Modified CpG dinucleotides for recombinant viral vector production

Abstract

The present disclosure generally relates to methods of producing recombinant viral vectors with reduced immunogenicity.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 413,196, filed on October 4, 2022, and the entire disclosure of that application is hereby incorporated by reference in its entirety for all purposes.

[0003] Summary of the Disclosure

[0004] In some embodiments, nucleic acids are described herein that comprise: an auxiliary polynucleotide comprising a reduced number of CpG dinucleotides compared to a parental equivalent. In some embodiments, nucleic acids are described herein that comprise: an auxiliary polynucleotide comprising increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the auxiliary polynucleotide comprises a promoter, enhancer, intron, microRNA, linker, splicing element, or polyadenylation signal. In some embodiments, the auxiliary polynucleotide comprises a promoter. 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, CaMKII, GRK1, mini-promoter, inducible promoter, and derivatives thereof. In some embodiments, the auxiliary polynucleotide comprises a prokaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises a eukaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from yeast. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from plants. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from adenovirus, adeno-associated virus (AAV), alphavirus, parvovirus, baculovirus, Denguevirus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus.In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is Herpes Simplex Virus (HSV) type 1 (HSV-1), HSV type 2 (HSV-2), Varicella Zoster Virus (VZV), Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), human herpesvirus (HHV) type 6 (HHV-6), HHV-7 or HHV-8. In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A and VA RNA. In some embodiments, Rep comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Cap comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the lentiviral helper polynucleotide is selected from the group consisting of: Gag, Pol, Tat, Rev, Env and VSV-G. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.In some embodiments, the nucleic acid is provided within a plasmid, phagemid, phage derivative, virus, bacmid, bacterial artificial chromosome (BAC), minicircle, doggybone, yeast artificial chromosome (YAC), or cosmid. In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises increased methylation of CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, glycopeptide, macrolide, polypeptide, tetracycline, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, geneticin, or a derivative thereof.In some embodiments, the kanamycin comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is selected from the group consisting of: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and combinations or derivatives thereof. In some embodiments, the CpG dinucleotides are reduced by at least about 10% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 50% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 75% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 10% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 50% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 75% compared to the parental equivalent. In some embodiments, the nucleic acid does not comprise inverted terminal repeats (ITRs). In some embodiments, the nucleic acid does not comprise a payload of interest. In some embodiments, the nucleic acid further comprises an accessory polynucleotide, the accessory polynucleotide comprising an increased methylation of CpG dinucleotides compared to the parental equivalent.

[0005] In some embodiments, described herein is a recombinant AAV vector comprising: a) a first nucleic acid comprising a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:13 or 14; and b) a second nucleic acid comprising an accessory polynucleotide, the accessory polynucleotide comprising a nucleotide sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10 or 11.

[0006] In some embodiments, described herein is a recombinant AAV vector comprising: a) a first nucleic acid comprising a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:14; and b) a second nucleic acid comprising one or more accessory polynucleotides, wherein a first accessory polynucleotide of the one or more accessory polynucleotides comprises a nucleotide sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10, and a second accessory polynucleotide of the one or more accessory polynucleotides comprises a nucleotide sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO:11.

[0007] In some embodiments, recombinant AAV vectors are described herein that comprise: a) a first nucleic acid comprising a payload of interest; and b) a second nucleic acid comprising an auxiliary polynucleotide, wherein the second nucleic acid has a reduced number of CpG dinucleotides compared to a parental equivalent. In some embodiments, recombinant AAV vectors are described herein that comprise: a) a first nucleic acid comprising a payload of interest; and b) a second nucleic acid comprising an auxiliary polynucleotide, wherein the second nucleic acid has methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the auxiliary polynucleotide is selected from the group consisting of: a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, and a polyadenylation signal. In some embodiments, the auxiliary polynucleotide comprises a promoter. 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, CaMKII, GRK1, a minimal promoter, an inducible promoter, and derivatives thereof. In some embodiments, the auxiliary polynucleotide comprises a prokaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises a eukaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from yeast. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from plants. In some embodiments, the auxiliary polynucleotide comprises one or more components derived from adenovirus, AAV, alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus.In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7 or HHV-8. In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A and VA RNA. In some embodiments, Rep comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Cap comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the lentiviral helper polynucleotide is selected from the group consisting of: Gag, Pol, Tat, Rev, Env and VSV-G. In some embodiments, the first nucleic acid or the second nucleic acid is DNA. In some embodiments, the first nucleic acid or the second nucleic acid is RNA. In some embodiments, the first nucleic acid or the second nucleic acid is provided within a plasmid, phagemid, phage derivative, virus, bacmid, BAC, minicircle, doggybone, YAC or cosmid. In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus or retrovirus.In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7 or HHV-8. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises increased methylation of CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a glycopeptide, a macrolide, a polypeptide, a tetracycline or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, geneticin or a derivative thereof. In some embodiments, the kanamycin comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is selected from the group consisting of pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC and combinations or derivatives thereof.In some embodiments, the CpG dinucleotides are reduced by at least about 10% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 50% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 75% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 10% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 50% compared to the parental equivalent. In some embodiments, the methylation of the CpG dinucleotides is increased by at least about 75% compared to the parental equivalent. In some embodiments, the second nucleic acid does not contain an ITR. In some embodiments, the payload of interest comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 or 14.

[0008] In some embodiments, the present disclosure describes compositions comprising: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs target a sequence for methylating cytosine in a CpG dinucleotide. In some embodiments, the CRISPR-associated endonuclease is Cas9. In some embodiments, the CRISPR-associated endonuclease is catalytically inactive. In some embodiments, the CRISPR-associated endonuclease is catalytically inactive Cas9 (dCas9) or a derivative thereof. In some embodiments, the RNA methyltransferase is a prokaryotic RNA methyltransferase. In some embodiments, the prokaryotic RNA methyltransferase is Fmu, YebU, RsmF or a derivative thereof. In some embodiments, the RNA methyltransferase is a eukaryotic RNA methyltransferase. In some embodiments, the eukaryotic RNA methyltransferase is TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7 or a derivative thereof. In some embodiments, the RNA methyltransferase is a 5-methylcytosine (m5C) RNA methyltransferase. In some embodiments, the sequence is part of the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the sequence comprises at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement. In some embodiments, the sequence comprises any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement. In some embodiments, one or more guide RNAs are encoded by a sequence according to any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement. In some embodiments, at least 100 CpG dinucleotides are methylated. In some embodiments, at least 500 CpG dinucleotides are methylated. In some embodiments, at least 1000 CpG dinucleotides are methylated. In some embodiments, one or more CpG dinucleotides within up to about 200 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within up to about 1000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within up to about 5000 base pairs of the sequence are methylated.In some embodiments, one or more CpG dinucleotides within at most about 10,000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 20,000 base pairs of the sequence are methylated.

[0009] In some embodiments, the present disclosure describes compositions comprising: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement. In some embodiments, the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease, a homing endonuclease, or a CRISPR-associated endonuclease. In some embodiments, the CRISPR-associated endonuclease is Cas9. In some embodiments, the CRISPR-associated endonuclease is catalytically inactive. In some embodiments, the CRISPR-associated endonuclease is dCas9 or a derivative thereof. In some embodiments, the methyltransferase is a DNA methyltransferase. In some embodiments, the DNA methyltransferase is a prokaryotic DNA methyltransferase. In some embodiments, the prokaryotic DNA methyltransferase is CcrM, Dcm, M.HhaI, M.HaeIII, or a derivative thereof. In some embodiments, the DNA methyltransferase is a eukaryotic DNA methyltransferase. In some embodiments, the eukaryotic DNA methyltransferase is a MET1, CMT, or DRM methyltransferase. In some embodiments, the eukaryotic DNA methyltransferase is a METI, METIIa, METIII, METIIb, PMET, CMET5, CMET21, ZMET1, Masc1, or a derivative thereof. In some embodiments, the DNA methyltransferase is DNMT1 or DNMT3a. In some embodiments, the methyltransferase is an RNA methyltransferase. In some embodiments, the RNA methyltransferase is a prokaryotic RNA methyltransferase. In some embodiments, the prokaryotic RNA methyltransferase is Fmu, YebU, RsmF, or a derivative thereof. In some embodiments, the RNA methyltransferase is a eukaryotic RNA methyltransferase. In some embodiments, the eukaryotic RNA methyltransferase is a TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, or a derivative thereof. In some embodiments, the RNA methyltransferase is a 5-methylcytosine (m5C) RNA methyltransferase. In some embodiments, the sequence is part of the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the sequence has at least about 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement. In some embodiments, the sequence comprises any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement. In some embodiments, the nucleic acid is a guide RNA (gRNA).In some embodiments, the gRNA is encoded by a sequence comprising at least about 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, the gRNA is encoded by a sequence of any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof.

[0010] In some embodiments, cells are described herein that comprise a vector or composition of any of the foregoing embodiments. 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. 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 engineered cells. In some embodiments, the cells are stable cells. In some embodiments, the vector or composition is transiently expressed. In some embodiments, the vector or composition is stably expressed. In some embodiments, the vector or composition is stably expressed from an inducible system.

[0011] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: a) providing a cell with a nucleic acid comprising a payload of interest; and b) culturing the cell of step a) in a growth medium comprising cytosine or a derivative thereof to generate a recombinant AAV vector that is methylated and contains at least about 25% CpG dinucleotides. In some embodiments, the concentration of cytosine or its derivative is in the range of about 100 micromoles per liter (μM) to about 5 millimoles per liter (mM). In some embodiments, the concentration of cytosine or its derivative is up to 3 mM. In some embodiments, cytosine or its derivative is selected from the group consisting of methylcytosine, phosphorylated cytosine, and phosphorothioate cytosine. In some embodiments, the nucleic acid comprises reduced CpG dinucleotides. In some embodiments, the nucleic acid comprises increased methylation of CpG dinucleotides. In some embodiments, the nucleic acid comprises reduced CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, the method further comprises providing, in step a), a nucleic acid comprising: a helper polynucleotide comprising reduced CpG dinucleotides compared to a parental equivalent. In some embodiments, the method further comprises providing, in step a), a nucleic acid comprising: a helper polynucleotide comprising increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the method further comprises providing, in step a), a composition comprising: a) a CRISPR-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, the one or more guide RNAs targeting a sequence for methylating cytosine in a CpG dinucleotide. In some embodiments, the method further comprises providing, in step a), a composition comprising: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence having at least 97% sequence identity to a sequence comprising any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement. In some embodiments, in step a), the cell is transfected with a polynucleotide. In some embodiments, in step a), the cell is infected with a polynucleotide. In some embodiments, the cell is in suspension. In some embodiments, the cell is adherent. In some embodiments, the cell is an engineered stable cell line. In some embodiments, the payload of interest comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 or 14.

[0012] In some embodiments, the present disclosure describes pharmaceutical compositions comprising: a recombinant AAV vector comprising a payload of interest; more than one helper polynucleotide comprising reduced CpG dinucleotides; and a pharmaceutically acceptable excipient.

[0013] In some embodiments, the present disclosure describes pharmaceutical compositions comprising: a) a recombinant AAV vector comprising a payload of interest; b) more than one helper polynucleotide comprising increased methylation of CpG dinucleotides; and c) a pharmaceutically acceptable excipient.

[0014] In some embodiments, described herein are pharmaceutical compositions having less than about 50% CpG dinucleotides compared to parental equivalents, the pharmaceutical compositions comprising a) a recombinant AAV vector comprising a payload of interest; b) more than one helper polynucleotide; and c) a pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises less than about 50% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 30% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 20% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 10% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 15% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 10% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 5% CpG dinucleotides. In some embodiments, the more than one helper polynucleotide comprises less than about 25% CpG dinucleotides. In some embodiments, the more than one helper polynucleotide comprises less than about 40% CpG dinucleotides. In some embodiments, the more than one helper polynucleotide comprises less than about 50% CpG dinucleotides. In some embodiments, the helper polynucleotide is selected from the group consisting of promoters, enhancers, introns, microRNAs, linkers, splicing elements, and polyadenylation signals. In some embodiments, the helper polynucleotide comprises a promoter. 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, CaMKII, GRK1, minimal promoters, inducible promoters, and derivatives thereof. In some embodiments, the helper polynucleotide comprises a prokaryotic helper polynucleotide. In some embodiments, the helper polynucleotide comprises a eukaryotic helper polynucleotide. In some embodiments, the helper polynucleotide comprises one or more components derived from yeast. In some embodiments, the helper polynucleotide comprises one or more components derived from plants. In some embodiments, the helper polynucleotide comprises one or more components derived from adenovirus, adeno-associated virus (AAV), alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus.In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is herpes simplex virus (HSV) type 1 (HSV-1), HSV type 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV) type 6 (HHV-6), HHV-7 or HHV-8. In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A and VA RNA. In some embodiments, Rep comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Cap comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 8 or 11. In some embodiments, the lentiviral helper polynucleotide is selected from the group consisting of: Gag, Pol, Tat, Rev, Env and VSV-G. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises increased methylation of CpG dinucleotides. In some embodiments, the helper polynucleotide comprises a backbone polynucleotide that comprises reduced CpG dinucleotides and increased methylation of CpG dinucleotides. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene.In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, glycopeptide, macrolide, polypeptide, tetracycline, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, hygromycin, or a derivative thereof. In some embodiments, kanamycin comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is selected from the group consisting of pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and combinations or derivatives thereof. In some embodiments, the CpG dinucleotides are reduced by at least about 10% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 50% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by at least about 75% compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 10% compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 50% compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 75% compared to the parental equivalent. In some embodiments, the payload of interest comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:13 or 14. Brief Description of the Drawings

[0016] The present disclosure may be more fully understood with reference to the following drawings.

[0017] Figure 1 is a table of the standard human genetic code.

[0018] Figure 2 is a table of the standard Escherichia coli (E. coli) genetic code.

[0019] Figure 3 is a DNA map of the wild-type adeno-associated virus (AAV) replication (Rep) gene in an exemplary plasmid. The Rep gene encodes four Rep proteins involved in viral replication ("Rep78", "Rep68", "Rep52", and "Rep40"), which are flanked by inverted terminal repeats (ITRs; not shown) and three promoters "p5" (not shown), "p19", and "p40".

[0020] Figure 4Is a DNA map of the wild-type AAV capsid (Cap) gene in an exemplary plasmid. The Cap gene encodes three Cap proteins (viral proteins (VP) 1 (VP1), VP2, and VP3) involved in capsid formation and is flanked by inverted terminal repeats (ITRs; not shown) and three promoters (p5, p19, and p40; not shown).

[0021] Figure 5 Is a DNA map of the wild-type kanamycin resistance gene in an exemplary plasmid.

[0022] Figure 6 Is a schematic diagram of a recombinant adeno-associated virus (rAAV) plasmid (“pRep_Cap_KanR_CpG-depleted”) generated from CpG-depleted AAV Rep, CpG-depleted AAV Cap, and CpG-depleted KanR in a packaging plasmid.

[0023] Figure 7 Is a schematic diagram of an rAAV plasmid (“pRep_CpG-depleted_Cap_KanR”) generated from CpG-depleted AAV Rep and CpG-depleted KanR in a packaging plasmid.

[0024] Figure 8 Is a schematic diagram of an rAAV plasmid (“pRep_Cap_CpG-depleted_KanR”) generated from CpG-depleted AAV Cap and CpG-depleted KanR in a packaging plasmid.

[0025] Figure 9 Is a set of Figure 6 (“Plasmid 1”), Figure 7 (“Plasmid 2”), and Figure 8 (“Plasmid 3”) described in three experimental plasmids or micrographs of VPC2.0 cells expressing GFP observed after transfection with a control plasmid (“pRep_Cap_KanR”, also referred to as “control”) having endogenous CpG in Rep, Cap, and KanR.

[0026] Details

[0027] Viral vectors are commonly used to deliver therapeutic genes to humans. In particular, adeno-associated virus (AAV) has been used in recombinant viral vector systems for therapeutic delivery. Wild-type AAV is a small, non-enveloped human parvovirus that is non-pathogenic to humans. The AAV genome contains two open reading frames, Rep and Cap, flanked by two inverted terminal repeats (ITRs). Typically, recombinant adeno-associated virus (rAAV) vectors retain the ITRs located on either side of a custom expression cassette containing the payload of interest. To generate rAAV, additional vectors, such as helper polynucleotides that mediate AAV replication in vitro, are required.

[0028] Currently, there are over 200 ongoing human clinical trials using rAAV, which contain drugs ultimately for use in humans or veterinary animals. Many clinical trials have demonstrated the safety, non-toxicity, and efficacy of recombinant viral vectors in animal models and humans. However, recent clinical data have led to a renewed focus on rAAV toxicity and immunogenicity. In particular, elements within the rAAV genome, in combination with process-derived contaminants and impurities from different production platforms, have raised concerns because once administered to patients, they are potential triggers of the innate immune response.

[0029] One major immunostimulatory molecular pattern identified for AAV-induced immune responses involves CpG. CpG refers to cytosine (C) linked by a phosphate bond (p) to guanine (G) in a CG dinucleotide within a DNA genome. The frequency of CpG and the methylation status of cytosine in CpG vary across species. CpG plays a key role in human immune responses, and their methylation status is an important trigger for human immune responses. In humans, unmethylated C in CpG is recognized by the Toll-like receptor 9 pathway as a DNA signature of a pathogenic origin. This triggers an immune response against hypomethylated DNA.

[0030] CpG, particularly unmethylated CpG, can trigger immune responses in humans, which results in rAAV toxicity and immunogenicity. In some embodiments, the source of unmethylated CpG in rAAV preparations is found in the final packaged rAAV vectors, which are accidentally packaged from various sources such as from helper polynucleotides. Another source of unmethylated CpG comes from unpackaged impurities from components (e.g., helper polynucleotides) necessary for generating rAAV in the final rAAV preparation. Thus, there is a need to improve recombinant viral vectors and preparations for use in therapeutic gene therapy and genome editing.

[0031] Definitions

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In general, the nomenclature and techniques described herein in connection with immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well known and commonly used in the art. Units of measure not otherwise defined conform to The International System of Units (SI), NIST Special Publication 330, 2019 Edition.

[0033] Unless the context clearly indicates otherwise, as used herein, all numerical values or ranges of numerical values include all integers within such ranges or covering such ranges, and fractions of the values or integers within or covering the ranges. Thus, for example, reference to a range of 90%-100% includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, reference to a range of 1-5,000 times includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4 or 1.5 times, etc., 2.1, 2.2, 2.3, 2.4 or 2.5 times, etc., and so on.

[0034] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an" and "the" are also intended to include the plural forms. It should also be understood that the terms "comprises" and / or "comprising", when used in this specification, specify the presence of the stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] As used herein, the term "adeno-associated virus vector" or "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16. The AAV vector may have one or more AAV wild-type genes that are completely or partially deleted, for example, the Rep and / or Cap genes, but retain the functional flanking inverted terminal repeat (ITR) sequences. The functional ITR sequences facilitate the rescue, replication, and packaging of AAV virions. Thus, an AAV vector is defined herein as containing at least those sequences (e.g., functional ITRs) that are required in cis for the replication and packaging of the virus. The ITR only needs to provide for functional rescue, replication, and packaging, and the sequence does not need to be a wild-type polynucleotide sequence and may be altered, for example, by insertion, deletion, or substitution of nucleotides, in some embodiments.

[0036] The term "adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the regions flanking each end of the AAV genome that function together in cis as an origin of DNA replication and as a packaging signal for the virus. The AAV ITR, together with the AAV Rep coding region, can also provide for efficient excision and integration of a polynucleotide sequence inserted between two flanking ITRs into the mammalian genome. As used herein, "AAV ITR" does not necessarily encompass a wild-type polynucleotide sequence and, in some embodiments, is altered, for example, by insertion, deletion, or substitution of nucleotides. Additionally, the AAV ITR is derived from any of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16, etc. Further, the 5' and 3' ITRs flanking a selected polynucleotide sequence in an AAV vector need not be the same or derived from the same AAV serotype or isolate, so long as they function as expected, for example, to permit the desired therapeutic or genome editing effect.In addition, the AAV ITR is derived from any one of several AAV serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, and AAV-HSC16.

[0037] Unless specifically stated or apparent from the context, as used herein, the term "about" when referring to a number or a range of numbers shall be understood to mean the recited number and numbers ±10% thereof, or for values listed in a range, 10% less than the listed lower limit and 10% more than the listed upper limit.

[0038] As used herein, "capsid gene" or "Cap gene" or "Cap" refers to the wild-type or recombinant gene of a protein that encodes the capsid protein of any viral vector (e.g., AAV) that is a component of the viral particle. In some embodiments, the Cap gene has a reduced number of CpG dinucleotides, increased methylation of CpG dinucleotides, or both.

[0039] As used herein, "complementary" generally refers to a polynucleotide comprising a nucleotide sequence capable of selectively annealing, under certain conditions, to an identified region of a target polynucleotide. As used herein, the terms "substantially complementary" and grammatical equivalents are intended to mean a polynucleotide comprising a nucleotide sequence capable of specifically annealing, under certain conditions, to an identified region of a target polynucleotide. Annealing refers to the nucleobase pairing interaction of one nucleic acid with another nucleic acid that results in the formation of a duplex, triplex, or other higher-order structure. Through Watson-Crick and Hoogsteen-type hydrogen bonding, the primary interactions are generally nucleobase-specific, e.g., A:T, A:U, and G:C. In some embodiments, base stacking and hydrophobic interactions can also contribute to duplex stability. Hybridization generally refers to the process by which two single-stranded polynucleotides non-covalently bind to form a stable double-stranded polynucleotide. The resulting double-stranded polynucleotide is a "hybrid" or "duplex". In some cases, hybridization does not require 100% sequence identity, and in some embodiments, hybridization occurs with approximately greater than 70%, 75%, 80%, 85%, 90%, or 95% sequence identity. In some embodiments, sequence identity includes sequences containing insertions and / or deletions in addition to non-identical nucleobases.

[0040] A "CpG" site refers to a region in DNA where a cytosine nucleotide appears next to a guanine nucleotide along the length of the linear nucleic acid sequence of the nucleotides, e.g., -C-phosphate-G-, i.e., the cytosine and guanine are separated by only one phosphate, or the cytosine is on the 5' side of the guanine nucleotide.

[0041] "Helper polynucleotide" refers to a polynucleotide that permits the generation of a vector (e.g., rAAV). Exemplary helper polynucleotides of the present disclosure include, but are not limited to, promoters, enhancers, introns, microRNAs, linkers, splicing elements, polyadenylation signals, Rep, Cap, E1A, E1B, E4, E2A, ampicillin resistance gene, kanamycin resistance gene, VA, Gag, Pol, Tat, Rev, Env, and VSV-G. In some embodiments, the helper polynucleotide has a reduced number of CpG dinucleotides, increased methylation of CpG dinucleotides, or both. The helper polynucleotides of the present disclosure do not include an expression cassette containing a payload of interest, which includes a promoter operably linked to the payload of interest. "Helper polynucleotide" is also intended to include, but is not limited to, promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcriptional termination signals such as polyadenylation signals and poly-U sequences). Helper polynucleotides include nucleic acids that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). In some embodiments, helper polynucleotides, such as tissue-specific promoters, direct expression primarily in a desired tissue of interest such as muscle, neuron, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In some embodiments, helper polynucleotides also direct expression in a time-dependent manner, such as cell cycle-dependent or developmental stage-dependent, tissue-specific, environment-specific, or cell type-specific. In some embodiments, helper polynucleotides also direct expression in an inducible manner, such as will be controlled by the expression or administration of an appropriate inducer molecule. The term "helper polynucleotide" also encompasses enhancer elements such as WPRE (e.g., WPRE variants); CMV enhancer; the R-U5' segment in the LTR of HTLV-I; SV40 enhancer; and the intron sequence between exon 2 and exon 3 of rabbit β-globin.

[0042] "Helper virus" refers to a virus that permits replication of a co-infected virus that is otherwise defective and packaging by a host cell.

[0043] As used herein, the term "immunogenicity" refers to the ability of a substance to induce an immune response in a recipient. In some embodiments, an immune response is induced when the immune system of an organism or a certain type of immune cell is exposed to an immunogenic substance. The term "non-immunogenic" refers to the lack or absence of an immune response above a detectable threshold to a substance. In some embodiments, no immune response is detected when the immune system of an organism or a certain type of immune cell is exposed to a non-immunogenic substance. In some embodiments, provided herein are non-immunogenic compositions, vectors or nucleic acids of the present disclosure that do not induce an immune response above a predetermined threshold when measured by an immunogenicity assay. In some embodiments, provided herein are compositions, vectors or nucleic acids with reduced immunogenicity of the present disclosure that induce a reduced immune response below a predetermined threshold when measured by an immunogenicity assay. For example, when an immunogenicity assay is used to measure antibodies produced against an inflammatory marker, the non-immunogenic compositions or compositions with reduced immunogenicity provided herein result in the production of antibodies or markers at levels below a predetermined threshold. The predetermined threshold is, for example, at most 1.5-fold, 2-fold, 3-fold, 4-fold or 5-fold of the antibody or marker level produced by a control reference.

[0044] As used herein, the term "nuclease-deficient variant of a Cas nuclease", "Cas (dead Cas) without catalytic activity" or "dCas" refers to a Cas nuclease-inactive mutant protein generated by mutating both of the two cleavage domains of a wild-type Cas nuclease. For example, dCas will retain its ability to bind to genomic DNA through guide RNA:genomic DNA base pairing; however, unlike wild-type Cas nucleases in which permanent gene disruption is achieved through cleavage by the nuclease, dCas does not introduce cleavage.

[0045] As used herein, the term "parvovirus" encompasses the family Parvoviridae, including but not limited to the autonomous parvoviruses and the dependoviruses. Autonomous parvoviruses include, for example, the genus Bocavirus, the genus Dependovirus, the genus Erythrovirus, the genus Amdovirus, the genus Parvovirus, the genus Densovirus, the genus Iterovirus, the genus Containvirus, the genus Avarporvovirus, the genus Copiparvovirus, the genus Protoparvovirus, the genus Tetrapivorvirus, the genus Ambidensovirus, the genus Brevinnovovirus, the genus Hepdensovirus, and the genus Pendensovirus. Exemplary autonomous parvoviruses include but are not limited to porcine parvovirus, murine parvovirus, canine parvovirus, mink enteritis virus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus (feline panleukemia virus), feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus.

[0046] As used herein, the terms "RNA-guided", "guide RNA", and "gRNA" refer to any RNA molecule that facilitates the targeting of a polynucleotide-guided protein (e.g., dCas) to a target nucleic acid as described herein. For example, a gRNA is a molecule that recognizes (e.g., binds to) a target nucleic acid. The RNA guidance is designed to be complementary to the target nucleic acid. Exemplary "guide RNAs" include but are not limited to PEgRNA, dRNA, CRISPR RNA (crRNA), pre-crRNA, mature crRNA, or a crRNA combined with a homologous tracrRNA. In some embodiments, the latter is an independent RNA or fused to a single RNA using a linker. As used herein, the term "CRISPR RNA" or "crRNA" refers to a 17-20 nucleotide sequence that is complementary to the target DNA. As used herein, the term "trans-activating RNA" or "tracrRNA" refers to a ribonucleic acid sequence that acts as a scaffold to link the crRNA to a Cas nuclease.

[0047] "Percent identity", "% identity", or "sequence identity" refers to the degree to which two sequences (nucleotide or amino acid) have the same residue at the same position in an alignment. For example, "a nucleotide sequence is X% identical to SEQ ID NO:Y" refers to the % identity of the nucleotide sequence to SEQ ID NO:Y and is detailed as X% of the residues in the nucleotide sequence being identical to the corresponding residues of the sequence disclosed in SEQ ID NO:Y. A sequence said to be X% identical to a reference sequence may contain more nucleotide or amino acid residues than specified in the reference sequence, but must contain a sequence corresponding to the reference sequence. In most cases, the sequences under discussion will contain sequences corresponding to all of the specified reference sequences. Usually, computer programs are applied to perform such calculations. Exemplary programs for comparing and aligning sequence pairs include ALIGN, FASTA, BLAST with gaps, BLASTP, BLASTN, or GCG.

[0048] The term "plasmid" refers to an extrachromosomal element that carries genes that can replicate independently of the cell's chromosome. A plasmid can be in the form of a circular double-stranded DNA molecule. Such elements can include autonomous replication sequences, genomic integration sequences, phage, or nucleotide sequences, as well as linear, circular, or supercoiled, single-stranded or double-stranded DNA or RNA from any source. Exemplary plasmids include, but are not limited to, minicircle plasmids and doggybone plasmids. In some embodiments, the plasmid (e.g., transfer plasmid or packaging plasmid) has reduced CpG dinucleotides, increased methylation of CpG dinucleotides, or both.

[0049] "Polynucleotide" or "nucleic acid" is used interchangeably herein and refers to a nucleotide chain of any length and includes DNA and RNA. In some embodiments, the nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate incorporated into the chain by a DNA polymerase or an RNA polymerase. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. If present, the modification to the nucleotide structure is imparted before or after chain assembly. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, the polynucleotide is further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capping"; replacing one or more naturally occurring nucleotides with analogs; internucleotide modifications such as, for example, those having uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates) and charged linkages (e.g., phosphorothioates, dithiophosphates); those containing side group moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine); those having intercalating agents (e.g., acridine, psoralen); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides); those containing alkylating agents; those having modified linkages (e.g., α-anomeric nucleic acids) and the unmodified forms of polynucleotides. In some embodiments, any hydroxyl group normally present in the sugar is replaced, for example, by a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides or conjugated to a solid support. In some embodiments, the 5' and 3' terminal OHs are phosphorylated or replaced by an amine or organic capping group of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. In some embodiments, the polynucleotide also contains analog forms of ribose or deoxyribose sugars, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-ribose or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars or β-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranoses, furanoses, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. In some embodiments, one or more phosphodiester linkages are replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate ester is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NRi (“amidate”), P(O)R, P(O)OR’, CO or CH2 (“formacetal”), where each R or R’ is independently H or an optionally substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl optionally containing an ether (-O-) linkage. Not all linkages in the polynucleotide need to be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0050] “Promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence that controls the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3’ to the promoter sequence. A promoter may be derived from a native gene, or may include different elements derived from different promoters found in nature, or a synthetic DNA segment. In some embodiments, different promoters direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions or inducer molecules. A promoter that causes a gene to be expressed in most cell types most of the time is often referred to as a “constitutive promoter”. A promoter that causes a gene to be expressed in a specific cell and tissue type is often referred to as a “cell-specific promoter” or a “tissue-specific promoter”, respectively. A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is often referred to as a “development-specific promoter” or a “cell differentiation-specific promoter”. A promoter that is induced and causes gene expression after exposure or treatment of cells with an agent, biomolecule, chemical, ligand, light, etc. that induces the promoter is often referred to as an “inducible promoter” or a “regulatable promoter”. It is also recognized that in some embodiments, different lengths of DNA fragments have the same promoter activity since the exact boundaries of regulatory sequences have not been fully defined in most cases. In some embodiments, the promoter has a reduced number of CpG dinucleotides, increased methylation of CpG dinucleotides, or both.

[0051] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable for mammals, particularly human or animal patients.

[0052] As used herein, the term "protospacer" refers to a DNA sequence of approximately 20 base pairs adjacent to a protospacer adjacent motif sequence. The protospacer shares the same sequence as the spacer sequence of the gRNA. The gRNA anneals to the complement of the protospacer sequence on the target DNA (specifically, on one of its strands, i.e., the "target strand" versus the "non-target strand" of the target DNA sequence). For the Cas nuclease to function, it also requires a specific protospacer adjacent motif, which varies depending on the bacterial species of the Cas gene. One of ordinary skill in the art will understand that the literature in the prior art sometimes refers to the "protospacer" as the ~20 nucleotide target-specific guide sequence on the gRNA itself, rather than using the term "spacer" to refer to it. The context of the description surrounding the occurrence of either "protospacer" or "spacer" will help inform the reader whether the term refers to the gRNA or the DNA target.

[0053] As used herein, the term "protospacer adjacent sequence" or "PAM" refers to a DNA sequence of approximately 2-6 base pairs, which is an important targeting component of the Cas nuclease. Generally, the PAM sequence is located on either strand and downstream of the Cas nuclease cleavage site in the 5' to 3' direction. A typical PAM sequence (i.e., the PAM sequence associated with the Cas9 nuclease of Streptococcus pyogenes or SpCas9) is 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. Different PAM sequences are associated with different Cas nucleases or equivalent proteins from different organisms. Additionally, in some embodiments, any given Cas nuclease is modified to alter the PAM specificity of the polynucleotide-guided nuclease such that the polynucleotide-guided nuclease recognizes an alternative PAM sequence. It should also be understood that Cas nucleases from different bacterial species (i.e., orthologs) can have different PAM specificities.

[0054] 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 of non-AAV origin) flanked by at least one AAV ITR. In some embodiments, such rAAV vectors are replicated and packaged into virus particles when present in a host cell having a suitable helper polynucleotide or virus (or expressing suitable helper functions) and expressing the AAV Rep and Cap gene products (i.e., the AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the rAAV vector is referred to as a "provirus" and is "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.

[0055] The term "transgene" refers to a polynucleotide that is introduced into a cell and that can be transcribed by the cell into RNA and optionally translated into protein and / or expressed under appropriate conditions. A transgene can confer desired properties on the cell into which it is introduced, or otherwise result in a desired therapeutic or diagnostic outcome.

[0056] The term "payload of interest" refers to a polynucleotide that is introduced into a cell and that can perform an intended function (such as gene repair or gene editing). In some embodiments, the payload of interest confers desired properties on the cell into which it is introduced, or otherwise results in a desired therapeutic or diagnostic outcome.

[0057] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors or another suitable replicon (e.g., viral vector). A variety of vectors have been developed for delivering polynucleotides encoding exogenous polynucleotides or proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO1994 / 011026; incorporated herein by reference as it relates to vectors suitable for expressing nucleic acid molecules of interest. Expression vectors suitable for the compositions and methods described herein contain polynucleotide sequences and, for example, additional sequence elements for expressing heterologous nucleic acid material (e.g., nucleic acid molecules) in a cell. Certain vectors for expressing the nucleic acid molecules described herein contain plasmids containing regulatory sequences such as promoters and enhancer regions that direct gene transcription. In some embodiments, the compact bidirectional promoter does not contain an enhancer. Other useful vectors for expressing the nucleic acid agents disclosed herein contain polynucleotide sequences that enhance the translation rate of these polynucleotides or improve the stability or nuclear export of the RNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signals (polyA) to direct efficient transcription of the genes carried on the expression vector. In some embodiments, the expression vectors suitable for the compositions and methods described herein contain backbone polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, neomycin, geneticin, kanamycin, nourseothricin, aminoglycoside, beta-lactam, glycopeptide, macrolide, polypeptide, tetracycline, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, chloramphenicol or derivatives thereof.

[0058] As used herein, the term "wild type" or "wild-type" refers to the typical form of an organism, strain, gene, nucleic acid, vector, or vector component that exists in nature as distinguished from mutant or variant forms.

[0059] The polynucleotides of the present disclosure

[0060] Unmethylated CpGs can trigger an immune response in humans and result in vector toxicity and immunogenicity. Reducing the number of CpGs, increasing the methylation of unmethylated CpGs, or both, can reduce such toxicity and immunogenicity, particularly in components involved in the production of recombinant viral vectors (e.g., rAAV). Such components are described herein as helper polynucleotides that are involved in the production of recombinant viral vectors and provide assistance during the production of recombinant viral vectors, such as by providing genes for helper replication or packaging. A helper polynucleotide as described herein refers to any component that is not the transgene or payload of interest and is not the corresponding component for the expression or function of the transgene or payload of interest.

[0061] In some embodiments, nucleic acids are described herein that comprise: a helper polynucleotide comprising a reduced number of CpG dinucleotides compared to a parental equivalent. In some embodiments, nucleic acids are also described herein that comprise: a helper polynucleotide comprising an increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the nucleic acid comprises a reduced number of CpG dinucleotides and an increased methylation of CpG dinucleotides.

[0062] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0063] In some embodiments, the helper polynucleotide is within a plasmid (e.g., a circular DNA molecule that can replicate autonomously within a cell), cosmid (e.g., pWE or sCos vector), artificial chromosome, human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), minicircle, doggybone, P1-derived artificial chromosome (PAC), phagemid, phage derivative, telomere, or virus. In some embodiments, the helper polynucleotide is within a mammalian vector selected from the list consisting of: pSF-CMV-NEO-NH2-PPT-3XFLAG, pSF-CMV-NEO-COOH-3XFLAG, pSF-CMV-PURO-NH2-GST-TEV, pSF-OXB20-COOH-TEV-FLAG(R)-6His, pCEP4 pDEST27, pSF-CMV-Ub-KrYFP, pSF-CMV-FMDV-daGFP, pEF1a-mCherry-N1 vector, pEF1a-tdTomato vector, pSF-CMV-FMDV-Hygro, pSF-CMV-PGK-Puro, pMCP-tag(m), and pSF-CMV-PUBO-NH2-CMYC; bacterial expression vectors: pSF-OXB20-BetaGal, pSF-OXB20-Fluc, pSF-OXB20, and pSF-Tac; plant expression vectors: pRI 101-ANDNA and pCambia2301; and yeast expression vectors: pTYB21 and pKLAC2, and insect vectors: pAc5.1 / V5-His A and pDEST8.

[0064] In some embodiments, the helper polynucleotide is within a virus. In some embodiments, the virus is alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the virus is alphavirus. In some embodiments, the virus is parvovirus. In some embodiments, the virus is adenovirus. In some embodiments, the virus is AAV. In some embodiments, the virus is baculovirus. In some embodiments, the virus is dengue virus. In some embodiments, the virus is lentivirus. In some embodiments, the virus is herpes virus. In some embodiments, the virus is poxvirus. In some embodiments, the virus is circovirus. In some embodiments, the virus is bocavirus. In some embodiments, the virus is vaccinia virus. In some embodiments, the virus is retrovirus.

[0065] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is herpes simplex virus type 1 (HSV 1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7) or human herpesvirus 8 (HHV-8).

[0066] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof.In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0067] 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.

[0068] In some embodiments, the helper polynucleotide comprises a reduced number of CpG dinucleotides compared to the parental equivalent. In some embodiments, the number of CpG dinucleotides is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 99% compared to the parental equivalent. In some embodiments, the CpG dinucleotides are reduced by about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the CpG dinucleotides are reduced by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0069] In some embodiments, the helper polynucleotide comprises an increased methylation of CpG dinucleotides compared to the parental equivalent. In some embodiments, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95% compared to the parental equivalent. In some embodiments, the number of CpG dinucleotides 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the number of CpG dinucleotides is increased by about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%.

[0070] In some embodiments, the helper polynucleotide does not contain inverted terminal repeats (ITRs).

[0071] In some embodiments, the helper polynucleotide does not contain a payload of interest.

[0072] In some embodiments, the helper polynucleotide comprises a promoter, enhancer, intron, microRNA, linker, splicing element, polyadenylation signal sequence, or a combination thereof. In some embodiments, the helper polynucleotide comprises a promoter. In some embodiments, the helper polynucleotide comprises an enhancer. In some embodiments, the helper polynucleotide comprises an intron. In some embodiments, the helper polynucleotide comprises a microRNA. In some embodiments, the helper polynucleotide comprises a linker. In some embodiments, the helper polynucleotide comprises a splicing element. In some embodiments, the helper polynucleotide comprises a polyadenylation signal sequence.

[0073] In some embodiments, the auxiliary polynucleotide comprises a promoter. 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, CaMKII, GRK1, a minimal promoter, an inducible promoter, 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 the synapsin promoter or a derivative thereof. In some embodiments, the promoter is the CaMKII promoter or a derivative thereof. In some embodiments, the promoter is the GRK1 promoter or a derivative thereof. In some embodiments, the promoter is a minimal promoter or a derivative thereof. In some embodiments, the promoter is an inducible promoter.

[0074] In some embodiments, the auxiliary polynucleotide comprises a prokaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises a eukaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide consists of a eukaryotic auxiliary polynucleotide and a prokaryotic auxiliary polynucleotide.

[0075] In some embodiments, the auxiliary polynucleotide comprises one or more components derived from yeast (e.g., an autonomously replicating sequence, a centromere, or a telomere). In some embodiments, the auxiliary polynucleotide comprises one or more components derived from plants (e.g., virA, virB, virD, virG, or virE).

[0076] In some embodiments, the helper polynucleotide comprises one or more components derived from a virus (e.g., a promoter, Rep, Cap). In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is HSV type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8. 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 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 a circovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0077] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof.In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0078] 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.

[0079] In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A, and VA RNA. For example, in some embodiments, the adenovirus or AAV helper polynucleotide is Rep. In some embodiments, the adenovirus or AAV helper polynucleotide is Cap. In some embodiments, the adenovirus or AAV helper polynucleotide is E1A. In some embodiments, the adenovirus or AAV helper polynucleotide is E1B. In some embodiments, the adenovirus or AAV helper polynucleotide is E4. In some embodiments, the adenovirus or AAV helper polynucleotide is E2A. In some embodiments, the adenovirus or AAV helper polynucleotide is VA RNA.

[0080] In some embodiments, Rep is derived from AAV1 or a derivative thereof. In some embodiments, Rep is derived from AAV2 or a derivative thereof. In some embodiments, Rep is derived from AAV3 or a derivative thereof. In some embodiments, Rep is derived from AAV4 or a derivative thereof. In some embodiments, Rep is derived from AAV5 or a derivative thereof. In some embodiments, Rep is derived from AAV6 or a derivative thereof. In some embodiments, Rep is derived from AAV7 or a derivative thereof. In some embodiments, Rep is derived from AAV8 or a derivative thereof. In some embodiments, Rep is derived from AAV9 or a derivative thereof. In some embodiments, Rep is derived from AAV10 or a derivative thereof. In some embodiments, Rep is derived from AAV11 or a derivative thereof. In some embodiments, Rep is derived from AAV12 or a derivative thereof. In some embodiments, Rep is derived from AAV13 or a derivative thereof. In some embodiments, Rep is derived from AAV14 or a derivative thereof. In some embodiments, Rep is derived from AAV15 or a derivative thereof. In some embodiments, Rep is derived from AAV16 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh8 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh10 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh20 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh39 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh74 or a derivative thereof. In some embodiments, Rep is derived from AAV-rhM4-1 or a derivative thereof. In some embodiments, Rep is derived from AAV-hu37 or a derivative thereof. In some embodiments, Rep is derived from AAV-Anc80 or a derivative thereof. In some embodiments, Rep is derived from AAV-Anc80L65 or a derivative thereof. In some embodiments, Rep is derived from AAV-7m8 or a derivative thereof. In some embodiments, Rep is derived from AAV-PHP-B or a derivative thereof. In some embodiments, Rep is derived from AAV-PHP-EB or a derivative thereof. In some embodiments, Rep is derived from AAV-2.5 or a derivative thereof. In some embodiments, Rep is derived from AAV-2tYF or a derivative thereof. In some embodiments, Rep is derived from AAV-3B or a derivative thereof. In some embodiments, Rep is derived from AAV-LK03 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC1 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC2 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC3 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC4 or a derivative thereof.In some embodiments, Rep is derived from AAV-HSC5 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC6 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC7 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC8 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC9 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC10 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC11 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC12 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC13 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC14 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC15 or a derivative thereof. In some embodiments, Rep is derived from AAV-TT or a derivative thereof. In some embodiments, Rep is derived from AAV-DJ / 8 or a derivative thereof. In some embodiments, Rep is derived from AAV-Myo or a derivative thereof. In some embodiments, Rep is derived from AAV-NP40 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP59 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP22 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP66 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC16 or a derivative thereof.

[0081] In some embodiments, Rep comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98% or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7 or 10. In some embodiments, Rep comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 7 or 10.

[0082] In some embodiments, Cap comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98% or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, Cap comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:8 or 11.

[0083] In some embodiments, the lentiviral accessory polynucleotide is selected from the group consisting of: Gag, Pol, Tat, Rev, Env, and VSV-G. In some embodiments, the lentiviral accessory polynucleotide is Gag. In some embodiments, the lentiviral accessory polynucleotide is Pol. In some embodiments, the lentiviral accessory polynucleotide is Tat. In some embodiments, the lentiviral accessory polynucleotide is Rev. In some embodiments, the lentiviral accessory polynucleotide is Env. In some embodiments, the lentiviral accessory polynucleotide is VSV-G.

[0084] In some embodiments, the accessory polynucleotide comprises a backbone polynucleotide that contains fewer CpG dinucleotides compared to a parental equivalent. In some embodiments, the backbone polynucleotide contains increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the backbone polynucleotide contains fewer CpG dinucleotides and increased methylation of CpG dinucleotides compared to a parental equivalent.

[0085] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, an open reading frame, or a combination thereof. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, and an open reading frame.

[0086] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, glycopeptide, macrolide, polypeptide, tetracycline, or a derivative thereof. In some embodiments, the backbone polynucleotide comprises an aminoglycoside or a derivative thereof. In some embodiments, the backbone polynucleotide comprises a β-lactam or a derivative thereof. In some embodiments, the backbone polynucleotide comprises a glycopeptide or a derivative thereof. In some embodiments, the backbone polynucleotide comprises a macrolide or a derivative thereof. In some embodiments, the backbone polynucleotide comprises a polypeptide or a derivative thereof. In some embodiments, the backbone polynucleotide comprises a tetracycline or a derivative thereof.

[0087] In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, gougerotin, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin.

[0088] In some embodiments, kanamycin comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 75% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:9 or 12.

[0089] In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, a combination thereof, or a derivative. 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.

[0090] In some embodiments, the accessory polynucleotide of the present disclosure comprises a post-transcriptional element of woodchuck hepatitis virus or a derivative thereof.

[0091] In some embodiments, the nucleic acids described herein comprise reporter sequences for co-expression, such as, but not limited to, lacZ, GFP, CFP, YFP, RFP, mCherry, mCardinal, firefly luciferase, Renilla luciferase, NanoLuc luciferase, and tdTomato. In some embodiments, the vector comprises a selectable marker.

[0092] In some embodiments, accessory polynucleotides are described herein, wherein the accessory polynucleotide comprises two or more of a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, a polyadenylation signal sequence. In some embodiments, accessory polynucleotides are described herein, wherein the accessory polynucleotide comprises two or more of a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, a polyadenylation signal, a viral life cycle-encoding gene or a fragment thereof such as Rep, Cap, E1A, E1B, E4, E2A, ampicillin resistance gene, kanamycin resistance gene, VA, Gag, Pol, Tat, Rev, Env, and VSV-G.

[0093] In some embodiments, nucleic acids are described herein that comprise accessory polynucleotides, wherein any two or more of the accessory polynucleotides of the present disclosure are provided in the same plasmid or in different plasmids (e.g., a packaging plasmid, a transfer plasmid, or an accessory plasmid).

[0094] Recombinant viral vector

[0095] Viral genomes provide a rich source of vectors for the efficient delivery of exogenous polynucleotides into cells. Examples of viral vectors are parvoviruses (e.g., AAV), retroviruses (e.g., retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus. Examples of retroviruses are avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentiviruses, alpharetrovirus, gammaretrovirus, spumavirus.Other examples are murine leukemia viruses, murine sarcoma viruses, murine mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0096] The present disclosure contemplates a variety of recombinant viral vectors. In some embodiments, the recombinant viral vector is a recombinant herpesvirus, which is herpes simplex virus (HSV), such as HSV type 1 (HSV-1), HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV) type 6 (HHV-6), HHV-7, or HHV-8. In some embodiments, the HSV is HSV-1. In some embodiments, the HSV is HSV-2. In some embodiments, the HSV is VZV. In some embodiments, the HSV is EBV. In some embodiments, the HSV is CMV. In some embodiments, the HSV is HHV-6. In some embodiments, the HSV is HHV-7. In some embodiments, the HSV is HHV-8.

[0097] In some embodiments, the recombinant viral vector is an AAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16.

[0098] In some embodiments, the recombinant AAV is AAV1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV4 or a derivative thereof. In some embodiments, the recombinant AAV is AAV5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV16 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-7m8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, the recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC4 or a derivative thereof.In some embodiments, the recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, the recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Myo or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC16 or a derivative thereof.

[0099] Recombinant AAV vector

[0100] Recombinant adeno-associated virus vectors (rAAV) can be used for the incorporation of genes to facilitate the introduction of genes into cells, such as target cells. Described herein are recombinant AAV (rAAV) vectors that comprise: a first nucleic acid comprising a payload of interest; and a second nucleic acid comprising an accessory polynucleotide, wherein the second nucleic acid has a reduced number of CpG dinucleotides compared to a parental equivalent. Also described herein are recombinant AAV vectors that comprise: a first nucleic acid comprising a payload of interest; and a second nucleic acid comprising an accessory polynucleotide, wherein the second nucleic acid has increased methylation of CpG dinucleotides compared to a parental equivalent.

[0101] In some embodiments, the first nucleic acid or the second nucleic acid is DNA. In some embodiments, the first nucleic acid or the second nucleic acid is RNA.

[0102] In some embodiments, the payload of interest is interferon beta (IFNβ). In some embodiments, the IFNβ comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98% or 99%) sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 75% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 95% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 97% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 98% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having at least 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14. In some embodiments, the IFNβ comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO: 13 or 14.

[0103] In some embodiments, the payload of interest is IFNα, IFNβ, IFNγ, or a combination thereof. In some embodiments, the payload of interest is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, and IFNα-IFNβ-IFNγ. In some embodiments, the payload of interest is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, and IFNα-IFNβ-IFNγ and the IFN is murine IFN. In some embodiments, the payload of interest is IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, and IFNα-IFNβ-IFNγ and the IFN is human IFN. In some embodiments, the payload of interest is a combination of human or murine IFNα, IFNβ, IFNγ, IFNα-IFNβ, IFNα-IFNγ, IFNβ-IFNγ, and IFNα-IFNβ-IFNγ. In some embodiments, the payload of interest is human IFNα, human IFNβ, human IFNγ, or a combination thereof. In some embodiments, the payload of interest is murine IFNα, murine IFNβ, murine IFNγ, or a combination thereof. In some embodiments, the payload of interest is human IFNα, human IFNβ, human IFNγ, murine IFNα, murine IFNβ, murine IFNγ, or a combination thereof.

[0104] In some embodiments, the rAAV comprises a backbone having a reduced number of CpG dinucleotides, an increased number of methylated CpG dinucleotides, or both. In some embodiments, the nucleic acid comprises those sequences of AAV that are cis-required for replication and packaging of DNA (e.g., functional ITRs) into virions. In some embodiments, the rAAV vector contains a marker or reporter gene.

[0105] Useful rAAV vectors include those having all or part of one or more native AAV genes deleted but retaining functional flanking ITR sequences. 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, it is any suitable AAV serotype, including any currently known or later discovered serotype or any engineered, evolved, selected, or chimeric capsid serotype.

[0106] The capsid proteins of AAV constitute the outer non-nucleic acid portion of the virion and are encoded by the AAV Cap gene. In some embodiments, the Cap gene encodes VP1, VP2, VP3, MAAP, AP, or a combination thereof.

[0107] In some embodiments, the helper polynucleotide comprises Rep, Cap, or helper functions required for production of the rAAV of the present disclosure. In some embodiments, the Rep, Cap, or helper polynucleotide is 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 acid encoding the capsid proteins is delivered to the packaging host cell via two vectors; a first vector comprising a first nucleic acid encoding two capsid proteins (e.g., VP1 and VP2) and a second vector comprising a second nucleic acid encoding a single capsid protein (e.g., VP3). In some embodiments, three vectors each comprising a nucleic acid encoding a different capsid protein are delivered to the packaging host cell.

[0108] In some embodiments, a single nucleic acid encoding more than one replication protein (e.g., Rep78, Rep68, Rep52, and Rep40) is delivered to the packaging host cell in a single vector. In some embodiments, the nucleic acid encoding the replication protein is delivered to the packaging host cell via two vectors; a first vector comprising a first nucleic acid encoding one to three replication proteins and a second vector comprising a second nucleic acid encoding one to three replication proteins. In some embodiments, four vectors each comprising a nucleic acid encoding a different replication protein are delivered to the packaging host cell.

[0109] In some embodiments, a single nucleic acid encoding more than one adenovirus helper protein (e.g., E1A, E1B, E4, E2A, and VARNA) is delivered to the packaging host cell in a single vector. In some embodiments, the nucleic acid encoding the adenovirus helper protein is delivered to the packaging host cell via two vectors. In some embodiments, more than two vectors each comprising a nucleic acid encoding a different capsid protein are delivered to the packaging host cell. The selected genetic element is delivered by any suitable method, including those described herein. Methods for constructing any embodiment of the present disclosure include genetic engineering, recombineering, and synthetic techniques.

[0110] In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16.

[0111] In some embodiments, the recombinant AAV is AAV1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV4 or a derivative thereof. In some embodiments, the recombinant AAV is AAV5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV16 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh20 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh39 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rh74 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-rhM4-1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-hu37 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Anc80L65 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-7m8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-PHP-EB or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2.5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-2tYF or a derivative thereof. In some embodiments, the recombinant AAV is AAV-3B or a derivative thereof. In some embodiments, the recombinant AAV is AAV-LK03 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC1 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC2 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC3 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC4 or a derivative thereof.In some embodiments, the recombinant AAV is AAV-HSC5 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC6 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC7 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC9 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC10 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC11 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC12 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC13 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC14 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC15 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-TT or a derivative thereof. In some embodiments, the recombinant AAV is AAV-DJ / 8 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-Myo or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP40 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP59 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP22 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-NP66 or a derivative thereof. In some embodiments, the recombinant AAV is AAV-HSC16 or a derivative thereof.

[0112] In some embodiments, the rAAV vector is a pseudotyped rAAV vector. A pseudotyped vector includes an AAV vector of a given serotype that is pseudotyped with a Cap gene from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, etc.). For example, a representative pseudotyped vector is an AAV2 vector pseudotyped with a Cap gene from AAV serotype 8 or AAV serotype 9.

[0113] In some embodiments, the AAV has a mutation within the virion capsid that is used to more effectively transduce a particular cell type compared to an unmutated capsid virion. In some embodiments, suitable AAV mutants have ligand insertion mutations for promoting targeting of AAV to a particular cell type. In some embodiments, construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants are used.

[0114] In some embodiments, artificial AAV capsids are used. Such artificial capsids are generated by any suitable technique using selected AAV sequences (e.g., fragments of the VP1 capsid protein) in combination with heterologous sequences obtained from different selected AAV serotypes, non - contiguous portions of the same AAV serotype, from non - AAV viral sources, or from non - viral sources. Artificial AAV serotypes include but are not limited to pseudotyped AAV, chimeric AAV capsids, recombinant AAV capsids, or “humanized” AAV capsids.

[0115] Other rAAV virions used in the compositions and methods of the present disclosure include but are not limited to those capsid hybrids generated by molecular breeding of viruses, by exon shuffling, or by using artificial intelligence or machine learning.

[0116] In some embodiments, the capsid is modified to improve therapy. In some embodiments, the capsid is modified to obtain minimized immunogenicity, better stability and particle persistence, efficient degradation, and / or accurate delivery of the heterologous coding sequence or its functional fragment or variant to the cell nucleus. In some embodiments, the modification or mutation is an amino acid deletion, insertion, substitution, or any combination thereof 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, and 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.

[0117] In another embodiment, the modified capsid polypeptide of the present disclosure contains a modified sequence, wherein such modification can include both conservative and non - conservative substitutions, deletions, and / or additions, and generally contains a peptide sharing 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 the corresponding wild - type capsid protein.

[0118] In some embodiments, the helper polynucleotide contains fewer CpG dinucleotides compared to the parental equivalent. In some embodiments, compared to the parental equivalent, 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 95%. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the CpG dinucleotides are reduced by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0119] In some embodiments, the helper polynucleotide comprises increased methylation of CpG dinucleotides compared to the parental equivalent. In some embodiments, compared to the parental equivalent, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95%. In some embodiments, the number of CpG dinucleotides 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the number of CpG dinucleotides is increased by about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is in the range of about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%.

[0120] In some embodiments, the auxiliary polynucleotide comprises a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, a polyadenylation signal sequence, or a combination thereof. In some embodiments, the auxiliary polynucleotide comprises a promoter. In some embodiments, the auxiliary polynucleotide comprises an enhancer. In some embodiments, the auxiliary polynucleotide comprises an intron. In some embodiments, the auxiliary polynucleotide comprises a microRNA. In some embodiments, the auxiliary polynucleotide comprises a linker. In some embodiments, the auxiliary polynucleotide comprises a splicing element. In some embodiments, the auxiliary polynucleotide comprises a polyadenylation signal sequence.

[0121] In some embodiments, the auxiliary polynucleotide comprises a promoter. 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, CaMKII, GRK1, minimal promoter, inducible promoter, 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 the synapsin promoter or a derivative thereof. In some embodiments, the promoter is the CaMKII promoter or a derivative thereof. In some embodiments, the promoter is the GRK1 promoter or a derivative thereof. In some embodiments, the promoter is the minimal promoter or a derivative thereof. In some embodiments, the promoter is the inducible promoter or a derivative thereof.

[0122] In some embodiments, the auxiliary polynucleotide comprises a prokaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises a eukaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide consists of a eukaryotic auxiliary polynucleotide and a prokaryotic auxiliary polynucleotide.

[0123] In some embodiments, the auxiliary polynucleotide comprises one or more components derived from yeast (e.g., autonomously replicating sequence, centromere, or telomere). In some embodiments, the auxiliary polynucleotide comprises one or more components derived from plants (e.g., virA, virB, virD, virG, or virE).

[0124] In some embodiments, the helper polynucleotide comprises one or more components derived from yeast (e.g., an autonomous replication sequence, a centromere, or a telomere). In some embodiments, the helper polynucleotide comprises one or more components derived from a plant (e.g., virA, virB, virD, virG, or virE).

[0125] In some embodiments, the helper polynucleotide comprises one or more components derived from a virus (e.g., a promoter, Rep, Cap). In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is herpes simplex virus type 1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8. 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 a circovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0126] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof.In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0127] 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.

[0128] In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A, and VA RNA. In some embodiments, the adenovirus or AAV helper polynucleotide is Rep. In some embodiments, the adenovirus or AAV helper polynucleotide is Cap. In some embodiments, the adenovirus or AAV helper polynucleotide is E1A. In some embodiments, the adenovirus or AAV helper polynucleotide is E1B. In some embodiments, the adenovirus or AAV helper polynucleotide is E4. In some embodiments, the adenovirus or AAV helper polynucleotide is E2A. In some embodiments, the adenovirus or AAV helper polynucleotide is VA RNA.

[0129] In some embodiments, Rep is derived from AAV1 or a derivative thereof. In some embodiments, Rep is derived from AAV2 or a derivative thereof. In some embodiments, Rep is derived from AAV3 or a derivative thereof. In some embodiments, Rep is derived from AAV4 or a derivative thereof. In some embodiments, Rep is derived from AAV5 or a derivative thereof. In some embodiments, Rep is derived from AAV6 or a derivative thereof. In some embodiments, Rep is derived from AAV7 or a derivative thereof. In some embodiments, Rep is derived from AAV8 or a derivative thereof. In some embodiments, Rep is derived from AAV9 or a derivative thereof. In some embodiments, Rep is derived from AAV10 or a derivative thereof. In some embodiments, Rep is derived from AAV11 or a derivative thereof. In some embodiments, Rep is derived from AAV12 or a derivative thereof. In some embodiments, Rep is derived from AAV13 or a derivative thereof. In some embodiments, Rep is derived from AAV14 or a derivative thereof. In some embodiments, Rep is derived from AAV15 or a derivative thereof. In some embodiments, Rep is derived from AAV16 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh8 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh10 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh20 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh39 or a derivative thereof. In some embodiments, Rep is derived from AAV-rh74 or a derivative thereof. In some embodiments, Rep is derived from AAV-rhM4-1 or a derivative thereof. In some embodiments, Rep is derived from AAV-hu37 or a derivative thereof. In some embodiments, Rep is derived from AAV-Anc80 or a derivative thereof. In some embodiments, Rep is derived from AAV-Anc80L65 or a derivative thereof. In some embodiments, Rep is derived from AAV-7m8 or a derivative thereof. In some embodiments, Rep is derived from AAV-PHP-B or a derivative thereof. In some embodiments, Rep is derived from AAV-PHP-EB or a derivative thereof. In some embodiments, Rep is derived from AAV-2.5 or a derivative thereof. In some embodiments, Rep is derived from AAV-2tYF or a derivative thereof. In some embodiments, Rep is derived from AAV-3B or a derivative thereof. In some embodiments, Rep is derived from AAV-LK03 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC1 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC2 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC3 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC4 or a derivative thereof.In some embodiments, Rep is derived from AAV-HSC5 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC6 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC7 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC8 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC9 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC10 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC11 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC12 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC13 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC14 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC15 or a derivative thereof. In some embodiments, Rep is derived from AAV-TT or a derivative thereof. In some embodiments, Rep is derived from AAV-DJ / 8 or a derivative thereof. In some embodiments, Rep is derived from AAV-Myo or a derivative thereof. In some embodiments, Rep is derived from AAV-NP40 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP59 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP22 or a derivative thereof. In some embodiments, Rep is derived from AAV-NP66 or a derivative thereof. In some embodiments, Rep is derived from AAV-HSC16 or a derivative thereof.

[0130] In some embodiments, Rep comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98% or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:7 or 10. In some embodiments, Rep comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:7 or 10.

[0131] In some embodiments, the Cap comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98% or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:8 or 11. In some embodiments, the Cap comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:8 or 11.

[0132] In some embodiments, the lentiviral accessory polynucleotides are selected from the group consisting of: Gag, Pol, Tat, Rev, Env, and VSV-G. In some embodiments, the lentiviral accessory polynucleotide is Gag. In some embodiments, the lentiviral accessory polynucleotide is Pol. In some embodiments, the lentiviral accessory polynucleotide is Tat. In some embodiments, the lentiviral accessory polynucleotide is Rev. In some embodiments, the lentiviral accessory polynucleotide is Env. In some embodiments, the lentiviral accessory polynucleotide is VSV-G.

[0133] In some embodiments, the accessory polynucleotide comprises a backbone polynucleotide that contains fewer CpG dinucleotides compared to a parental equivalent. Also described herein are recombinant AAV vectors comprising an accessory polynucleotide, wherein the accessory polynucleotide comprises a backbone polynucleotide that contains increased methylation of CpG dinucleotides compared to a parental equivalent. Also described herein are recombinant AAV vectors comprising an accessory polynucleotide, wherein the accessory polynucleotide comprises a backbone polynucleotide that contains fewer CpG dinucleotides and increased methylation of CpG dinucleotides compared to a parental equivalent.

[0134] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, an open reading frame, or a combination thereof. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, and an open reading frame.

[0135] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a glycopeptide, a macrolide, a polypeptide, a tetracycline, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a β-lactam or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a glycopeptide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a macrolide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a polypeptide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a tetracycline or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, gougerotin, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin or a derivative thereof.

[0136] In some embodiments, kanamycin comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:9 or 12.

[0137] In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, a combination thereof, or a derivative 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.

[0138] Recombinant AAV vectors comprising helper polynucleotides are described herein, wherein any two or more of the helper polynucleotides of the present disclosure are provided in the same plasmid or in different plasmids (e.g., packaging plasmids, transfer plasmids, or helper plasmids).

[0139] Methyltransferase composition

[0140] In some embodiments, methods and compositions for increasing methylation of CpG dinucleotides are described herein. In some embodiments, the methods and compositions for increasing methylation of CpG dinucleotides comprise a methyltransferase. In some embodiments, compositions are described herein that comprise: a nuclease fused to a methyltransferase (e.g., an RNA methyltransferase or a DNA methyltransferase) for generating a vector having increased methylation of unmethylated CpGs. In some embodiments, compositions are also described herein that comprise: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement. In some embodiments, compositions are also described herein that comprise: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, the one or more guide RNAs targeting a sequence for methylating cytosine in a CpG dinucleotide.

[0141] This disclosure describes compositions that comprise a nuclease fused to a methyltransferase, wherein the methyltransferase is an RNA methyltransferase. In some embodiments, the RNA methyltransferase is a 5-methylcytosine (m5C) RNA methyltransferase. In some embodiments, the RNA methyltransferase is a prokaryotic RNA methyltransferase (e.g., Fmu, YebU, RsmF, or a derivative thereof). In some embodiments, the prokaryotic RNA methyltransferase is Fmu, YebU, RsmF, or a derivative thereof. In some embodiments, the prokaryotic RNA methyltransferase is Fmu or a derivative thereof. In some embodiments, the prokaryotic RNA methyltransferase is YebU or a derivative thereof. In some embodiments, the prokaryotic RNA methyltransferase is RsmF or a derivative thereof.

[0142] In some embodiments, the RNA methyltransferase is a eukaryotic RNA methyltransferase (e.g., TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, or a derivative thereof). In some embodiments, the eukaryotic RNA methyltransferase is TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is TRM4B or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is TRDMT1 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NOP2A(OL12) or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN1(NOP2) or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN2 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN3 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN4 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN5 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN6 or a derivative thereof. In some embodiments, the eukaryotic RNA methyltransferase is NSUN7 or a derivative thereof.

[0143] In some embodiments, the methyltransferase is a DNA methyltransferase. In some embodiments, the DNA methyltransferase is DNMT1, DNMT2, or DNMT3a. In some embodiments, the DNA methyltransferase is DNMT1. In some embodiments, the DNA methyltransferase is DNMT2. In some embodiments, the DNA methyltransferase is DNMT3a. In some embodiments, the DNA methyltransferase is a prokaryotic DNA methyltransferase (e.g., CcrM, Dcm, M.HhaI methyltransferase, or derivatives thereof). In some embodiments, the prokaryotic DNA methyltransferase is the CcrM methyltransferase or derivatives thereof. In some embodiments, the prokaryotic DNA methyltransferase is the Dcm methyltransferase or derivatives thereof. In some embodiments, the prokaryotic DNA methyltransferase is the M.HhaI methyltransferase or derivatives thereof. In some embodiments, the prokaryotic DNA methyltransferase is the M.HhaI methyltransferase or derivatives thereof.

[0144] In some embodiments, the DNA methyltransferase is a eukaryotic DNA methyltransferase (e.g., MET1, CMT, or DRM methyltransferase). In some embodiments, the eukaryotic DNA methyltransferase is the MET1 methyltransferase. In some embodiments, the eukaryotic DNA methyltransferase is the CMT methyltransferase. In some embodiments, the eukaryotic DNA methyltransferase is the DRM methyltransferase.

[0145] In some embodiments, the eukaryotic DNA methyltransferase is METI, METIIa, METIII, METIIb, PMET, CMET5, CMET21, ZMET1, Masc1, or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is METI or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is METIIa or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is METIII or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is METIIb or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is PMET or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is CMET5 or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is CMET21 or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is ZMET1 or derivatives thereof. In some embodiments, the eukaryotic DNA methyltransferase is Masc1 or derivatives thereof.

[0146] In some embodiments, the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease, or a CRISPR-associated endonuclease.

[0147] Exemplary CRISPR-associated endonucleases include, but are not limited to, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, Casio, CaslOd, CasF, CasG, CasH, CasX, CasΦ, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966. In some embodiments, the CRISPR-associated endonuclease is Cas1, CaslB, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Casio, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cas9, Casl2 (e.g., Casl2a, Casl2b, Casl2c, Casl2d, Casl2k, Casl2J, Casl2L, etc.), Casl3 (e.g., Casl3a, Casl3b (e.g., Casl3b-t1, Casl3b-t2, Casl3b-t3), Casl3c, Casl3d, etc.), Casl4, CasX, CasY, or an engineered form of a Cas protein. In some embodiments, the CRISPR-associated endonuclease is Cas9. In some embodiments, the CRISPR-associated endonuclease is catalytically inactive. In some embodiments, the CRISPR-associated endonuclease is catalytically inactive Cas9 (dCas9) or a derivative thereof.

[0148] In some embodiments, the CRISPR-associated endonuclease (e.g., Cas9) is from or derived from Staphylococcus aureus, Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.) Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidates Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus or Acaryochloris marina.

[0149] In some embodiments, the CRISPR-associated endonuclease (e.g., Cas9) is wild-type, modified, or a fragment thereof. In some embodiments, the CRISPR-associated endonuclease (e.g., Cas9) comprises an amino acid sequence identical to that of wild-type Streptococcus pyogenes or Staphylococcus aureus. In some embodiments, the CRISPR-associated endonuclease (e.g., Cas9) is modified to alter one or more properties of the protein (e.g., nuclease activity, affinity, stability, etc.) relative to the wild-type or another Cas protein. In some embodiments, domains of the CRISPR-associated endonuclease (e.g., Cas9) that are not involved in RNA-guided cleavage are eliminated from the protein such that the modified Cas protein is smaller than the wild-type Cas protein.

[0150] In some embodiments, the CRISPR-associated endonuclease comprises at least one RNA recognition and / or RNA binding domain. In some embodiments, the RNA recognition and / or RNA binding domain interacts with a guide RNA (gRNA). In some embodiments, the CRISPR-associated endonuclease comprises a nuclease domain (e.g., a DNase or RNase domain), a DNA binding domain, a helicase domain, an RNase domain, a protein-protein interaction domain, a dimerization domain, or a combination thereof.

[0151] In some embodiments, the CRISPR-associated endonuclease used herein is substantially homologous to the Cas proteins disclosed herein (e.g., Cas9, saCas9, spCas9). In some embodiments, a "substantially homologous" protein is about 50%, about 70%, about 80%, about 90%, about 95%, or about 99% homologous to the amino acid sequence of the Cas proteins disclosed herein.

[0152] In some embodiments, the CRISPR-associated endonuclease is Cas9.

[0153] In some embodiments, the CRISPR-associated endonuclease is catalytically inactive (e.g., a nuclease-deficient variant (dCas) of a Cas nuclease).

[0154] In some embodiments, the CRISPR-associated endonuclease is catalytically inactive Cas9 (dCas9) or a derivative thereof. For example, a nuclease-deficient variant of a Cas nuclease (dCas) (which is an engineered nuclease-deficient variant of a Cas nuclease that can pair with a guide RNA (gRNA) (e.g., dCas9, dCas9-KRAB, or dCs9-SID4X)) is fused to an RNA methyltransferase to methylate unmethylated CpGs.

[0155] In some embodiments, the present disclosure describes a composition comprising: a CRISPR-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase; and one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, wherein the one or more guide RNAs target a sequence for methylating cytosine in a CpG dinucleotide, and the sequence comprises a portion of the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the sequence is a portion of the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 nucleotides of the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the sequence comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the sequence comprises from about 15 nucleotides to about 28 nucleotides. In some embodiments, the sequence comprises at least about 15 nucleotides. In some embodiments, the sequence comprises at most about 28 nucleotides.In some embodiments, the sequence comprises from about 15 to about 18 nucleotides, from about 15 to about 19 nucleotides, from about 15 to about 20 nucleotides, from about 15 to about 21 nucleotides, from about 15 to about 22 nucleotides, from about 15 to about 23 nucleotides, from about 15 to about 24 nucleotides, from about 15 to about 25 nucleotides, from about 15 to about 28 nucleotides, from about 16 to about 21 nucleotides, from about 16 to about 22 nucleotides, from about 16 to about 23 nucleotides, from about 16 to about 24 nucleotides, from about 16 to about 25 nucleotides, from about 16 to about 28 nucleotides, from about 17 to about 22 nucleotides, from about 17 to about 23 nucleotides, from about 17 to about 24 nucleotides, from about 17 to about 25 nucleotides, from about 17 to about 28 nucleotides, from about 18 to about 23 nucleotides, from about 18 to about 24 nucleotides, from about 18 to about 25 nucleotides, from about 18 to about 28 nucleotides, from about 19 to about 24 nucleotides, from about 19 to about 25 nucleotides, from about 19 to about 28 nucleotides, from about 20 to about 21 nucleotides, from about 20 to about 22 nucleotides, from about 20 to about 25 nucleotides, from about 20 to about 28 nucleotides, or from about 21 to about 28 nucleotides of the nucleic acid sequence of SEQ ID NO:1.

[0156] This disclosure describes a composition comprising: a nuclease fused to a methyltransferase; and a nucleic acid targeting a sequence having at least 97% (e.g., 98% or 99%) sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence has at least 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the sequence is any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement.

[0157] Table 1. Sequences

[0158]

[0159] In some embodiments, the composition and method comprise one or more guide RNAs. In some embodiments, the guide RNA comprises a sense sequence. In some embodiments, the guide RNA comprises an antisense sequence. In some embodiments, the guide RNA comprises a nucleotide sequence other than a region complementary or substantially complementary to a region of the target sequence. For example, the guide RNA is part of or is considered part of or is included in a crRNA, e.g., a crRNA:tracrRNA chimera.

[0160] In some embodiments, the guide RNA (e.g., gRNA) comprises synthetic nucleotides or modified nucleotides. In some embodiments, the guide RNA comprises one or more internucleotide linkers modified from natural phosphodiesters. In some embodiments, all internucleotide linkers of the guide RNA or its contiguous nucleotide sequence are modified. For example, in some embodiments, the internucleotide linkage comprises sulfur (S), such as a phosphorothioate internucleotide linkage.

[0161] In some embodiments, the guide RNA (e.g., gRNA) comprises a modification to the ribose or nucleobase. In some embodiments, the guide RNA comprises one or more nucleosides that comprise a modified sugar moiety, where the modified sugar moiety is a modification of the sugar moiety as compared to the ribose sugar moiety found in deoxyribonucleic acid (DNA) and RNA. In some embodiments, the modification is within the ribose ring structure. Exemplary modifications include, but are not limited to, substitution with a hexose ring (HNA), a bicyclic (e.g., locked nucleic acid (LNA)) having a biradical bridge between the C2 and C4 carbons of the ribose ring, or an unlinked ribose ring (e.g., unlocked nucleic acid (UNA)) that generally lacks a bond between the C2 and C3 carbons. In some embodiments, the sugar-modified nucleoside comprises bicyclohexose nucleic acid or tricyclic nucleic acid. In some embodiments, the modified nucleoside comprises a nucleoside in which the sugar moiety is replaced with a non-sugar moiety, such as in the case of peptide nucleic acid (PNA) or morpholino nucleic acid.

[0162] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the sugar modification comprises a modification by changing a substituent group on the ribose ring to a group other than hydrogen or the 2'-OH group naturally present in DNA and RNA nucleosides. In some embodiments, a substituent is introduced at the 2', 3', 4', 5' position or a combination thereof. In some embodiments, the nucleoside having a modified sugar moiety comprises a 2'-modified nucleoside, e.g., a 2'-substituted nucleoside. In some embodiments, the 2'-sugar-modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2' position (a 2'-substituted nucleoside) or comprises a 2'-linked biradical, and includes 2'-substituted nucleosides and LNA (2'-4' biradical bridged) nucleosides. Examples of 2'-substituted modified nucleosides include, but are not limited to, 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. In some embodiments, the modification in the ribose group comprises a modification at the 2' position of the ribose group. In some embodiments, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-deoxy, and 2'-O-(2-methoxyethyl).

[0163] In some embodiments, the guide RNA comprises one or more modified sugars. In some embodiments, the guide RNA comprises only modified sugars. In certain embodiments, the guide RNA comprises greater than about 10%, 25%, 50%, 75%, or 90% modified sugars. In some embodiments, the modified sugar is a bicyclic sugar. In some embodiments, the modified sugar comprises a 2'-O-methoxyethyl group. In some embodiments, the guide RNA comprises both internucleoside linker modifications and nucleoside modifications.

[0164] In some embodiments, the guide RNA comprises from about 15 nucleotides to about 28 nucleotides. In some embodiments, the guide RNA comprises at least about 15 nucleotides. In some embodiments, the guide RNA comprises at most about 28 nucleotides. In some embodiments, the guide RNA comprises from about 15 nucleotides to about 16 nucleotides, from about 15 nucleotides to about 17 nucleotides, from about 15 nucleotides to about 18 nucleotides, from about 15 nucleotides to about 19 nucleotides, from about 15 nucleotides to about 20 nucleotides, from about 15 nucleotides to about 21 nucleotides, from about 15 nucleotides to about 22 nucleotides, from about 15 nucleotides to about 23 nucleotides, from about 15 nucleotides to about 24 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 15 nucleotides to about 28 nucleotides, from about 16 nucleotides to about 17 nucleotides, from about 16 nucleotides to about 18 nucleotides, from about 16 nucleotides to about 19 nucleotides, from about 16 nucleotides to about 20 nucleotides, from about 16 nucleotides to about 21 nucleotides, from about 16 nucleotides to about 22 nucleotides, from about 16 nucleotides to about 23 nucleotides, from about 16 nucleotides to about 24 nucleotides, from about 16 nucleotides to about 25 nucleotides, from about 16 nucleotides to about 28 nucleotides, from about 17 nucleotides to about 18 nucleotides, from about 17 nucleotides to about 19 nucleotides, from about 17 nucleotides to about 20 nucleotides, from about 17 nucleotides to about 21 nucleotides, from about 17 nucleotides to about 22 nucleotides, from about 17 nucleotides to about 23 nucleotides, from about 17 nucleotides to about 24 nucleotides, from about 17 nucleotides to about 25 nucleotides, from about 17 nucleotides to about 28 nucleotides, from about 18 nucleotides to about 19 nucleotides, from about 18 nucleotides to about 20 nucleotides, from about 18 nucleotides to about 21 nucleotides, from about 18 nucleotides to about 22 nucleotides, from about 18 nucleotides to about 23 nucleotides, from about 18 nucleotides to about 24 nucleotides, from about 18 nucleotides to about 25 nucleotides, from about 18 nucleotides to about 28 nucleotides, from about 19 nucleotides to about 20 nucleotides, from about 19 nucleotides to about 21 nucleotides, from about 19 nucleotides to about 22 nucleotides, from about 19 nucleotides to about 23 nucleotides, from about 19 nucleotides to about 24 nucleotides, from about 19 nucleotides to about 25 nucleotides, from about 19 nucleotides to about 28 nucleotides, from about 20 nucleotides to about 21 nucleotides, from about 20 nucleotides to about 22 nucleotides, from about 20 nucleotides to about 23 nucleotides, from about 20 nucleotides to about 24 nucleotides, from about 20 nucleotides to about 25 nucleotides, from about 20 nucleotides to about 28 nucleotides, from about 21 nucleotides to about 22 nucleotides, from about 21 nucleotides to about 23 nucleotides, from about 21 nucleotides to about 24 nucleotides, from about 21 nucleotides to about 25 nucleotides, from about 21 nucleotides to about 28 nucleotides,from about 22 nucleotides to about 23 nucleotides, from about 22 nucleotides to about 24 nucleotides, from about 22 nucleotides to about 25 nucleotides, from about 22 nucleotides to about 28 nucleotides, from about 23 nucleotides to about 24 nucleotides, from about 23 nucleotides to about 25 nucleotides, from about 23 nucleotides to about 28 nucleotides, from about 24 nucleotides to about 25 nucleotides, from about 24 nucleotides to about 28 nucleotides, or from about 25 nucleotides to about 28 nucleotides. In some embodiments, the guide RNA comprises about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, or about 28 nucleotides.

[0165] In some embodiments, the guide RNA (e.g., gRNA) is encoded by any one of the nucleic acid sequences of SEQ ID NO: 2-6 or a sequence having at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence having at least about 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement. In some embodiments, the guide RNA is encoded by a sequence according to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement.

[0166] In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 80% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 85% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 90% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 95% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 98% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, one or more guide RNAs are encoded by a sequence comprising at least about 99% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or the reverse complement thereof. In some embodiments, the guide RNA is encoded by the sequence of any one of the nucleic acid sequences according to SEQ ID NOs: 2-6 or the reverse complement thereof.

[0167] In some embodiments, the present disclosure describes a composition comprising: a nuclease (e.g., Cas9 or dCas9) fused to a methyltransferase (e.g., an RNA methyltransferase), wherein at least 100 (e.g., at least 200, 300, 400, 500, or 1000) CpG dinucleotides are methylated. In some embodiments, at least 200 CpG dinucleotides are methylated. In some embodiments, at least 300 CpG dinucleotides are methylated. In some embodiments, at least 400 CpG dinucleotides are methylated. In some embodiments, at least 500 CpG dinucleotides are methylated. In some embodiments, at least 1000 CpG dinucleotides are methylated.

[0168] In some embodiments, one or more CpG dinucleotides within at most about 200 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 1000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 5000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 10000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 20000 base pairs of the sequence are methylated.

[0169] In some embodiments, at least 100 (e.g., at least 200, 300, 400, 500, or 1000) CpG dinucleotides are methylated. In some embodiments, at least 200 CpG dinucleotides are methylated. In some embodiments, at least 300 CpG dinucleotides are methylated. In some embodiments, at least 400 CpG dinucleotides are methylated. In some embodiments, at least 500 CpG dinucleotides are methylated. In some embodiments, at least 1000 CpG dinucleotides are methylated.

[0170] In some embodiments, one or more methylated CpG dinucleotides within at most about 200 base pairs of the sequence are methylated. In some embodiments, one or more methylated CpG dinucleotides within at most about 1000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 5000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 10000 base pairs of the sequence are methylated. In some embodiments, one or more CpG dinucleotides within at most about 20000 base pairs of the sequence are methylated.

[0171] The methods of the present disclosure

[0172] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: providing a cell with a nucleic acid comprising a payload of interest; and culturing the cell in a growth medium comprising cytosine or a derivative thereof to generate a recombinant AAV vector comprising at least about 25% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90%) methylated CpG dinucleotides.

[0173] This document describes methods for generating rAAV vectors, which include culturing cells in a growth medium containing cytosine or its derivatives, where the cytosine or its derivatives are selected from the group consisting of: methylcytosine, phosphorylated cytosine, and phosphorothioate cytosine. In some embodiments, the cytosine or its derivative is methylcytosine. In some embodiments, the cytosine or its derivative is phosphorylated cytosine. In some embodiments, the cytosine or its derivative is phosphorothioate cytosine.

[0174] In some embodiments, the cells are cultured in a growth medium containing various concentrations of cytosine or its derivatives. In some embodiments, the concentration of the cytosine or its derivative ranges from about 100 μM to about 5 mM (e.g., about 200 μM to about 4 mM, about 300 μM to about 3 mM, about 400 μM to about 2 mM, or about 500 μM to about 1 mM). In some embodiments, the concentration of the cytosine or its derivative ranges from about 200 μM to about 4 mM. In some embodiments, the concentration of the cytosine or its derivative ranges from about 300 μM to about 3 mM. In some embodiments, the concentration of the cytosine or its derivative ranges from about 400 μM to about 2 mM. In some embodiments, the concentration of the cytosine or its derivative ranges from about 500 μM to about 1 mM.

[0175] In some embodiments, the concentration of the cytosine or its derivative is at most 3 mM (e.g., at most 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 1 mM, 2 mM, or 3 mM). In some embodiments, the concentration of the cytosine or its derivative is at most 100 μM. In some embodiments, the concentration of the cytosine or its derivative is at most 200 μM. In some embodiments, the concentration of the cytosine or its derivative is at most 300 μM. In some embodiments, the concentration of the cytosine or its derivative is at most 400 μM. In some embodiments, the concentration of the cytosine or its derivative is at most 500 μM. In some embodiments, the concentration of the cytosine or its derivative is at most 1 mM. In some embodiments, the concentration of the cytosine or its derivative is at most 2 mM.

[0176] In some embodiments, the nucleic acid contains reduced CpG dinucleotides. In some embodiments, the nucleic acid contains increased methylation of CpG dinucleotides. In some embodiments, the nucleic acid contains reduced CpG dinucleotides and increased methylation of CpG dinucleotides.

[0177] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: providing a nucleic acid comprising a payload of interest to a cell, wherein the nucleic acid comprises reduced CpG dinucleotides, and further comprising providing to the cell a nucleic acid comprising: a helper polynucleotide comprising reduced CpG dinucleotides compared to a parental equivalent.

[0178] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: providing a nucleic acid comprising a payload of interest to a cell, wherein the nucleic acid comprises reduced CpG dinucleotides, and further comprising providing to the cell a nucleic acid comprising: a methylated helper polynucleotide comprising increased CpG dinucleotides compared to a parental equivalent.

[0179] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: providing a nucleic acid comprising a payload of interest to a cell, wherein the nucleic acid comprises reduced CpG dinucleotides, and further comprising providing a composition comprising: a) a CRISPR-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding one or more guide RNAs, the one or more guide RNAs targeting a sequence for methylating cytosine in a CpG dinucleotide.

[0180] In some embodiments, methods for generating recombinant AAV vectors are described herein, the methods comprising: providing a nucleic acid comprising a payload of interest to a cell, wherein the nucleic acid comprises reduced CpG dinucleotides, and further comprising providing a composition comprising: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NO: 2-6 or its reverse complement.

[0181] Methods for generating rAAV vectors are described herein, the methods comprising: providing a nucleic acid to a cell, wherein the cell is transfected with the nucleic acid. In some embodiments, the cell is infected with the nucleic acid. In some embodiments, the cell is in suspension. In some embodiments, the cell is adherent. In some embodiments, the cell is an engineered stable cell line.

[0182] In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungal cell), a mammalian cell (Chinese hamster ovary (CHO) cell, baby hamster kidney (BHK), human embryonic kidney (HEK), mouse myeloma (NS0), or human retinal cell), an immortalized cell (e.g., HeLa cell, COS cell, HEK-293T cell, MDCK cell, 3T3 cell, PC12 cell, Huh7 cell, HepG2 cell, K562 cell, N2a cell, or SY5Y cell), an insect cell (e.g., Spodoptera frugiperda cell, Trichoplusia ni cell, Drosophila melanogaster cell, S2 cell, or Heliothis virescens cell), a yeast cell (e.g., Saccharomyces cerevisiae cell, Cryptococcus cell, or Candida cell), a plant cell (e.g., parenchyma cell, collenchyma cell, or sclerenchyma cell), a fungal cell (e.g., Saccharomyces cerevisiae cell, Cryptococcus cell, or Candida cell), or a prokaryotic cell (e.g., Escherichia coli cell, streptococcus bacterium cell, streptomyces soil bacterium cell, or archaea cell). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a fungal cell. In some embodiments, the cell is a prokaryotic cell.

[0183] In some embodiments, the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell or a derivative thereof. In some embodiments, the cell is an A549 cell or a derivative thereof. In some embodiments, the cell is a HEK-293 cell or a derivative thereof. In some embodiments, the cell is a HEK-293T cell or a derivative thereof. In some embodiments, the cell is a BHK cell or a derivative thereof. In some embodiments, the cell is a CHO cell or a derivative thereof. In some embodiments, the cell is a HeLa cell or a derivative thereof. In some embodiments, the cell is an MRC5 cell or a derivative thereof. In some embodiments, the cell is an Sf9 cell or a derivative thereof. In some embodiments, the cell is a Cos-1 cell or a derivative thereof. In some embodiments, the cell is a Cos-7 cell or a derivative thereof. In some embodiments, the cell is a Vero cell or a derivative thereof. In some embodiments, the cell is a BSC 1 cell or a derivative thereof. In some embodiments, the cell is a BSC 40 cell or a derivative thereof. In some embodiments, the cell is a BMT10 cell or a derivative thereof. In some embodiments, the cell is a WI38 cell or a derivative thereof. In some embodiments, the cell is a HeLa cell or a derivative thereof. In some embodiments, the cell is a Saos cell or a derivative thereof. In some embodiments, the cell is a C2C12 cell or a derivative thereof. In some embodiments, the cell is an L cell or a derivative thereof. In some embodiments, the cell is an HT1080 cell or a derivative thereof. In some embodiments, the cell is a HepG2 cell or a derivative thereof. In some embodiments, the cell is a Huh7 cell or a derivative thereof. In some embodiments, the cell is a K562 cell or a derivative thereof. In some embodiments, the cell is a primary cell.

[0184] In some embodiments, the nucleic acids or compositions described herein are transiently expressed. In some embodiments, the nucleic acids or compositions are transiently expressed from an inducible system. In some embodiments, the nucleic acids or compositions are stably expressed. In some embodiments, the nucleic acids or compositions are stably expressed from an inducible system.

[0185] In some embodiments, the recombinant viral vector is produced by the producer cell line method. Briefly, for example, a cell line (e.g., the HeLa 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 recombinant vector production, which in some embodiments is amplified to a production bioreactor and infected with an auxiliary polynucleotide (e.g., wild-type adenovirus) as an aid to initiate vector production. In some embodiments, the recombinant viral vector (e.g., rAAV) is subsequently harvested, inactivated (e.g., by heating) and / or the adenovirus is removed, and the viral particles are purified. In some embodiments, the recombinant viral vector is purified and formulated.

[0186] In some embodiments, suitable media are used for the production of the recombinant vector. These media include, but are not limited to, media produced by Hyclone Laboratories and JRH, which include Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations, particularly custom media formulations for use in the production of recombinant vectors.

[0187] In some embodiments, the suitable production media of the present disclosure are supplemented with serum or serum-derived recombinant proteins at levels of 0.5 - 20 (v / v or w / v). In some embodiments, the vector is produced under serum-free conditions, which are also referred to as media that do not contain animal-derived products. In some embodiments, commercial or custom media are designed to support the production of the vector, including but not limited to supplementation with glucose, vitamins, amino acids, and / or growth factors to increase the titer and / or yield of the vector in the production culture.

[0188] The vector production culture encompasses a variety of conditions (over a wide temperature range, different time lengths, etc.) suitable for the particular host cell utilized. The vector production culture includes adherent-dependent cultures, which are cultured in suitable adherent-dependent containers such as, for example, plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed bed bioreactors or fluidized bed bioreactors. In some embodiments, the vector production culture includes suspension-adapted host cells such as HeLa, HEK-293, and SF-9 cells, which are cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, single-use bioreactors such as Cytiva Xcellerex and Sartorius, and disposable systems such as the Wave bag system.

[0189] In some embodiments, the viral particles of the present disclosure are harvested from a vector production culture by lysing the host cells of the production culture or by harvesting the spent medium from the production culture, provided that the cells are cultured under conditions that cause the viral particles to be released from the intact cells into the medium. Suitable methods for lysing cells include, for example, multiple freeze-thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0190] In additional embodiments, the viral particles are purified. As used herein, the term "purified" includes a preparation of viral particles that is free of at least some of the other components that are naturally present or present at the place of initial preparation of the viral particles. Thus, for example, in some embodiments, purified techniques are used to prepare isolated viral particles to enrich the isolated viral particles from a source mixture (such as a culture lysate or production culture supernatant). In some embodiments, enrichment is measured in a variety of ways, such as, for example, by the ratio of deoxyribonuclease-resistant particles (DRP) or genomic copies (gc) present in solution, or by infectivity, or with respect to a second potential interfering substance present in the source mixture (such as a contaminant, including production culture contaminants or process contaminants, including helper viruses, medium components, etc.).

[0191] In some embodiments, the vector production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters, including, for example, a DOHC grade Millipore Millistak+HC Pod filter, an A1HC grade Millipore Millistak+HC Pod filter, and a 0.2 μm filter Opticap XL 10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques, such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or greater.

[0192] In some embodiments, the vector production culture harvest is also treated with to digest any high molecular weight DNA present in the production culture. In some embodiments, the digestion is carried out under standard conditions, including, for example, a final concentration of 1-2.5 units / ml of over a temperature range from ambient temperature to 37 °C for a period of 30 minutes to several hours.

[0193] In some embodiments, one or more of the following purification steps are used to isolate or purify viral particles: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating viral particles; capture on a support by hydroxyapatite chromatography; heat inactivation of helper virus; capture on a support by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and capture on a support by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. In some embodiments, these steps are used alone, in various combinations, or in different orders. In some embodiments, the method includes all of the steps in the order described below.

[0194] Methods for generating recombinant vectors are described herein, where the method includes providing a cell transfected with a helper polynucleotide. In some embodiments, the cell is transfected with a helper polynucleotide that provides helper functions to AAV. In some embodiments, the helper polynucleotide provides adenovirus functions, including, for example, Ela, Elb, E2a, E4ORF6. In some embodiments, the sequences of the adenovirus genes providing these functions are obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12, and 40, and also include any currently identified human types. In some embodiments, the method involves transfecting a cell with a vector that expresses one or more genes necessary for AAV replication, AAV gene transcription, and / or AAV packaging.

[0195] Methods for generating recombinant vectors are described herein, where the method includes providing a cell transfected with a helper polynucleotide under the control of a promoter. In some embodiments, the cell is a stable host cell that contains the desired components under the control of an inducible promoter. In some embodiments, the cell is a stable host cell that contains the desired components under the control of a constitutive promoter. In some embodiments, the cell is a stable host cell that contains the selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a stable host cell is generated that is derived from HEK-293 cells (which contain El helper functions under the control of a constitutive promoter), but the host cell contains Rep protein and / or Cap protein under the control of an inducible promoter.

[0196] In some embodiments, the small gene, Rep sequence, Cap sequence, and helper functions required to produce the rAAV of the present disclosure are delivered to the packaging host cell in the form of any genetic element of the transfer sequence. In some embodiments, the selected genetic elements are delivered by any suitable method.

[0197] In some embodiments, the production of recombinant vectors (such as rAAV vectors) includes transfection, stable cell line production, and infectious hybrid virus production systems, the infectious hybrid virus production systems including adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids.

[0198] In some embodiments, methods including transfection, stable cell line production, and infectious hybrid virus production systems are used for the production of rAAV vectors. In some embodiments, the methods described herein include adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. All rAAV production cultures for producing rAAV viral particles require: 1) a suitable host cell, including, for example, cell lines of human origin such as HeLa, A549, or 293 cells, or cell lines of insect origin such as SF-9 (in the case of the baculovirus production system) or transgenic plant cells; 2) suitable helper virus functions, provided by wild-type or mutant adenoviruses (such as 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 a heterologous sequence encoding any desired sequence or fragment or variant thereof); and 5) a suitable culture medium and culture medium components that support rAAV production. Suitable culture media for producing rAAV vectors include, but are not limited to, media produced by Hyclone Laboratories and JRH, including Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 II SFM medium as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to the custom culture medium formulations for the production of recombinant AAV vectors.

[0199] In some embodiments, recombinant AAV particles are produced by transfecting producer cells with a plasmid (cis plasmid) containing the rAAV genome, the rAAV genome comprising a payload flanked by 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, E40RF6, and VARNA are provided by adenovirus infection or by transfecting a third plasmid that provides the adenovirus helper polynucleotide into the producer cells. In some embodiments, the producer cells are HEK - 293 cells. In some embodiments, the helper polynucleotides provided will vary depending on the producer cells used and whether the producer cells already carry some of these helper polynucleotides.

[0200] 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 and a helper adenovirus plasmid are co - transfected (e.g., using electroporation, lipofection, optical transfection, calcium phosphate method, or polyethylenimine (PEI)) into a cell line (e.g., HEK - 293 cells), and the virus is collected and optionally purified.

[0201] In some embodiments, the rAAV vectors are produced by a producer cell line method, such as the exemplary producer cell line method provided below. Briefly, a cell line (e.g., HeLa cell line) is stably transfected with a plasmid containing the Rep gene, the Cap gene, and a promoter - payload sequence. The cell line is screened to select lead clones for rAAV production, which are then expanded into a production bioreactor and infected with an adenovirus (e.g., wild - type adenovirus) as a helper to initiate rAAV production. Subsequently, the virus is harvested, the adenovirus can be inactivated (e.g., by heating) and / or removed, and the rAAV vector is purified.

[0202] In some embodiments, methods are provided for generating any rAAV vector as disclosed herein, comprising (a) culturing a host cell under conditions for generating an rAAV vector, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication and / or encapsidation protein; (ii) an rAAV provector, comprising a nucleic acid encoding a therapeutic polypeptide and / or nucleic acid as described herein flanked by at least one AAV ITR; and (iii) AAV helper functions; and (b) recovering the rAAV vector produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the group consisting of the following AAV ITRs: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, caprine AAV, bovine AAV, or murine AAV, etc. In some embodiments, the encapsidation protein is an AAV2 encapsidation protein.

[0203] This disclosure describes methods for generating rAAV vectors, the methods comprising: providing a cell with a nucleic acid comprising a payload of interest; and culturing the cell in a growth medium comprising cytosine or a derivative thereof to generate an rAAV vector of the present disclosure to generate a recombinant AAV vector comprising methylation of at least about 30% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 35% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 40% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 45% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 50% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 60% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 70% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 80% of CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises methylation of at least about 90% of CpG dinucleotides.

[0204] Pharmaceutical composition

[0205] In some embodiments, this disclosure describes a pharmaceutical composition comprising: a recombinant AAV vector comprising a payload of interest; more than one helper polynucleotide comprising reduced CpG dinucleotides; and a pharmaceutically acceptable excipient. In some embodiments, this disclosure also describes a pharmaceutical composition comprising: a recombinant AAV vector comprising a payload of interest; more than one helper polynucleotide comprising increased methylation of CpG dinucleotides; and a pharmaceutically acceptable excipient. In some embodiments, this disclosure also describes a pharmaceutical composition having less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) CpG dinucleotides, the pharmaceutical composition comprising a recombinant AAV vector comprising a payload of interest; more than one helper polynucleotide; and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises less than about 50% CpG dinucleotides.

[0206] The compositions or medicaments of the present disclosure are in a form suitable for administration to an individual in need thereof.

[0207] According to certain embodiments, the pharmaceutically acceptable excipient compositions of the present disclosure are suitably selected from the group consisting of: injectable excipient liquids such as sterile water for injection; and aqueous solutions such as saline.

[0208] Acceptable excipients are physiologically acceptable to the subject to which they are administered and retain the therapeutic properties of the compound administered therewith / therein. Acceptable excipients and their formulations are generally described, for example, in Remington’s Pharmaceutical Sciences, supra. An exemplary excipient is physiological saline. The phrase “pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject compound from the site of administration in one organ or part of the body to another organ or part of the body or in an in vitro assay system. Each excipient is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject to which it is administered. An acceptable excipient should also not alter the specific activity of the subject compound.

[0209] In another embodiment, the pharmaceutical compositions disclosed herein further comprise acceptable additives to improve the stability of the compounds in the composition and / or control the release rate of the composition. Acceptable additives do not alter the specific activity of the subject compound. 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. Optionally, exemplary acceptable additives include, but are not limited to, surfactants such as polysorbate 20 or polysorbate 80 to increase the stability of the peptide and reduce the gelling of the solution. In some embodiments, the surfactant is added to the composition in an amount of 0.01% to 5% of the solution. The addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0210] Suspension, lyophilized, and crystalline forms of the nucleic acids or compositions herein are also contemplated herein; methods of preparing suspension, lyophilized, and crystalline forms are known to those of skill in the art.

[0211] In some embodiments, the pharmaceutical compositions disclosed herein are sterile. In some embodiments, the pharmaceutical compositions disclosed herein are sterilized by conventional, well-known sterilization techniques. For example, sterilization is readily accomplished by filtration through a sterile filtration membrane. In some embodiments, the resulting solution is packaged for use or filtered and lyophilized under sterile conditions, and the lyophilized product is combined with a sterile solution prior to administration.

[0212] In some embodiments, such as when the polypeptide is relatively unstable in a liquid composition, lyophilization is employed to stabilize the polypeptide for long-term storage.

[0213] In some embodiments, excipients such as, for example, polyols (including mannitol, sorbitol, and glycerol); sugars (including glucose and sucrose); and amino acids (including alanine, glycine, and glutamic acid) act as stabilizers for lyophilized products. In some embodiments, polyols and sugars are also used to protect polypeptides from freeze- and drying-induced damage and to enhance stability during storage in the dry state. In some embodiments, sugars are effective both during the lyophilization process and during storage. Other classes of molecules, including monosaccharides and disaccharides, as well as polymers such as PVP, have also been reported as stabilizers for lyophilized products.

[0214] For injection, in some embodiments, the pharmaceutical compositions disclosed herein are in a powder suitable for reconstitution with a suitable solution as described above. Examples of these include, but are not limited to, lyophilized powders, rotary-dried powders, or spray-dried powders, amorphous powders, granules, precipitates, or microparticles. For injection, the composition optionally contains stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0215] In some embodiments, sustained-release articles are prepared. Suitable examples of sustained-release articles include semipermeable matrices of solid hydrophobic polymers containing the pharmaceutical compositions herein, where the matrix is in the form of a shaped article, e.g., a film or a microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), poly(lactide) (see, e.g., U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as Lupron Depot TM (including injectable microspheres of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of the molecule to exceed 100 days, while certain hydrogels enable release of the protein for a shorter period of time.

[0216] In some embodiments, the pharmaceutical compositions disclosed herein are designed to be short-acting, fast-release, long-acting, or sustained-release as described herein. In one embodiment, the pharmaceutical compositions disclosed herein are formulated for controlled release or for slow release.

[0217] In some embodiments, the pharmaceutical composition is contained in a container, package, or dispenser together with instructions for administration.

[0218] In some embodiments, the pharmaceutical composition comprises less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 49% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 48% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 47% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 46% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 45% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 40% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 35% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 30% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 25% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 20% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 15% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 10% CpG dinucleotides. In some embodiments, the pharmaceutical composition comprises less than about 5% CpG dinucleotides.

[0219] In some embodiments, the pharmaceutical composition comprises a recombinant AAV vector, wherein the recombinant AAV vector comprises less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 49% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 48% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 47% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 46% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 45% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 40% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 35% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 30% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 25% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 20% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 15% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 10% CpG dinucleotides. In some embodiments, the recombinant AAV vector comprises less than about 5% CpG dinucleotides.

[0220] This text describes a pharmaceutical composition comprising more than one auxiliary polynucleotide, said auxiliary polynucleotide comprising a reduced number of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 49% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 48% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 47% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 46% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 45% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 40% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 35% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 30% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 25% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 20% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 15% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 10% of CpG dinucleotides. In some embodiments, the more than one auxiliary polynucleotide comprises less than about 5% of CpG dinucleotides.

[0221] In some embodiments, more than one accessory polynucleotide contains fewer CpG dinucleotides compared to a parental equivalent. In some embodiments, compared to a parental equivalent, 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 95%. In some embodiments, the CpG dinucleotides are reduced in the range of about 5% to about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 15% to about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 20% to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced in the range of about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the CpG dinucleotides are reduced by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the CpG dinucleotides are reduced by at least about 50%. In some embodiments, the CpG dinucleotides are reduced by at least about 75%.

[0222] In some embodiments, more than one auxiliary polynucleotide contains increased methylation of CpG dinucleotides compared to the parental equivalent. In some embodiments, compared to the parental equivalent, the methylation of CpG dinucleotides is increased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95%. In some embodiments, the number of CpG dinucleotides 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased 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 number of CpG dinucleotides is increased in the range of about 25% to about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 30% to about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased in the range of about 40% to about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.In some embodiments, the number of CpG dinucleotides is increased by about 50% to about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 60% to about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 70% to about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 80% to about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the number of CpG dinucleotides is increased by about 90% to about 95%, 96%, 97%, 98%, 99% or 100%.

[0223] In some embodiments, the auxiliary polynucleotide comprises a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, a polyadenylation signal sequence, or a combination 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 the synapsin promoter or a derivative thereof. In some embodiments, the promoter is the CaMKII promoter or a derivative thereof. In some embodiments, the promoter is the GRK1 promoter or a derivative thereof. In some embodiments, the promoter is a micro promoter or a derivative thereof. In some embodiments, the promoter is an inducible promoter or a derivative thereof.

[0224] In some embodiments, the auxiliary polynucleotide comprises a prokaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide comprises a eukaryotic auxiliary polynucleotide. In some embodiments, the auxiliary polynucleotide consists of a eukaryotic auxiliary polynucleotide and a prokaryotic auxiliary polynucleotide.

[0225] In some embodiments, the auxiliary polynucleotide comprises one or more components derived from yeast (e.g., an autonomously replicating sequence, a centromere, or a telomere). In some embodiments, the auxiliary polynucleotide comprises one or more components derived from plants (e.g., virA, virB, virD, virG, or virE).

[0226] In some embodiments, the helper polynucleotide comprises one or more components derived from a virus (e.g., a promoter, Rep, Cap). In some embodiments, the virus is an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16 or a derivative thereof. In some embodiments, the herpesvirus is herpes simplex virus type 1 (HSV 1), HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, or HHV-8. 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 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 a circovirus. In some embodiments, the virus is a bocavirus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a retrovirus.

[0227] In some embodiments, the virus is AAV1 or a derivative thereof. In some embodiments, the virus is AAV2 or a derivative thereof. In some embodiments, the virus is AAV3 or a derivative thereof. In some embodiments, the virus is AAV4 or a derivative thereof. In some embodiments, the virus is AAV5 or a derivative thereof. In some embodiments, the virus is AAV6 or a derivative thereof. In some embodiments, the virus is AAV7 or a derivative thereof. In some embodiments, the virus is AAV8 or a derivative thereof. In some embodiments, the virus is AAV9 or a derivative thereof. In some embodiments, the virus is AAV10 or a derivative thereof. In some embodiments, the virus is AAV11 or a derivative thereof. In some embodiments, the virus is AAV12 or a derivative thereof. In some embodiments, the virus is AAV13 or a derivative thereof. In some embodiments, the virus is AAV14 or a derivative thereof. In some embodiments, the virus is AAV15 or a derivative thereof. In some embodiments, the virus is AAV16 or a derivative thereof. In some embodiments, the virus is AAV-rh8 or a derivative thereof. In some embodiments, the virus is AAV-rh10 or a derivative thereof. In some embodiments, the virus is AAV-rh20 or a derivative thereof. In some embodiments, the virus is AAV-rh39 or a derivative thereof. In some embodiments, the virus is AAV-rh74 or a derivative thereof. In some embodiments, the virus is AAV-rhM4-1 or a derivative thereof. In some embodiments, the virus is AAV-hu37 or a derivative thereof. In some embodiments, the virus is AAV-Anc80 or a derivative thereof. In some embodiments, the virus is AAV-Anc80L65 or a derivative thereof. In some embodiments, the virus is AAV-7m8 or a derivative thereof. In some embodiments, the virus is AAV-PHP-B or a derivative thereof. In some embodiments, the virus is AAV-PHP-EB or a derivative thereof. In some embodiments, the virus is AAV-2.5 or a derivative thereof. In some embodiments, the virus is AAV-2tYF or a derivative thereof. In some embodiments, the virus is AAV-3B or a derivative thereof. In some embodiments, the virus is AAV-LK03 or a derivative thereof. In some embodiments, the virus is AAV-HSC1 or a derivative thereof. In some embodiments, the virus is AAV-HSC2 or a derivative thereof. In some embodiments, the virus is AAV-HSC3 or a derivative thereof. In some embodiments, the virus is AAV-HSC4 or a derivative thereof. In some embodiments, the virus is AAV-HSC5 or a derivative thereof. In some embodiments, the virus is AAV-HSC6 or a derivative thereof. In some embodiments, the virus is AAV-HSC7 or a derivative thereof. In some embodiments, the virus is AAV-HSC8 or a derivative thereof.In some embodiments, the virus is AAV-HSC9 or a derivative thereof. In some embodiments, the virus is AAV-HSC10 or a derivative thereof. In some embodiments, the virus is AAV-HSC11 or a derivative thereof. In some embodiments, the virus is AAV-HSC12 or a derivative thereof. In some embodiments, the virus is AAV-HSC13 or a derivative thereof. In some embodiments, the virus is AAV-HSC14 or a derivative thereof. In some embodiments, the virus is AAV-HSC15 or a derivative thereof. In some embodiments, the virus is AAV-TT or a derivative thereof. In some embodiments, the virus is AAV-DJ / 8 or a derivative thereof. In some embodiments, the virus is AAV-Myo or a derivative thereof. In some embodiments, the virus is AAV-NP40 or a derivative thereof. In some embodiments, the virus is AAV-NP59 or a derivative thereof. In some embodiments, the virus is AAV-NP22 or a derivative thereof. In some embodiments, the virus is AAV-NP66 or a derivative thereof. In some embodiments, the virus is AAV-HSC16 or a derivative thereof.

[0228] 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.

[0229] In some embodiments, the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A, and VA RNA. For example, in some embodiments, the adenovirus or AAV helper polynucleotide is Rep. In some embodiments, the adenovirus or AAV helper polynucleotide is Cap. In some embodiments, the adenovirus or AAV helper polynucleotide is E1A. In some embodiments, the adenovirus or AAV helper polynucleotide is E1B. In some embodiments, the adenovirus or AAV helper polynucleotide is E4. In some embodiments, the adenovirus or AAV helper polynucleotide is E2A. In some embodiments, the adenovirus or AAV helper polynucleotide is VA RNA.

[0230] In some embodiments, the lentiviral accessory polynucleotides are selected from the group consisting of: Gag, Pol, Tat, Rev, Env, and VSV-G. For example, in some embodiments, the lentiviral accessory polynucleotide is Gag. In some embodiments, the lentiviral accessory polynucleotide is Pol. In some embodiments, the lentiviral accessory polynucleotide is Tat. In some embodiments, the lentiviral accessory polynucleotide is Rev. In some embodiments, the lentiviral accessory polynucleotide is Env. In some embodiments, the lentiviral accessory polynucleotide is VSV-G.

[0231] The present disclosure describes pharmaceutical compositions comprising: a recombinant AAV vector comprising a payload of interest; and more than one accessory polynucleotide, wherein the accessory polynucleotide comprises a backbone polynucleotide having a reduced number of CpG dinucleotides compared to a parental equivalent. In some embodiments, the backbone polynucleotide comprises an increased methylation of CpG dinucleotides compared to a parental equivalent. In some embodiments, the backbone polynucleotide comprises a reduced number of CpG dinucleotides and an increased methylation of CpG dinucleotides compared to a parental equivalent.

[0232] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, an open reading frame, or a combination thereof. In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene, an origin of replication, and an open reading frame.

[0233] In some embodiments, the backbone polynucleotide comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a glycopeptide, a macrolide, a polypeptide, a tetracycline, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a β-lactam or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a glycopeptide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a macrolide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a polypeptide or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes a tetracycline or a derivative thereof.

[0234] In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, geneticin, or a derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin.

[0235] In some embodiments, kanamycin comprises a nucleotide sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:9 or 12. In some embodiments, kanamycin comprises a nucleotide sequence having the nucleic acid sequence of SEQ ID NO:9 or 12.

[0236] In some embodiments, the backbone polynucleotide comprises an origin of replication. In some embodiments, the origin of replication is pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, a combination thereof, or a derivative 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.

[0237] In some embodiments, the accessory polynucleotide of the present disclosure comprises a woodchuck hepatitis virus post-transcriptional element.

[0238] In some embodiments, the nucleic acids described herein contain reporter sequences for co-expression, such as but not limited to lacZ, GFP, CFP, YFP, RFP, mCherry, mCardinal, RLuc, FLuc, NanoLuc luciferase, and tdTomato. In some embodiments, the vector contains a selectable marker.

[0239] In some embodiments, nucleic acids are described herein that contain accessory polynucleotides, wherein any two or more of the accessory polynucleotides of the present disclosure are provided in the same plasmid or in different plasmids (e.g., packaging plasmids, transfer plasmids, or accessory plasmids). Examples

[0240] The following are examples of specific embodiments for practicing 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. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental error and deviation should be allowed.

[0241] Example 1. Reduction of CpG Content in rAAV

[0242] This example demonstrates how the CpG content of the genes, backbone polynucleotides, and accessory polynucleotides in rAAV is reduced.

[0243] First, each CpG present in the nucleotide sequences of wild-type AAV Rep, AAV Cap, and the kanamycin resistance (KanR) gene was identified on the exemplary plasmid pRep_Cap_KanR (Table 2).

[0244] Table 2. Sequences for CpG Reduction or Methylation

[0245]

[0246]

[0247] Then, maps of the Rep and Cap genes were generated (see Figure 3 and Figure 4 ), and each C or G of the CpG duplex in the Rep or Cap gene was removed and replaced with appropriate nucleotides using the human codon usage table depicted in Figure 1 to maintain the amino acid codon sequence. Similarly, for the kanamycin resistance gene, a map was generated ( Figure 5 ), and each C or G of the CpG duplex in the kanamycin resistance gene was removed and replaced with appropriate nucleotides using the Escherichia coli codon usage table depicted in Figure 2 to maintain the amino acid codon sequence.

[0248] For AAV Rep, three regions containing several key regulatory sequences, namely the AAV p19 and p40 promoters and the AAV splice donor site, were excluded from CpG depletion, as provided in Table 3 below. As indicated in each row, nucleotide changes are provided by uppercase or lowercase letters, respectively. For AAV Cap, four regions containing several regulatory sequences, namely AAP, MAAP, and splice acceptor sites A1 and A2, were excluded from CpG depletion. For KanR, no regions were excluded.

[0249] Table 3. Nucleotide Sequences with Reduced CpG

[0250]

[0251]

[0252] Using CpG-depleted genes, three plasmids were generated in Escherichia coli strains JM108, NEB Stable, and Stbl3. The three generated plasmids included the following: 1) an rAAV plasmid generated from CpG-depleted AAV Rep and CpG-depleted AAV Cap in the packaging plasmid and CpG-depleted KanR in the packaging plasmid (“pRep_Cap_KanR_CpG-depleted”; “Plasmid 1”, Figure 6 ), 2) a plasmid with CpG-depleted AAV Rep and CpG-depleted KanR in the packaging plasmid (“pRep_CpG-depleted_Cap_KanR”; “Plasmid 2”, Figure 7 ), and 3) a plasmid with CpG-depleted AAV Cap and CpG-depleted KanR in the packaging plasmid (“pRep_Cap_CpG-depleted_KanR”; “Plasmid 3”, Figure 8 ). The yield, concentration, identity (by restriction digestion of each plasmid preparation), and quality of each plasmid were confirmed. A control plasmid with endogenous CpG in Rep, Cap, and KanR (“pRep_Cap_KanR”) was also generated. The calculation of CpG reduction for each plasmid is provided in Table 4.

[0253] Table 4. CpG Reduction in Experimental Plasmids

[0254]

[0255] Example 2. Packaging AAV-GFP Using CpG-Depleted pRepCap Plasmids

[0256] This example demonstrates the functional verification of the novel CpG-depleted pRepCap plasmids of Example 1.

[0257] Materials and Methods

[0258] To verify the function of each novel plasmid with CpG depletion of Example 1, AAV vectors expressing an enhanced green fluorescent protein (eGFP) reporter gene payload were packaged. Briefly, according to the manufacturer's protocol, 1.5 x 10 8 suspension-adapted HEK293 cells were transfected in 50 mL with a combination of 3 plasmids to support AAV packaging. The 3 plasmids included pHelper and pAAV and each had pRep_Cap_KanR_CpG-depleted, pRep_CpG-depleted_Cap_KanR, pRep_Cap_CpG-depleted_KanR, or pRep_Cap_KanR. Approximately 48 hours after incubation, samples were obtained from each culture and placed in a 96-well plate for fluorescence imaging. Lysis buffer was added to the remaining portion of the cell culture to lyse the cells. The lysate was then digested with nuclease and clarified by centrifugation (30 minutes, 4198 x G, 4 °C). The clarified crude lysate was stored frozen at -80 °C before analytical evaluation.

[0259] The AAV9-GFP titer was estimated using a digital polymerase chain reaction (PCR) protocol. Briefly, each AAV crude lysate was treated with DNase-I, lysed, and partitioned; then PCR amplification was performed using a CMV primer / probe set with a dPCR instrument.

[0260] The capsid titer was roughly estimated by a semi-quantitative method using an AAV9 antibody coated on a strip.

[0261] Results

[0262] Micrographs of VPC2.0 cells expressing GFP were observed 48 hours after transfection with CpG-depleted plasmids, and no difference in transfection efficiency was observed when using CpG-depleted pRepCap plasmids compared to the unmodified wild-type form ( Figure 9 ). The percentage of GFP-expressing cells and the intensity of GFP expression in the cells were similar.

[0263] The results of the capsid titer are shown in Table 5.

[0264] Table 5. AAV titers after production with novel plasmids

[0265] pRepCap plasmid vg / mL Total vg pRep_Cap_KanR +++ +++ pRep_Cap_KanR CpG-depleted + + pRep_CpG-depleted_Cap_KanR + + pRep_Cap_CpG-depleted_KanR ++ ++

[0266] Example 3. Reduction of CpG content of the transgene in rAAV

[0267] This example demonstrates how the CpG content of the transgene in rAAV is reduced.

[0268] Using the method described in Example 1, the CpG content of the transgene in rAAV was adjusted by incorporating codon substitutions that reduce the CpG content of the gene. For example, each CpG present in the nucleotide sequence of the wild-type human interferon beta 1 (IFNβ) gene was identified on an exemplary plasmid (Table 6). Each C or G of the CpG duplex in the gene was removed and replaced with an appropriate nucleotide to maintain the amino acid codon sequence. As represented in Table 6, the nucleotide changes are provided in capital letters.

[0269] Table 6. CpG Reduction and Exemplary Payload Genes with CpG Reduction

[0270]

[0271] Example 4. Reduction of the CpG Content of Genes of Interest in the Helper Plasmid

[0272] This example demonstrates how the CpG content of genes of interest in the helper plasmid of rAAV is reduced.

[0273] Using the method described in Example 1, the CpG content of the gene in the rAAV helper plasmid was adjusted by incorporating codon substitutions that reduce the CpG content of the gene. For example, each CpG present in the nucleotide sequence of a gene of interest such as wild-type E4, E2A, or VA was identified. Each C or G of the CpG duplex in the gene was removed and replaced with an appropriate nucleotide to maintain the amino acid codon sequence.

[0274] Example 5. Increased Methylation Content of CpG Dinucleotides in rAAV

[0275] This example demonstrates how the methylation content of CpG dinucleotides in rAAV, packaging plasmids, and helper plasmids is increased.

[0276] For example, one of ordinary skill in the art can design the methylated CpG content of an accessory polynucleotide (e.g., a regulatory polynucleotide) or a fragment thereof by methylating unmethylated CpGs. Such accessory polynucleotides include, for example, promoters, enhancers, introns, microRNAs, linkers, splicing elements, and polyadenylation signals in rAAV, or viral life cycle-encoding genes or fragments thereof such as Rep, Cap, E1A, E1B, E4, E2A, ampicillin resistance gene, kanamycin resistance gene, VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G in a packaging plasmid or an accessory plasmid. For example, starting from a wild-type gene sequence, a catalytically inactive CRISPR-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding a CRISPR-associated endonuclease fused to an RNA methyltransferase is introduced. Such a methyltransferase can be coordinated with a guide RNA targeting, for example, an accessory polynucleotide (such as a regulatory polynucleotide or a fragment thereof in rAAV); a viral life cycle-encoding gene or a fragment thereof in a packaging plasmid; or a polynucleotide in an accessory plasmid. This coordination of the methyltransferase with the gRNA can help increase the methylation of CpG dinucleotides of the respective genes while preserving the sequence identity of the encoded proteins. After design, the final methylation-optimized sequence is prepared.

[0277] Example 6. Increased Methylation Content of CpG Dinucleotides in Viral Vectors

[0278] This example demonstrates how the methylation content of CpG dinucleotides in rAAV, a packaging plasmid, or an accessory plasmid is increased.

[0279] For example, one of ordinary skill in the art can design the methylated CpG content of an accessory polynucleotide (e.g., a regulatory polynucleotide) or a fragment thereof by methylating unmethylated CpGs. Such accessory polynucleotides include, for example, promoters, enhancers, introns, microRNAs, linkers, splicing elements, and polyadenylation signals in rAAV, or viral life cycle-encoding genes or fragments thereof such as Rep, Cap, E1A, E1B, E4, E2A, ampicillin resistance gene, kanamycin resistance gene, VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G in a packaging plasmid or an accessory plasmid. For example, a polynucleotide comprising a payload of interest can be provided to a cell (e.g., a host cell), and the cell can be cultured in a growth medium containing cytosine or a derivative thereof to generate a recombinant AAV vector having increased methylation of CpG dinucleotides compared to a recombinant AAV vector generated in a control growth medium.

[0280] In addition, after production with CpG-depleted plasmids, the total methylation profile of the AAV genome can be determined. For example, methylation sequencing methods such as bisulfite sequencing can be used to determine the methylation levels between AAV genome preparations made with CpG-containing plasmids and those made with CpG-depleted plasmids. Briefly, genomic DNA is extracted from the AAV preparation and prepared into a library for sequencing, and then bisulfite sequencing is performed.

[0281] In addition, ELISA can be used for the rapid assessment of DNA methylation.

[0282] Example 7. Evaluation of the effect of CpG depletion or increased CpG dinucleotides on the methylation content in viral vectors

[0283] Vectors are evaluated after design and generation, such as any such viral vectors generated by the methods described in any one of Examples 1-4. Characteristics for evaluation include identifying vector genome titer (vg / mL), transduction units or infectious titer, Rep protein expression and ratio, Cap protein expression and ratio, total genomic methylation level, residual plasmid DNA (e.g., KanR, Rep, Cap; such as residual plasmid DNA in copy number or by next-generation sequencing). In addition, the vector immunogenicity is evaluated in vitro, for example using TLR9 assays, and in vivo.

[0284] The TLR9 assay involves the use of a reporter cell line in vitro. Briefly, the iLite TLR9 assay prepares cells for the development of a functional assay to evaluate the response of TLR9 to AAV preparations made with CpG-containing plasmids and those made with CpG-depleted plasmids. The cells consist of genetically modified HEK293 reporter cells that quantitatively express firefly luciferase in response to TLR9 activation. The cells are mixed with various dilutions of AAV (both with and without CpG sequences), and the resulting output firefly luciferase expression is quantified on a luminometer. A decrease in the firefly luciferase expression level indicates a decrease in CpG-triggered signal transduction via TLR9, thus demonstrating that the AAV is less immunogenic.

[0285] In vivo assays for evaluating immunogenicity include AAV products made with CpG-containing plasmids and CpG-depleted plasmids, transducing wild-type or TLR9 mutant (TLR9 - / - ) mice, and the TLR9 mutant (TLR9 - / - ) mice are used as controls because TLR9 - / - mice as well as TLR9 antagonists can prevent innate immune responses, and the proteins of interest (e.g., infiltrating neutrophils, Mac-1+ Macrophages and CD335 + Differential responses of natural killer cells).

[0286] In addition, in vivo assays for assessing immunogenicity include IFN-γ ELISpot assays, which can be used to measure broad-spectrum cellular immune responses against viral antigens such as CpG sequences in the payload genome.

Claims

1. A nucleic acid, the nucleic acid comprising: an accessory polynucleotide comprising a reduced number of CpG dinucleotides compared to a parental equivalent.

2. A nucleic acid, the nucleic acid comprising: an accessory polynucleotide comprising increased methylation of CpG dinucleotides compared to a parental equivalent.

3. The nucleic acid according to claim 1 or 2, wherein the accessory polynucleotide comprises a promoter, an enhancer, an intron, a microRNA, a linker, a splicing element, or a polyadenylation signal.

4. The nucleic acid according to any one of claims 1-3, wherein the accessory polynucleotide comprises one or more components derived from an adenovirus, an adeno-associated virus (AAV), an alphavirus, a parvovirus, a baculovirus, a dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus.

5. The nucleic acid according to claim 4, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, or AAV-HSC16 or a derivative thereof.

6. The nucleic acid according to claim 4, wherein the adenovirus or AAV accessory polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A, and VARNA.

7. The nucleic acid according to claim 6, wherein the Rep comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO:7 or 10.

8. The nucleic acid according to claim 6, wherein the Rep comprises a nucleotide sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

10.

9. The nucleic acid according to claim 6, wherein the Rep comprises a nucleotide sequence having 100% sequence identity with the nucleic acid sequence of SEQ ID NO:

10.

10. The nucleic acid according to claim 6, wherein the Cap comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 8 or 11.

11. The nucleic acid according to claim 6, wherein the Cap comprises a nucleotide sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

11.

12. The nucleic acid according to claim 6, wherein the Cap comprises a nucleotide sequence having at least about 100% sequence identity with the nucleic acid sequence of SEQ ID NO:

11.

13. The nucleic acid according to claim 4, wherein the lentiviral accessory polynucleotide is selected from the group consisting of: Gag, Pol, Tat, Rev, Env, and VSV-G.

14. The nucleic acid according to any one of claims 1-13, wherein the accessory polynucleotide comprises a backbone polynucleotide, and the backbone polynucleotide comprises a reduced number of CpG dinucleotides, an increased methylation of CpG dinucleotides, or both a reduced number of CpG dinucleotides and an increased methylation of CpG dinucleotides.

15. The nucleic acid according to claim 14, wherein the backbone polynucleotide comprises an antibiotic resistance gene.

16. The nucleic acid according to claim 15, wherein the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, hygromycin, or a derivative thereof.

17. The nucleic acid according to claim 16, wherein the kanamycin comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 9 or 12.

18. The nucleic acid according to claim 16, wherein the kanamycin comprises a nucleotide sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO:

12.

19. The nucleic acid according to claim 16, wherein the kanamycin comprises a nucleotide sequence having 100% sequence identity with the nucleic acid sequence of SEQ ID NO:

12.

20. A recombinant AAV vector, the recombinant AAV vector comprising: a) a first nucleic acid comprising a nucleotide sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14; and b) a second nucleic acid comprising an accessory polynucleotide, the accessory polynucleotide comprising a nucleotide sequence having at least about 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 10 or 11.

21. A recombinant AAV vector, the recombinant AAV vector comprising: a) a first nucleic acid comprising a nucleotide sequence having at least 90% sequence identity with the nucleic acid sequence of SEQ ID NO: 14; and A second nucleic acid comprising one or more helper polynucleotides, wherein a first helper polynucleotide of the one or more helper polynucleotides comprises a nucleotide sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO:10, and a second helper polynucleotide of the one or more helper polynucleotides comprises a nucleotide sequence having at least about 90% sequence identity to the nucleic acid sequence of SEQ ID NO:

11.

22. A recombinant AAV vector, the recombinant AAV vector comprising: a) a first nucleic acid comprising a payload of interest; and b) a second nucleic acid comprising helper polynucleotides, wherein the second nucleic acid has a reduced number of CpG dinucleotides compared to a parental equivalent.

23. A recombinant AAV vector, the recombinant AAV vector comprising: a) a first nucleic acid comprising a payload of interest; and b) a second nucleic acid comprising helper polynucleotides, wherein the second nucleic acid has an increased methylation of CpG dinucleotides compared to a parental equivalent.

24. The recombinant AAV vector according to claim 22 or 23, wherein the helper polynucleotides are selected from the group consisting of: promoters, enhancers, introns, microRNAs, linkers, splicing elements, and polyadenylation signals.

25. The recombinant AAV vector according to claim 22 or 23, wherein the helper polynucleotide comprises a promoter.

26. The recombinant AAV vector according to any one of claims 22-25, wherein the helper polynucleotide comprises one or more components derived from adenovirus, AAV, alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus.

27. The recombinant AAV vector according to claim 26, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP-B, AAV-PHP-EB, AAV-2.5, AAV-2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66 or AAV-HSC16 or a derivative thereof.

28. The recombinant AAV vector according to claim 26, wherein the adenovirus or AAV helper polynucleotide is selected from the group consisting of: Rep, Cap, E1A, E1B, E4, E2A, and VARNA.

29. The recombinant AAV vector according to claim 28, wherein the Rep comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 7 or 10.

30. The recombinant AAV vector according to claim 28, wherein the Cap comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 8 or 11.

31. The recombinant AAV vector according to any one of claims 22-30, wherein the helper polynucleotide comprises a backbone polynucleotide, and the backbone polynucleotide comprises reduced CpG dinucleotides, increased methylation of CpG dinucleotides, or both reduced CpG dinucleotides and increased methylation of CpG dinucleotides.

32. The recombinant AAV vector according to claim 31, wherein the backbone polynucleotide comprises an antibiotic resistance gene.

33. The recombinant AAV vector according to claim 32, wherein the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a glycopeptide, a macrolide, a polypeptide, a tetracycline, or a derivative thereof.

34. The recombinant AAV vector according to claim 33, wherein the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, hygromycin or a derivative thereof.

35. The recombinant AAV vector according to any one of claims 14-34, wherein the kanamycin comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 9 or 12.

36. The recombinant AAV vector according to any one of claims 22-35, wherein the payload of interest comprises a nucleotide sequence having at least about 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 13 or 14.

37. A composition, the composition comprising: a) a clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding the CRISPR-associated endonuclease fused to the RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding the one or more guide RNAs, the one or more guide RNAs targeting a sequence for methylating cytosine in CpG dinucleotides.

38. The composition according to claim 37, wherein the CRISPR-associated endonuclease is Cas9.

39. The composition according to claim 37, wherein the CRISPR-associated endonuclease is catalytically inactive.

40. The composition according to any one of claims 37-39, wherein the CRISPR-associated endonuclease is catalytically inactive Cas9 (dCas9) or a derivative thereof.

41. The composition according to any one of claims 37-40, wherein the RNA methyltransferase is a prokaryotic RNA methyltransferase.

42. The composition according to claim 41, wherein the prokaryotic RNA methyltransferase is Fmu, YebU, RsmF or a derivative thereof.

43. The composition according to any one of claims 37-40, wherein the RNA methyltransferase is a eukaryotic RNA methyltransferase.

44. The composition according to claim 43, wherein the eukaryotic RNA methyltransferase is TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7 or a derivative thereof.

45. The composition according to any one of claims 37-40, wherein the RNA methyltransferase is a 5-methylcytosine (m5C) RNA methyltransferase.

46. The composition according to any one of claims 37-45, wherein the sequence is a part of the nucleic acid sequence of SEQ ID NO:

1.

47. The composition according to any one of claims 37-45, wherein the sequence comprises at least 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement.

48. The composition according to any one of claims 37-45, wherein the sequence comprises any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement.

49. The composition according to any one of claims 37-48, wherein the one or more guide RNAs are encoded by a sequence comprising at least about 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement.

50. The composition according to any one of claims 37-48, wherein the one or more guide RNAs are encoded by a sequence according to any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement.

51. A composition comprising: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence comprising at least 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 2-6 or its reverse complement.

52. The composition according to claim 51, wherein the nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease, a homing endonuclease or a CRISPR-associated endonuclease.

53. The composition according to claim 52, wherein the CRISPR-associated endonuclease is Cas9.

54. The composition according to claim 52, wherein the CRISPR-associated endonuclease has no catalytic activity.

55. The composition according to any one of claims 52-54, wherein the CRISPR-associated endonuclease is dCas9 or a derivative thereof.

56. The composition according to any one of claims 51-55, wherein the methyltransferase is a DNA methyltransferase.

57. The composition according to claim 56, wherein the DNA methyltransferase is a prokaryotic DNA methyltransferase.

58. The composition according to claim 57, wherein the prokaryotic DNA methyltransferase is CcrM, Dcm, M.HhaI, M.HaeIII or a derivative thereof.

59. The composition according to claim 56, wherein the DNA methyltransferase is a eukaryotic DNA methyltransferase.

60. The composition according to claim 59, wherein the eukaryotic DNA methyltransferase is a MET1, CMT or DRM methyltransferase.

61. The composition according to claim 59, wherein the eukaryotic DNA methyltransferase is METI, METIIa, METIII, METIIb, PMET, CMET5, CMET21, ZMET1, Masc1 or a derivative thereof.

62. The composition according to claim 56, wherein the DNA methyltransferase is DNMT1 or DNMT3a.

63. The composition according to any one of claims 51-55, wherein the methyltransferase is an RNA methyltransferase.

64. The composition according to claim 63, wherein the RNA methyltransferase is a prokaryotic RNA methyltransferase.

65. The composition according to claim 64, wherein the prokaryotic RNA methyltransferase is Fmu, YebU, RsmF or a derivative thereof.

66. The composition according to claim 63, wherein the RNA methyltransferase is a eukaryotic RNA methyltransferase.

67. The composition according to claim 66, wherein the eukaryotic RNA methyltransferase is TRM4B, TRDMT1, NOP2A (OL12), NSUN1 (NOP2), NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7 or a derivative thereof.

68. The composition according to claim 63, wherein the RNA methyltransferase is a 5-methylcytosine (m5C) RNA methyltransferase.

69. The composition according to any one of claims 51-68, wherein the sequence is a part of the nucleic acid sequence of SEQ ID NO:

1.

70. The composition according to any one of claims 51-68, wherein the sequence comprises at least about 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement.

71. The composition according to any one of claims 51-68, wherein the sequence comprises any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement.

72. The composition according to any one of claims 51-68, wherein the nucleic acid is a guide RNA (gRNA).

73. The composition according to claim 72, wherein the gRNA is encoded by a sequence having at least about 97% sequence identity with any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement.

74. The composition according to claim 72, wherein the gRNA is encoded by a sequence according to any one of the nucleic acid sequences of SEQ ID NO:2-6 or its reverse complement.

75. A cell comprising the nucleic acid according to any one of claims 1-19, the vector according to any one of claims 36-21 or the composition according to any one of claims 37-74.

76. The cell according to claim 75, wherein the cell is a eukaryotic cell.

77. The cell according to claim 75, wherein the cell is a mammalian cell.

78. The cell according to claim 75, wherein the cell is an immortalized cell.

79. The cell according to claim 75, wherein the cell is an insect cell.

80. The cell according to claim 75, wherein the cell is a yeast cell.

81. The cell according to claim 75, wherein the cell is a plant cell.

82. The cell according to claim 75, wherein the cell is a fungal cell.

83. The cell according to claim 75, wherein the cell is a prokaryotic cell.

84. The cell according to claim 75, wherein the cell is A549, HEK-293, HEK-293T, BHK, CHO, HeLa, MRC5, Sf9, Cos-1, Cos-7, Vero, BSC 1, BSC 40, BMT 10, WI38, HeLa, Saos, C2C12, L cell, HT1080, HepG2, Huh7, K562, a primary cell or a derivative thereof.

85. The cell according to claim 75, wherein the cell is an engineered cell.

86. The cell according to claim 75, wherein the cell is a stable cell.

87. A method for generating a recombinant AAV vector, the method comprising: a) providing a nucleic acid comprising a payload of interest to a cell; and b) culturing the cell of step a) in a growth medium comprising cytosine or a derivative thereof to generate the recombinant AAV vector methylated to comprise at least about 25% CpG dinucleotides.

88. The method according to claim 87, wherein the concentration of the cytosine or its derivative is in the range of about 100 micromoles per liter (μM) to about 5 millimoles per liter (mM).

89. The method according to claim 87, wherein the concentration of the cytosine or its derivative is up to 3 mM.

90. The method according to any one of claims 87-89, wherein the cytosine or its derivative is selected from the group consisting of methylcytosine, phosphorylated cytosine, and phosphorothioate cytosine.

91. The method according to any one of claims 87-90, wherein the nucleic acid comprises a reduced number of CpG dinucleotides, increased methylation of CpG dinucleotides, or both a reduced number of CpG dinucleotides and increased methylation of CpG dinucleotides.

92. The method according to any one of claims 87-91, further comprising providing, in step a), a nucleic acid comprising: a helper polynucleotide comprising a reduced number of CpG dinucleotides compared to a parental equivalent.

93. The method according to any one of claims 87-92, further comprising providing, in step a), a nucleic acid comprising: a helper polynucleotide comprising increased methylation of CpG dinucleotides compared to a parental equivalent.

94. The method according to any one of claims 87-93, further comprising providing, in step a), a composition comprising: a) a CRISPR-associated endonuclease fused to an RNA methyltransferase or a nucleic acid encoding the CRISPR-associated endonuclease fused to the RNA methyltransferase; and b) one or more guide RNAs or a nucleic acid encoding the one or more guide RNAs, the one or more guide RNAs targeting a sequence for methylating cytosine in a CpG dinucleotide.

95. The method according to any one of claims 87 - 94, further comprising providing, in step a), a composition comprising: a) a nuclease fused to a methyltransferase; and b) a nucleic acid targeting a sequence having at least 97% sequence identity to any one of the nucleic acid sequences of SEQ ID NOs: 2 - 6 or its reverse complement.

96. The method according to any one of claims 87 - 95, wherein the payload of interest comprises a nucleotide sequence having at least about 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 13 or 14.

97. A pharmaceutical composition, the pharmaceutical composition comprising: a) a recombinant AAV vector comprising a payload of interest; b) more than one helper polynucleotide comprising reduced CpG dinucleotides; and c) a pharmaceutically acceptable excipient.

98. A pharmaceutical composition, the pharmaceutical composition comprising: a) a recombinant AAV vector comprising a payload of interest; b) more than one helper polynucleotide comprising increased methylation of CpG dinucleotides; and c) a pharmaceutically acceptable excipient.

99. A pharmaceutical composition, the pharmaceutical composition having less than about 50% CpG dinucleotides compared to a parental equivalent, the pharmaceutical composition comprising a) a recombinant AAV vector comprising a payload of interest; b) more than one helper polynucleotide; and c) a pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises less than about 50% CpG dinucleotides.

100. The pharmaceutical composition according to claim 99, wherein the pharmaceutical composition comprises less than about 20% CpG dinucleotides.

101. The pharmaceutical composition according to any one of claims 99 - 100, wherein the recombinant AAV vector comprises less than about 10% CpG dinucleotides.

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