mCardinal polynucleotide using standard cloning techniques, with KpnI and EcoRI restriction enzymes instead of the mCardinal protein fluorescent marker ORF. To generate plasmid #12 (Figure 5L), two novel polynucleotide fragments were synthesized to cover both the bGH polyA sequence (SEQ ID NO: 34) and the CpG-depleted KanR via the CMV early enhancer sequence (SEQ ID NO: 35), and inserted into plasmid #1 using homologous recombination techniques. Plasmid #14 (Figure 5N) was constructed by inserting a 657 bp novel synthetic polynucleotide (SEQ ID NO: 54) into plasmid #12. Plasmids #18 (Figure 5R) and #16 (Figure 5P) were generated by substituting the hIFNβ-CpG ORF with a newly synthesized fragment for eGFP from plasmids #12 and #14, respectively, using BamHI / EcoRI. Plasmids #20 (Figure 5T) and #21 (Figure 5U) were obtained by substituting the hIFNβ-CpGdel ORF with a newly synthesized polynucleotide encoding mCardinal in plasmids #12 and #14, respectively. Plasmids #13 (Figure 5M) and #15 (Figure 5O) were constructed by inserting a newly synthesized polynucleotide fragment containing a splicing minigene (Xon) and a modified hIFNβ-CpGdel fragment that does not contain ATG and an alternative start codon into plasmids #12 and #14, respectively. Modified, newly synthesized eGFP fragments (without ATG and alternative start codon) were inserted in place of hIFNβ-CpGdel in plasmids #13 and #15 to construct plasmids #17 (Figure 5Q) and #19 (Figure 5S), respectively. Modified, newly synthesized mCardinal fragments (without ATG and alternative start codon) were inserted in place of hIFNβ-CpGdel in plasmids #13 and #15 to construct plasmids #22 (Figure 5V) and #23 (Figure 5W), respectively.

[0241] Example 2. Fabrication of an improved AAV This example describes the construction of an improved AAV with various combinations of regulatory elements for delivering the target gene to target cells.

[0242] In short, AAV strains were generated using a standard triple transfection protocol combined with pRepCap and an adenovirus helper plasmid. The platform process utilized the triple transfection protocol for adherent HEK293T cells, followed by one-step purification using density gradient ultracentrifugation to remove empty AAV capsids. The samples were then concentrated and formulated in a one-step process using a spin column. These research-grade lots were then formulated in AAV-compatible excipients (1X dPBS / 0.005% poloxamer). The vector genome (vg) titer of each lot was tested by RT-qPCR using an ITR primer set within the AAV ITR, and the results are provided in the certificate of analysis. AAV preparations #5, #6, #8, and #15 were produced using an established GMP-like large-scale manufacturing suspension platform (using suspension-adaptive HEK293 cells). The produced AAVs are listed in Table 2.

[0243] [Table 2]

[0244] These results confirm that all AAVs were produced with high viral genome titers (>1e13vg / mL), that the newly designed genome is suitable for AAV packaging and transduction experiments, and that it produces robust and clinically relevant levels of virus.

[0245] Example 3. Verification of various variants of therapeutic AAV candidates. This example describes the construction of an improved AAV for delivering the target gene to target cells, as well as the expression and payload activity regulated by various combinations of regulatory elements.

[0246] The productivity and functionality of various candidates of variations within the payload and different regulatory elements were evaluated. In four constructs, the variables tested included a CpG-depleted payload, two lengths of variation of the CAG promoter, and variable polyA sequences and antibiotic resistance cassettes. AAV preparations were tested in parallel in a series of assays to quantify viral potency using 1) vector genome titers by digital PCR (dPCR) using payload primers, 2) capsid titers using a commercially available ELISA kit, and 3) an in vitro cell-based assay measuring the expression of the payload after transduction in mammalian cells. The percentage of full capsids and volumetric yields of AAV were calculated using vg titers and capsid titers, and the data presented as relative ratios between various production lots are shown in Table 3, with AAV#6 used as an internal reference for all values tested simultaneously. For viral potency, ratios between production lots were calculated using values obtained at an MOI of 5e5 vg / cell for each construct.

[0247]

Table 3

[0248] These results demonstrated that the various elements used in combination within the AAV genomic plasmid did not have a significant impact on AAV productivity or activity.

[0249] Example 4. Large-scale AAV batches for evaluating clinical manufacturing suitability This example describes the production of large-scale batches of improved AAV with various combinations of regulatory elements for delivering a gene of interest to target cells.

[0250] Candidates with therapeutic payloads were produced using a large-scale suspension platform. AAV batches were produced in both 2L and 4L scales, fully purified, and evaluated against various quality criteria. AAV batches were purified through a multi-step process to ensure high levels of purity in the final AAV material. The process included tangential flow filtration (TFF), affinity capture chromatography, gradient ultracentrifugation or anion exchange chromatography, and final formulation using a centrifuge or another TFF. AAV strain genome titers were obtained using PCR-based protocols. These AAV preparations were used to evaluate various critical quality characteristics in parallel experiments.

[0251] As representative examples of AAVs described herein that have a therapeutic payload, plasmid 1 (Table 4) or plasmid 24 (Table 5) were used for the tests. AAV preparations were tested in parallel in a series of assays to quantify viral potency using 1) vector genome titer by digital PCR (dPCR) with primers located within the therapeutic payload, 2) capsid titer using a commercially available ELISA kit, and 3) in vitro cell-based assays measuring the expression of the transduced payload in mammalian cells. The percentage and volume yield of AAVs with full capsids were calculated using vg and capsid titers, and the collected data, expressed as relative ratios between various production lots, are shown in Tables 4 and 5, with AAV preparation #5 (Table 4) or AAV preparation #6 (tested in parallel without the capsid; Table 5) used as internal references, respectively. For viral potency, the ratios between production lots were calculated using values ​​obtained at an MOI of 5e5vg / cell for each construct in the experiments presented in Table 4. Regarding viral efficacy, the ratio between production lots was calculated using the values ​​obtained at an MOI of 3.9e5vg / cell for each construct in the experiments presented in Table 5.

[0252] [Table 4]

[0253] [Table 5]

[0254] The purity and presence of AAV capsid proteins were confirmed in the expected ratios in several AAV preparations. In short, a total of 1 e11 vg of each preparation was evaluated by gel electrophoresis under denaturing conditions, followed by Coomassie blue staining (Figure 7). In each preparation, the three capsid proteins (VP1, VP2, and VP3) were identified in the expected ratio of approximately 1:1:10, and no other proteins were detected, confirming high purity and the absence of detectable impurities or contaminants.

[0255] These results demonstrate orthogonal validation of the production capabilities of therapeutic AAV candidates (each candidate produced by a different manufacturing vendor) and highlight inter-facility availability and robust production processes across users and different stages of the process. All minor variations observed between different production service providers were within the expected range for small-batch production.

[0256] In summary, these successful cloning experiments and virus production demonstrated the high purity and uniformity of high-titer viral genomes (>1e13 vector genome (vg) / mL) in each AAV construct, the suitability of the newly designed genomes for AAV packaging and transduction experiments, and robust and clinically relevant levels of virus production.

[0257] Example 5: Parallel production of improved AAV variants for validation of AAV vectors with therapeutic payloads This embodiment describes a comparison of the functionality and packaging of different AAV vectors with therapeutic payloads.

[0258] In short, HEK293 cells (VPC2.0 cells, ThermoFisher Scientific) grown in suspension were individually transfected for each pAAV plasmid with pRepCap and pAd helper plasmids to support the packaging of the AAV genome. Upon production, the cells were lysed and subjected to nuclease digestion (benzonase), and the crude lysate was clarified by centrifugation to remove cell membranes and debris. The viral preparations were cryopreserved until vg titer testing was performed using a digital dPCR protocol with primers and probe sets specific to each payload (Figures 8A-8C).

[0259] In summary, these studies demonstrated successful virus production for each AAV construct at titers within the expected range (>1e10vg / mL), the suitability of the newly designed genome for AAV packaging and transduction experiments, and clinically relevant levels of virus production.

[0260] Example 6: Demonstration of the efficacy of an AAV with a therapeutic payload. This embodiment describes the effectiveness of payloads delivered by AAVs as described herein.

[0261] In short, HEK293T cells were seeded in 96-well plates and transduced using multiple variants of AAV-CAG-hIFNβ to achieve infection multiplicity ranging from approximately 3e4 to 1e6 vg / cell. 48 hours after transduction, the supernatant was collected for payload analysis. The amount of hIFNβ was measured in nanograms / milliliter (ng / mL; Figures 9A-9D) or picograms / milliliter (pg / mL; Figure 9E) using commercially available ELISA kits for human (h)IFNβ (Figures 9A-D) or mouse (m)IFNβ (Figure 9E).

[0262] Specifically, in the first experimental set, all preparations carried the hIFNβ payload and were packaged within the same AAV capsid serotype but were produced from plasmid #1 produced by four different vendors (Figure 9A). Analyzing the results, it was observed that when produced by a specific vendor (AAV preparation #6), the potency was lower. However, as shown previously, no other differences were detected. All other preparations (#5, #8, and #9) generated from plasmid 1 demonstrated similar potencies as measured by payload expression.

[0263] In a series of experiments, plasmid #7 (corresponding to AAV preparation #1 in Figure 9B or AAV preparation #3 in Figure 9D), plasmid #1 (corresponding to AAV preparation #9 in Figure 9B or AAV preparation #6 in Figure 9D), plasmid #12 (corresponding to AAV preparation #10 in Figure 9B), and plasmid number #14 (corresponding to AAV preparation #11 in Figure 9B), produced by the same vendor and internally packaged with the same AAV capsid serotype, showed no significant differences in their potencies among multiple variants of our AAV constructs and payloads, excluding preparation #6 shown in Figure 9A.

[0264] AAVs carrying payloads other than IFN did not result in measurable IFN expression, as expected. It was also demonstrated that AAV preparation #15 was one of those showing the highest level of potency among constructs and vendors (AAV preparation #15 was prepared by a process similar to the one used for clinical batch production).

[0265] In a series of experiments, comparing human (h) and mouse (m) cytokine payloads, it was demonstrated that mouse cytokines can be expressed in human cells (Figure 9E), thus demonstrating that such payloads can be used as controls in various in vitro and in vivo models.

[0266] In summary, these results demonstrate that the AAV described herein can successfully deliver functional therapeutic payloads, such as hIFNβ or mIFNβ, to mammalian cells.

[0267] Example 7: Demonstration of payload activity during delivery in mammalian cells This embodiment describes the activity of the payload delivered by the AAV described herein.

[0268] An activity assay using a reporter cell line was employed to measure the therapeutic transgene activity. This method utilized a reporter cell line (U937) of human premonocytes engineered to express Firefly Luciferase under the control of an IFNα / β-responsive promoter. In this model, when IFNα or IFNβ binds to the IFNα / β receptor (IFNAR1) on the cell surface, the IFNα / β-regulated Firefly Luciferase reporter gene construct is activated, resulting in a precisely quantifiable luminescence signal.

[0269] In short, AAV preparations were incubated with reporter cells at an MOI of 5e5vg / cell. AAV preparation #12 (expressing the reporter payload mCardinal) and cell culture medium (NC) were used as negative controls. Measurements were performed after an 18-hour incubation period at 37°C and 5% CO2. A standard curve was generated using commercially available recombinant hIFNβ1 cytokine (Prospect CYT-234) and expressed in infectious units / mL. Luciferase detection and measurement were performed using a reporter detection assay (Figure 10). These results demonstrated that various hIFNβ payloads were successfully expressed and biologically active in target cells. The data also correlated with efficacy measurements by payload expression, shown in Figures 9A–9E, with preparations #10 and #13 showing the highest activity and expression (Figure 9C), and preparation #6 showing the lowest activity and expression (Figures 9A and 9D).

[0270] In summary, these data demonstrate the functionality of the vector described herein in successfully delivering biologically active hIFNβ during transduction into human cells in an in vitro model.

[0271] Example 8: Therapeutic activity of AAV with therapeutic payload in cancer cells This embodiment describes the therapeutic activity of the payload delivered to cancer cells (e.g., brain tumor cells, e.g., brain tumors that have metastasized from primary cancer originating in other parts of the body) by the AAV described herein.

[0272] Patient-derived xenograft organoid (PXDO) models were established from secondary breast cancer that had metastasized to the brain, and the resulting organoids were used to monitor the antitumor effect of AAV with a therapeutic payload. Organoids were established by qualified manufacturing vendors using proprietary methods. Before seeding onto hydrogels, organoids were exposed to small amounts of culture medium for 2–4 hours with three doses of AAV preparation #6 (expressing hIFNβ) and an AAV-negative control expressing GFP (AAV-GFP). Untreated organoids were exposed to culture medium only. In addition to AAV administration, replication wells were included with 1 μm staurosporine as a positive killing control or AAV preparation buffer as a negative killing control. Six days after administration, cell viability was evaluated in four replication wells (n=4) under each treatment condition using a reporter assay (Promega G7570). Total viable cells were measured by generating a luminescence signal as luminescence (lux), which is directly proportional to the amount of ATP in culture and, consequently, the number of cells. Organoids obtained from the technical replication wells for each treatment condition were further collected, fixed, washed with PBS, and stained with Hoechst and Rhodamine Phalloidin to stain the cytoskeleton of the nucleus and eukaryotic cells, respectively. The stained organoids were imaged at 4x magnification and subsequently subjected to high-content imaging analysis using the 3D analysis platform Ominer® to quantify the effect of the compounds on tumor growth.

[0273] As shown in Figure 11, a dose-dependent decrease in organoid viability 6 days after infection was observed with AAV-hIFNβ infection, but not with AAV-GFP. In the AAV-hIFNβ-treated group, a time-dependent decrease in cell viability was observed at all tested viral concentrations. In the AAV-GFP-treated group, no significant changes were observed in any high-content imaging indicator at 6 days after treatment, indicating that AAV-GFP treatment did not have a therapeutic effect on this model at the tested doses. In organoids treated with AAV-hIFNβ, a dose- and time-dependent increase in the percentage of dead cells was observed, and furthermore, a significant decrease in total organoid size and number of nuclei per organoid was observed at all dose levels of AAV-hIFNβ. Compared to untreated and buffered organoids, AAV-IFNβ treatment showed a slight decrease in organoid number, number of nuclei, and total nuclear size at the highest viral dose tested.

[0274] These results demonstrated that the hIFNβ payload was normally expressed in target cancer cells (e.g., target brain tumor cells derived from primary cancer metastasized from other parts of the body), was biologically active, and therapeutically effective in treating cancer. Taken together, these results showed that AAV-hIFNβ induced an antitumor effect, primarily based on cytotoxicity, which was particularly pronounced at the highest dose levels tested. The data demonstrated that AAV-hIFNβ has the ability to induce antitumor activity in an ex vivo model of human tumor cells with brain metastases.

[0275] Example 9. Extended AAV delivery payload expression in GBM cancer cells This embodiment describes the therapeutic activity of the payload delivered to brain tumors by the AAV described herein.

[0276] In this example, immortalized glioblastoma (GBM) cells were exposed to either an AAV expressing an hIFNβ payload as described herein, or a recombinant hIFNβ cytokine (unvectorized). After exposure, hIFNβ levels were monitored in cell culture medium for 4 days.

[0277] In short, cells were seeded in 48-well plates and exposed to AAV preparation #13 (hIFNβ, 4e5vg / cell) or AAV preparation #12 (mCardinal, 4e5vg / cell) at a predetermined MOI. Complete culture medium changes were performed daily for a total of four days. Negative control wells were exposed to AAV expressing the fluorescent protein mCardinal (preparation #12) as a control, and to a medium without the activator. hIFNβ levels were measured from the collected supernatant using a high-sensitivity hIFNβ ELISA kit (PBL catalog number 41415).

[0278] The secreted hIFNβ levels were quantifiable as early as 10 hours after transduction, with peak expression observed at 72 hours and maintained up to 96 hours. AAV transduction alone did not stimulate therapeutic levels of hIFNβ secretion from GBM cells in vitro, as evidenced by the low levels of hIFNβ from AAV-mCardinal transduction cells. Indeed, the supernatant from AAV-mCardinal-treated cells was below the quantification limit of the measurement method in all four samples at 24 hours, all four samples at 48 hours, two samples at 72 hours, and three samples at 96 hours. On the other hand, none of the three copies that showed detectable levels in this experiment (plotted in Figure 12) exceeded 5 pg / mL.

[0279] In summary, the results demonstrate that the AAV described herein can be successfully transduced into human GBM cells to deliver a sustained hIFNβ payload.

[0280] Example 10. Production and testing of clinical-grade therapeutic AAV to initiate a Phase I / II clinical trial in brain tumor patients. This example describes the construction of clinical-grade AAVs with various combinations of regulatory elements for delivering the target gene.

[0281] AAVs containing a therapeutic payload were produced in plasmid batches in accordance with Good Manufacturing Practices (cGMP) regulations. Plasmid batches were used to produce the viral drug. An exemplary cGMP-compliant manufacturing process is shown in Figure 13.

[0282] To produce cGMP-compliant plasmid batches, master cell banks (MCBs) were created for the plasmids described herein, such as the long CMVie, short CBA, CpG-deleted hIFNβ payload, mutant WPRE, bGH polyA, and plasmid #24 with a CpG-deleted kanamycin resistance gene. Furthermore, to enable optimal AAV production by transfection, E. coli strains are selected to produce plasmids that are high-quality, highly uniform, highly stable, and predominantly supercoiled. The generated plasmids are used to produce various GLP (Good Laboratory Practice) and GMP (Good Manufacturing Practice) grade AAV batches to support preclinical development, including but not limited to toxicity and clinical trials to enable IND (Indication of Diagnosis).

[0283] A series of highly specific assays have been developed to test AAV GLP and GMP-compliant batches. These assays are used to evaluate AAV pharmaceuticals to assess safety, potency, titer, and purity criteria, as well as other appropriate critical quality characteristics.

Claims

1. From 5' to 3', a) CAG promoter and, b) A polynucleotide encoding a target payload, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent. A recombinant adeno-associated virus (rAAV) vector containing this vector.

2. From 5' to 3', a) CAG promoter and, b) Minigenes containing splice modulator binding sites, c) A polynucleotide encoding a target payload, wherein the polynucleotide comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent. A recombinant adeno-associated virus (rAAV) vector containing this vector.

3. The rAAV vector according to claim 1 or 2, wherein the payload for the purpose is a therapeutic polypeptide.

4. The rAAV vector according to claim 3, wherein the therapeutic polypeptide is a cytokine or an 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 to 6, wherein the methylation of the CpG nucleotide 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 99% or more compared to the parental equivalent.

9. The rAAV vector according to claim 8, wherein the CpG dinucleotide is completely 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%, or 99% or more compared to the parental equivalent.

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

12. From 5' to 3', a) CAG promoter and, b) Polynucleotides encoding interferon, c) Polynucleotides containing WPRE containing SEQ ID NO: 33 A recombinant adeno-associated virus (rAAV) vector containing this vector.

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

14. The rAAV vector according to claim 13, wherein the initial CMV enhancer element is derived from a wild-type CMV enhancer.

15. The rAAV vector according to claim 13 or 14, wherein the initial CMV enhancer element is cleaved with respect to the wild-type CMV enhancer.

16. The rAAV vector according to any one of claims 13 to 15, wherein the initial CMV 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 respect to sequence numbers 25 to 27.

17. The rAAV vector according to any one of claims 13 to 16, wherein the initial CMV enhancer element comprises the polynucleotide sequences described in SEQ ID NOs. 25 to 27.

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

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

52.

20. The rAAV vector according to any one of claims 13 to 18, wherein the promoter element comprises the polynucleotide sequence described in SEQ ID NO: 28 or 52.

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

22. The rAAV vector according to any one of claims 13 to 21, wherein the splice acceptor comprises a polynucleotide having at least 80% sequence identity with respect to SEQ ID NO: 30 or 53.

23. The rAAV vector according to any one of claims 13 to 21, wherein the splice acceptor comprises the polynucleotide sequence described in SEQ ID NO: 30 or 53.

24. The interleukin is IL-1α, 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-3 24. The rAAV vector according to any one of claims 6 to 23, which is 1, IL-32, IL-33, IL-34, IL-35, or IL-36.

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

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

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

30. The rAAV vector according to claim 29, wherein the interferon is the 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 respect to SEQ ID NO:

1.

32. The rAAV vector according to claim 29 or 30, wherein the human IFNβ comprises the amino acid sequence described 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 described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

35. The rAAV vector according to claim 29, wherein the interferon is the 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 respect to SEQ ID NO:

5.

37. The rAAV vector according to claim 29 or 35, wherein the human IFNα comprises the amino acid sequence described in Sequence 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 the polynucleotide described in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

8.

40. The rAAV vector according to claim 29, wherein the interferon is the 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 respect to SEQ ID NO:

9.

42. The rAAV vector according to claim 29 or 40, wherein the human IFNγ comprises the amino acid sequence described in Sequence 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 described 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 to 23, wherein the interferon is mouse IFNα, mouse IFNβ, mouse IFNγ, mouse IFNε, mouse IFNκ, mouse IFNω, mouse IFNλ, or a variant or derivative thereof.

46. The rAAV vector according to any one of claims 6 to 45, wherein the polynucleotide encoding the interferon comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to its parental equivalent.

47. The rAAV vector according to claim 46, wherein the methylation of the CpG nucleotide 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 according to claim 47, wherein the CpG dinucleotide is completely methylated.

49. The rAAV vector according to any one of claims 46 to 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 99% compared to the parental equivalent.

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

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

52. The rAAV vector, from 5' to 3', a) The first ITR sequence and b) Promoter and, c) The polynucleotide that codes for the target payload, d) The second ITR sequence and The rAAV vector according to claim 51, comprising:

53. The rAAV vector, from 5' to 3', a) The first ITR sequence and b) Promoter and, c) Minigenes and, d) The polynucleotide encoding the target payload, e) Second ITR sequence and The rAAV vector according to claim 51, comprising:

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

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

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

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

58. The first ITR sequence and / or the second ITR sequence are 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, A An rAAV vector according to any one of claims 51 to 57, comprising an ITR sequence derived from an AAV serotype selected from the group consisting of AV-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 to 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 to 59, wherein the first ITR sequence and / or the second ITR sequence comprises a polynucleotide having at least 80% sequence identity with SEQ ID NOs: 36 to 41.

61. The rAAV vector according to any one of claims 51 to 59, wherein the first ITR sequence and / or the second ITR sequence comprises the polynucleotide sequences described in SEQ ID NOs: 36 to 41.

62. The rAAV vector according to any one of claims 1 to 61, further comprising at least one modulating element.

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

64. The rAAV vector according to claim 63, wherein the regulatory element is a promoter.

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

66. The rAAV vector according to any one of claims 63 to 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, synapsin, GFAP, CaMKII, GRK1, and derivatives thereof.

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

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

69. The rAAV vector according to claim 68, wherein the WPRE is wild-type WPRE.

70. The rAAV vector according to claim 68, wherein the WPR comprises a polynucleotide having at least 80% sequence identity with respect to sequence number 32.

71. The rAAV vector according to claim 68, wherein the WPR comprises the polynucleotide described in Sequence ID No.

32.

72. The rAAV vector according to claim 68, wherein the WPRE is a modified WPRE.

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

33.

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

33.

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

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

77. The rAAV vector according to claim 76, wherein the SV40 sequence comprises a polynucleotide having at least 80% sequence identity with respect to sequence number 19.

78. The rAAV vector according to claim 76, wherein the SV40 sequence comprises the polynucleotide described in SEQ ID NO:

19.

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

80. The rAAV vector according to claim 79, wherein the bGH sequence comprises a polynucleotide having at least 80% sequence identity with respect to sequence number 34.

81. The rAAV vector according to claim 79, wherein the bGH sequence comprises the polynucleotide sequence described in SEQ ID NO:

34.

82. The rAAV vector according to any one of claims 1 or 3 to 81, further comprising a 5' minigene with respect to the polynucleotide encoding the payload of the objective.

83. The rAAV vector according to any one of claims 2 to 82, wherein the minigene encodes a splice modulator binding site.

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

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

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

87. The rAAV vector according to any one of claims 2 to 86, wherein the minigene encodes an in-frame translation termination codon.

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

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

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

91. The rAAV vector according to any one of claims 2 to 90, wherein the minigene 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 aforementioned small molecule splicing modifier 【Chemistry 1】 The rAAV vector according to claim 91, selected from the group consisting of the following.

94. The rAAV vector according to any one of claims 2 to 93, wherein the minigene comprises exons 6, 7, and 8 of SMN2, and the splice modulator binding site is recognized by LMI070.

95. The rAAV vector according to any one of claims 2 to 94, wherein the minigene contains at least about 80% sequence identity with respect to SEQ ID NO: 31 or 51.

96. The rAAV vector according to any one of claims 2 to 94, wherein the minigene comprises the polynucleotide described in SEQ ID NO: 31 or 51.

97. The rAAV vector according to any one of claims 2 to 96, wherein the minigene is regulated by the intracellular disease state.

98. The rAAV vector according to 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 to 99, wherein the minigene is regulated by cell type or tissue type.

101. The rAAV vector according to any one of claims 2 to 100, wherein the polynucleotide containing the minigene and the polynucleotide encoding the payload of the objective are linked by a polynucleotide sequence encoding a cleavable peptide.

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

103. The rAAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, 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-7 m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HS The rAAV vector according to any one of claims 1 to 102, which is C1, 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.

104. The rAAV vector according to any one of claims 1 to 103, further comprising 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, zeosin, or derivatives thereof.

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

108. The rAAV vector according to any one of claims 104 to 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 to 107, wherein the antibiotic resistance gene comprises the nucleic acid described 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 to 109, wherein the antibiotic resistance gene comprises a decrease in CpG dinucleotides and / or an increase in CpG dinucleotide methylation compared to the parental equivalent.

111. The rAAV vector according to claim 110, wherein the methylation of the CpG nucleotide 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 according to claim 110, wherein the CpG dinucleotide is completely methylated.

113. The rAAV vector according to any one of claims 110 to 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 99% compared to the parental equivalent.

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

115. The rAAV vector according to any one of claims 1 to 114, further comprising a replication origin.

116. The rAAV vector according to claim 115, wherein the replication origin 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 that requires treatment, a) A step of administering an rAAV vector as described in any one of claims 1 to 116, b) A step of administering a small molecule splicing modifier and Methods that include...

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

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

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

121. The method according to any one of claims 117 to 120, wherein the administration is directed to the ventricles of the brain.

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

123. The method according to any one of claims 117 to 121, wherein the administration is by intratumoral injection, intracranial injection, intracerebral injection, intraventricular, intraparenchymal, 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 to 123, wherein the small molecule splicing modifier is sudemycin, LMI070, RG7916, or RG7800.

125. The aforementioned small molecule splicing modifier 【Chemistry 2】 A method according to any one of claims 117 to 123, selected from the group consisting of the following.

126. From 5' to 3', a) The first ITR described in Sequence ID No. 40 or 41, b) A CAG promoter described in any one of sequence numbers 22-23, 48-50, and 64-65, c) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, d) The WPRE sequence described in Sequence ID No. 33, e) bGH polyA as described in Sequence ID No. 34, f) The second ITR described in Sequence ID No. 40 or 41 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

127. From 5' to 3', a) The first ITR described in Sequence ID No. 40 or 41, b) A CAG promoter described in any one of Sequence IDs 22 to 23, c) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, d) The WPRE sequence described in Sequence ID No. 33, e) SV40 PolyA as described in Sequence ID No. 19, f) The second ITR described in Sequence ID No. 40 or 41 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

128. From 5' to 3', a) A first ITR containing one of the items described in any one of sequence numbers 36 to 39, b) A CAG promoter described in any one of Sequence IDs 48-50 and 64-65, c) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, d) The WPRE sequence described in Sequence ID No. 33, e) bGH polyA as described in Sequence ID No. 34, f) The second ITR sequence described in any one of sequence numbers 36 to 39 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

129. From 5' to 3', a) The first ITR described in Sequence ID No. 40 or 41, b) A CAG promoter described in any one of Sequence IDs 22 to 23, c) A polynucleotide encoding a human interferon as described in Sequence ID No. 2 or Sequence ID No. 3, d) The WPRE sequence described in Sequence ID No. 33, e) SV40 PolyA as described in Sequence ID No. 19, f) The second ITR described in Sequence ID No. 40 or 41 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

130. From 5' to 3', a) The first ITR described in Sequence ID No. 40 or 41, b) A CAG promoter described in any one of Sequence IDs 22 to 23, c) The minigene described in Sequence ID No. 31 or 51, d) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, e) The WPRE sequence described in Sequence ID No. 33, f) SV40 PolyA as described in Sequence ID No. 19, g) The second ITR described in Sequence IDs 36-39 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

131. From 5' to 3', a) The first ITR described in Sequence ID No. 40 or 41, b) A CAG promoter described in any one of Sequence IDs 48-50 and 64-65, c) The minigene described in Sequence ID No. 31, d) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, e) The WPRE sequence described in Sequence ID No. 33, f) bGH polyA as described in Sequence ID No. 34, g) The second ITR described in Sequence ID No. 40 or 41 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

132. From 5' to 3', a) The first ITR described in Sequence IDs 36-39, b) A CAG promoter described in any one of Sequence IDs 48-50 and 64-65, c) The minigene described in Sequence ID No. 31 or 51, d) A polynucleotide encoding a CpG-depleted human interferon as described in Sequence ID No. 4, e) The WPRE sequence described in Sequence ID No. 33, f) bGH polyA as described in Sequence ID No. 34, g) The second ITR described in Sequence IDs 36-39 and A recombinant adeno-associated virus (rAAV) vector containing this vector.

133. From 5' to 3', a) The first ITR sequence and b) CAG promoter and, c) A coding sequence for human interferon with depleted CpG, d) WPRE sequence and, e) bGH polyA sequence, f) The second ITR sequence and A recombinant adeno-associated virus (rAAV) vector containing this vector.

134. From 5' to 3', a) The first ITR sequence and b) CAG promoter and, c) A coding sequence for human interferon with depleted CpG, d) WPRE sequence and, e) SV40 polyA sequence, f) The second ITR sequence and A recombinant adeno-associated virus (rAAV) vector containing this vector.

135. From 5' to 3', a) The first ITR sequence and b) CAG promoter and, c) The coding sequence for human interferon, d) WPRE sequence and, e) SV40 polyA sequence, f) The second ITR sequence and A recombinant adeno-associated virus (rAAV) vector containing this vector.

136. From 5' to 3', a) The first ITR sequence and b) CAG promoter and, c) The minigene described in Sequence ID No. 31 or 51, d) A coding sequence for human interferon with depleted CpG, e) WPRE sequence and, f) SV40 polyA sequence, g) Second ITR sequence and A recombinant adeno-associated virus (rAAV) vector containing this vector.

137. From 5' to 3', a) The first ITR sequence and b) CAG promoter and, c) The minigene described in Sequence ID No. 31 or 51, d) A coding sequence for human interferon with depleted CpG, e) WPRE sequence and, f) bGH polyA sequence, g) Second ITR sequence and A recombinant adeno-associated virus (rAAV) vector containing this vector.

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