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WO2025171001A1 - Compositions et procédés pour la production améliorée de vecteurs viraux aav - Google Patents

Compositions et procédés pour la production améliorée de vecteurs viraux aav

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Publication number
WO2025171001A1
WO2025171001A1 PCT/US2025/014586 US2025014586W WO2025171001A1 WO 2025171001 A1 WO2025171001 A1 WO 2025171001A1 US 2025014586 W US2025014586 W US 2025014586W WO 2025171001 A1 WO2025171001 A1 WO 2025171001A1
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WIPO (PCT)
Prior art keywords
acid sequence
aav
nucleic acid
seq
protein
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English (en)
Inventor
Gregory Thomas NACHTRAB
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Publication of WO2025171001A1 publication Critical patent/WO2025171001A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the disclosure is directed to molecular biology, gene therapy, and compositions and methods for improved production of adeno-associated virus (AAV) viral vectors.
  • AAV adeno-associated virus
  • viruses HSV-1, HCMV, HPV-16, and HBoVl have been shown to support AAV replication. Genes from these viruses can be used to supplement Ad genes to increase AAV viral vector production.
  • AAV replicase An additional method to boost AAV production is through engineering of AAV replicase.
  • the Rep gene via two promoters and alternative splicing, encodes four regulatory proteins Rep78, Rep68, Rep52 and Rep40, each of which are involved in AAV genome replication.
  • Each of the four Rep proteins include a replicase domain.
  • the standard AAV replicase used for rAAV production is derived from AAV2.
  • the N-terminal DNA-binding and endonuclease domain of AAV2 is distinct from AAV1 or AAV8 replicase. Accordingly, this region of the Rep gene was modified to produce modified replicase variants, and their impact on AAV production was evaluated.
  • nucleic acid sequences encoding the capsid protein can also improve AAV viral vector production by increasing the percentage of full capsids and/or production of capsids having higher genome integrity.
  • nucleic acid sequences encoding AAV cap genes were developed having been codon “deoptimized” such that less frequently occurring codons were used in place of the naturally occurring codon.
  • the disclosure provides an AAV RepCap plasmid comprising one or more of an additional viral gene, a modified replicase, and a codon deoptimized nucleic acid sequence encoding AAV cap genes. Because of the significant potential for AAV-based gene therapies, the disclosure provides compositions and methods for the improved production of AAV viral vectors.
  • the present disclosure provides an adeno-associated virus (AAV) RepCap plasmid comprising: a nucleic acid sequence encoding a replicase protein; a nucleic acid sequence encoding a capsid protein; a nucleic acid sequence encoding a viral promoter; and a nucleic acid sequence encoding at least one viral gene, wherein the at least one viral gene is an HSV- 1 viral gene, an HPV-16 gene, or an HCMV viral gene.
  • AAV adeno-associated virus
  • the viral gene is selected from the group consisting of ICP8, El, E2, UL97, ICP0, and E6, or a combination thereof. In some embodiments, the viral gene comprises UL97.
  • the nucleic acid sequence encoding the viral gene comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 1 l.
  • the UL97 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto
  • the ICP8 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80% identity thereto
  • the El gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto
  • the E2 gene encodes
  • the replicase protein is a wild-type replicase protein. In some embodiments, the replicase protein is a modified replicase protein. In some embodiments, the modified replicase protein is a modified AAV2 replicase protein.
  • the modified AAV2 replicase protein comprises one or more mutations selected from: L45R, T65V, E66Q, M91L, El 14D, Il 17V, R119T, and Q288K relative to the sequence of SEQ ID NO: 16. In some embodiments, the modified AAV2 replicase protein comprises all of L45R, T65V, E66Q, M91L, El 14D, Il 17V, R119T, and Q288K relative to the sequence of SEQ ID NO: 16. In some embodiments, replicase protein comprises an amino acid sequence of SEQID NO: 14, or a sequence at least 80% identical thereto.
  • the nucleic acid sequence encoding the replicase protein comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13.
  • the capsid protein is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrhlO, Anc80, PHP.eB, and an MyoAAV capsid protein, or a derivative or variant thereof.
  • the capsid protein is selected from the group consisting of an AAV9, AAVrhlO, and an MyoAAV capsid protein, or a derivative or variant thereof. In some embodiments, the capsid protein is a modified capsid protein.
  • the capsid protein is encoded by a codon deoptimized nucleic acid sequence.
  • the nucleic acid sequence encoding the capsid protein comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.
  • the viral promoter is a CMV promoter, SV40 promoter, CAG promoter, or EFS promoter.
  • the nucleic acid sequence encoding the viral promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.
  • the AAV RepCap plasmid comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NO: 42 - 49.
  • the disclosure provides a method of producing an AAV viral vector comprising (a) transfecting cells with: (i) a RepCap plasmid disclosed herein; (ii) an adenovirus helper plasmid; and (iii) a plasmid comprising an rAAV expression vector, (b) culturing the cells under conditions sufficient to produce the AAV viral vector; and (c) purifying the AAV viral vector from the cells.
  • the rAAV expression vector encodes a transgene, protein, or RNA sequence.
  • the RNA sequence is a small nuclear RNA sequence.
  • the protein comprises a therapeutic protein.
  • the RNA sequence comprises a non-coding RNA sequence.
  • the non-coding RNA comprises small nuclear RNA sequence, a short hairpin RNA (shRNA), an small interfering RNA (siRNA) or a microRNA (miRNA) or a guide RNA for a CRISPR/Cas protein.
  • the cells are adherent cells. In some embodiments, the cells are in a suspension culture. In some embodiments, the cells are selected from the group consisting of HEK293 cells, HEK293T cells, A549 cells, WEHI cells, 3T3 cells, 10T1/2 cells, BHK cells, MDCK cells, COS 1 cells, COS 7 cells, BSC 1 cells, BSC 40 cells, VERO cells, WI38 cells, HeLa cells and HepG2 cells. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells stably express an adenovirus El A. [0026] In some embodiments, the AAV viral vector is a single stranded AAV (ssAAV) viral vector. In some embodiments, the AAV viral vector is a self-complementary AAV (scAAV) viral vector.
  • ssAAV single stranded AAV
  • scAAV self-complementary AAV
  • the amount of the AAV viral vector is increased relative to production of an equivalent AAV viral vector using a RepCap plasmid lacking an HSV-1 viral gene, an HPV-16 viral gene, or an HCMV viral gene.
  • the amount is at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000% greater relative to production of the equivalent AAV viral vector using a RepCap plasmid lacking the HSV-1 viral gene, the HPV-16 viral gene, or the HCMV viral gene.
  • the genome integrity of the AAV viral vector is increased relative to production using a RepCap plasmid lacking an HSV-1 viral gene, an HPV-16 viral gene, or an HCMV viral gene.
  • the genome integrity of the AAV viral vector is at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000% greater relative to production of an equivalent viral vector using the RepCap plasmid lacking an HSV-1 viral gene, an HPV-16 viral gene, or an HCMV viral gene.
  • the AAV viral vector is isolated from the supernatant of the packaging cells. In some embodiments, the AAV viral vector is isolated from the cells by lysing.
  • the disclosure provides an AAV viral vector produced by any one of the methods disclosed herein.
  • the disclosure provides an AAV viral vector produced by the methods disclosed herein.
  • the disclosure provides an AAV producing cell expressing at least one viral gene.
  • the viral gene comprises ICP8, El, E2, UL97, ICP0, E6 or a combination thereof.
  • the viral gene is UL97.
  • the nucleic acid sequence encoding the viral gene comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.
  • the UL97 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 80% identity thereto;
  • the ICP8 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 80% identity thereto;
  • the El gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 80% identity thereto;
  • the E2 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 80% identity thereto;
  • the ICPO gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 80% identity thereto; and/or (f) the E6 gene encodes a protein comprising an amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80% identity thereto.
  • the disclosure provides a codon deoptimized nucleic acid sequence encoding an AAV capsid protein.
  • the nucleic acid sequence encoding the AAV capsid protein is codon deoptimized in a portion of the nucleic acid sequence that encodes a VP1, VP2, or VP3 capsid protein.
  • the capsid protein is a VP3 capsid protein.
  • the codon deoptimized nucleic acid sequence has a translation efficiency score lower than the translation efficiency score of a non-codon deoptimized or wild-type nucleic acid sequence encoding a capsid protein.
  • translation efficiency score is lower than about 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, or 0.2. In some embodiments, translation efficiency score is higher than about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.6.
  • the capsid protein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrhlO, Anc80, PHP.eB, or MyoAAV capsid protein.
  • the capsid protein is an AAV9, AAVrhlO, or MyoAAV capsid protein.
  • the nucleic acid sequence encoding the capsid protein comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO: 41.
  • the disclosure provides a modified AAV2 replicase protein comprising one or more mutations selected from: L45R, T65V, E66Q, M91L, El 14D, Il 17V, R119T, and Q288K relative to the sequence of SEQ ID NO: 16.
  • the nucleic acid sequence encoding the replicase protein comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13.
  • the replicase protein comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 14.
  • the disclosure provides an AAV RepCap plasmid comprising: a nucleic acid sequence encoding a replicase protein; a nucleic acid sequence encoding a capsid protein; a nucleic acid sequence encoding a viral promoter; and a nucleic acid sequence encoding UL97.
  • the disclosure provides a pharmaceutical composition comprising any one of the AAV RepCap plasmids disclosed herein, any one of the codon deoptimized nucleic acid sequences disclosed herein, or any one of the modified AAV2 replicase proteins disclosed herein.
  • the disclosure provides a kit comprising the AAV RepCap plasmid of the disclosure, and instructions for use.
  • FIG. 1A is a schematic depicting a standard AAV RepCap plasmid comprising sequences encoding replicase (Rep2) and AAV capsid (RhlO capsid, or RhlO) proteins and a RepCap plasmid comprising sequences encoding Rep, RhlO Cap and an expression cassette for the expression of a viral gene downstream of the capsid sequence.
  • FIG. IB is a series of graphs depicting relative rAAV production yield measured by DNAse-protected vector genomes at harvest utilizing a RepCap plasmid expressing viral genes El, E2, and E6 from HPV16 or viral genes UL97, IE1, and IE2 from CMV. Yield is relative to rAAV production using the standard RepCap plasmid.
  • FIG. 1C is a graph depicting relative rAAV production yield measured by DNAse- protected vector genomes at harvest utilizing a RepCap plasmid expressing UL97, El and E2, or El E2 and UL97.
  • FIG. ID is a graph depicting relative rAAV production yield measured by DNAse- protected vector genomes at harvest utilizing 1) a RepCap plasmid expressing UL97 (UL97) or 2) a RepCap plasmid expressing UL97 as well as a mutated replicase protein having mutations L45R, T65V, E66Q, M91L, El 14D, Il 17V, and R119T (modRep+UL97).
  • FIG. IE is a graph depicting strangle- stranded AAV (ssAAV) production utilizing a standard RepCap plasmid (Standard, or GOI1) or the mutant replicase and UL97 expressing RepCap plasmid (modRep+UL97). Genome titer was evaluated utilizing qPCR.
  • ssAAV strangle- stranded AAV
  • FIG. 2B is a series of graphs depicting genome integrity of self-complementary AAV (scAAV) encoding 2 snRNA copies produced with standard RepCap plasmids or modified RepCap plasmids (modRep+UL97) as measured by nanopore long read sequencing.
  • scAAV self-complementary AAV
  • FIG. 2C is a series of graphs depicting the distribution of read lengths for scAAV produced in FIG. 2B.
  • the y-axis depicts number of reads and the x-axis depicts read length. Arrows indicate peaks corresponding to full-length genomes.
  • FIG. 2D is a series of graphs depicting genome integrity of scAAV encoding 4 snRNA copies produced with standard RepCap plasmids or modified RepCap plasmids (modRep+UL97) as measured by nanopore long read sequencing.
  • FIG. 2E is a series of graphs depicting the distribution of read lengths for scAAV produced in FIG. 2D.
  • the y-axis depicts number of reads and the x-axis depicts read length. Arrows indicate peaks corresponding to full-length genomes.
  • FIG. 2F is a graph depicting genome integrity of ssAAV encoding 4 snRNA copies produced with modified RepCap plasmids as measured by nanopore long read sequencing.
  • FIG. 2G is a series of pie charts depicting the percentage of scAAV or ssAAV obtained from snRNA-encoding scAAV utilizing the modified RepCap plasmid.
  • FIG. 3A is a graph depicting rAAV production yield for capsid serotypes AAV9, MyoAAV4, and Rh74 produced using a standard RepCap or a modified RepCap plasmid encoding a mutant replicase and viral gene UL97 (modRep+UL97). Genome titer was evaluated utilizing ddPCR.
  • FIG. 3B is table evaluating AAV viral vector production yield following anion exchange chromatography (AEX) wherein production utilized the modified RepCap plasmid. Depicted are two runs, 0022-HQ-l and 0023-HQ-l.
  • FIG. 3C is a schematic depicting modified RepCap plasmids where the region of the nucleic acid sequence encoding the capsid (AAV9), specifically the C-terminal region after the AAP reading frame, was modified to alter codon usage by changing the native codons to rare human codons (“codon deoptimization”).
  • FIG. 3D is a graph depicting vector genome (vg/mL) and capsid (CP/mL) yield for rAAV viral vectors produced using a standard AAV9 RepCap plasmid or a codon deoptimized AAV9 RepCap plasmid (Cap9d375-736).
  • FIG. 3E is a table depicting vector genome (vg/mL) and capsid (CP/mL) yield for rAAV viral vectors produced using a standard AAV9 RepCap plasmid or codon deoptimized AAV9 RepCap plasmids, Cap9d375-736 or Cap9d375-600.
  • FIG. 4A is a graph depicting relative AAVRhlO rAAV production yield of vector genomes at harvest utilizing a modified RepCap plasmid expressing viral genes UL97, IE1, IE2 from CMV and combinations thereof measured by qPCR. Yield is relative to rAAV production using the standard RepCap plasmid.
  • FIG. 4B is a graph depicting relative AAVRhlO rAAV production yield of vector genomes at harvest utilizing a modified RepCap plasmid expressing viral genes El and E2 from HP VI 6 and ICP8 from HSV or viral gene LTL97 from CMV and combinations thereof measured by qPCR. Yield is relative to rAAV production using the standard RepCap plasmid.
  • FIG. 5 is a schematic depicting RepCap plasmids.
  • RepCap plasmid CO 1779 contains sequences encoding wild type AAV2 replicase and AAV9 capsid (although other capsid serotypes may be used).
  • RepCap plasmid C01831 contains sequences encoding wild type AAV2 replicase, AAV9 capsid (although other capsid serotypes may be used), and viral gene UL97 operably linked to a CMV promoter.
  • RepCap plasmid C02984 contains sequences encoding mutated AAV2 replicase, AAV9 capsid (although other capsid serotypes may be used), and viral gene UL97 operably linked to a CMV promoter, with modifications of the AAV2 replicase sequence.
  • FIG. 6 is a graph depicting relative yield of rAAV vector yield produced using RepCap vectors C01831 and C02984 in adherent HEK293T cells. The harvest yield was assessed by qPCR titering.
  • FIG. 7A is a table depicting vector yield of rAAV vectors (CO 1779 and C02984) encoding 4x A05178 snRNA molecules produced in adherent HEK293T. Two independent replicates were performed. The yield was assessed by ddPCR titering.
  • FIG. 7B is a table depicting vector yield of rAAV vectors (CO 1779 and C02984) encoding 2x A04569 snRNA molecules produced in adherent HEK293T. Two independent replicates were performed. The yield was assessed by ddPCR titering.
  • FIG. 8A is a graph depicting quantification of ssAAV vector (A02206) produced in suspension HEK293 -derived cell lines (sHEK293T cells) using the C01779 and C02984 RepCap plasmids. The harvest yield was assessed by qPCR titering. C02984 increased productivity 5-fold in suspension HEK293T cells.
  • FIG. 8B is a graph depicting quantification of ssAAV vectors (A02206 and A01475) produced in suspension HEK293 -derived cell lines (WuXi cells) using the C01779 and C02984 RepCap plasmids. The harvest yield was assessed by qPCR titering. C02984 increased productivity 5-fold in suspension HEK293T cells.
  • FIG. 9 is a graph depicting scAAV9 A04569 was produced in HEK293T (WuXi) cells in an AMBR250 bioreactor.
  • the harvest vector genome yield (left y-axis) was assessed by ddPCR titering.
  • the harvest capsid yield was assessed by AAV9 ELISA.
  • the percentage full particle (right y-axis) is calculated by dividing the vector genome titer by the capsid titer.
  • FIG. 10 is a TapeStation image assessing the packaging integrity of rAAV vectors using CO 1779 and C02984 RepCap plasmids. Two independent replicates were performed, and the genomic integrity profiles are consistent.
  • FIG. 11A is a graph depicting read length analysis from full-length sequencing of the scAAV vector A05178 produced with RepCap plasmid CO 1779.
  • FIG. 11B is a graph depicting read length analysis from full-length sequencing of the scAAV vector A05178 produced with RepCap plasmid C02984.
  • FIG. 12 is an image depicting a coverage analysis from full-length sequencing of the scAAV vector A05178 produced using RepCap plasmid C01779 (top panel) and scAAV A05178 produced using RepCap plasmid C02984 (bottom panel).
  • FIG. 13 is a graph depicting bioreactor harvest yields of both vector genomes (vg/mL) and capsids (cp/mL) from AAV9 scAAV production with standard or modified RepCap plasmid (C02984 which has a mutant replicase and encodes UL97).
  • the % full capsid is depicted on the right side of y-axis and the harvest vector yield (in vg/mL, filled bars, and cp/mL, open bars) is depicted on the left side of the y-axis.
  • FIG. 14 is a schematic depicting Rep and Cap gene open reading frames in the RepCap plasmid.
  • the Cap gene also encodes the viral assembly proteins MAAP and AAP in an alternate reading frame. Thus, only the C-terminal region downstream of the AAP is amenable to codon modification.
  • FIG. 15 is a schematic depicting modified RepCap plasmid and modified RepCap with codon modifications (codon deoptimized) (C05656).
  • the standard AAV9 VP3 Cap sequence has a predicted translation efficiency of 0.74 on a 0-1 scale and the score of C05656 was 0.53. Slightly less of the sequence was codon altered in C05768 and C05769. These have predicted translation efficiency scores of 0.6 and 0.68 respectively.
  • FIG. 16 is a graph depicting scAAV production in suspension flasks with codon deoptimized RepCap plasmid C05656. Yields of vector genome (vg) and capsid (cp) per mL are displayed on the y-axis.
  • FIG. 17 is a series of tables detailing scAAV produced using RepCap plasmids C05656, C05768, and C05769 in a 250 mL bioreactor.
  • the harvest vector genome yield was assessed by ddPCR and the capsid yield by ELISA.
  • Use of C05656 and C05768 result in approximately 80% full capsid particles at harvest, which is a substantial increase over the non-codon altered RepCap plasmids (C04991 - mutant replicase and UL97; C01779 - wildtype replicase and no viral helper gene).
  • FIG. 18 is a graph depicting relative Casl3d-expressing rAAV production yield of vector genomes at harvest utilizing a modified RepCap plasmid expressing viral gene UL97 measured by qPCR.
  • RepCap plasmids C01265, C01747, C01790, C01806, and C01807 all contain and express UL97.
  • the present disclosure provides adeno-associated virus (AAV) RepCap plasmids for the improved production of recombinant AAV (rAAV) vectors and rAAV viral vectors.
  • RepCap plasmids of the present disclosure can comprise a nucleic acid sequence encoding a mutant replicase protein.
  • RepCap plasmids of the present disclosure can comprise a nucleic acid sequence encoding at least one viral gene operably linked to a viral promoter.
  • the at least one viral gene is an HSV-1 viral gene, an HPV-16 viral gene, or an HCMV viral gene.
  • RepCap proteins of the present disclosure can be modified such that the nucleic acid sequence encoding the capsid protein utilize rare codons in place of the naturally occurring codons (“codon deoptimized”) in order to tune expression levels of the capsid proteins expressed from the RepCap plasmid.
  • These modifications to the RepCap plasmid can be used in isolation or in any combination in order to improve the yield of rAAV viral vectors produced during manufacture and/or improve the percentage of full AAV capsids produced during manufacture.
  • the disclosure provides vectors, such as AAV vectors, as well as methods of making same.
  • Plasmid refers to a circular double stranded DNA loop into which DNA segments in addition to the nucleotide of interest can be inserted, such as by standard molecular cloning techniques.
  • viral vector Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • virus e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • the vector is a lentiviral (such as an integration-deficient lentiviral vector) or adeno-associated viral (AAV) vector.
  • lentiviral such as an integration-deficient lentiviral vector
  • AAV adeno-associated viral
  • Vectors may be capable of autonomous replication in a host cell into which they are introduced such as e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors and other vectors such as, e.g., non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors such as e.g., expression vectors, are capable of directing the expression of genes they contain. Common expression vectors are often in the form of plasmids.
  • recombinant expression vectors comprise or encode a nucleic acid provided herein such as e.g., an snRNA or an RNA-targeting nucleic acid molecule comprising the same in a form suitable for expression of an RNA molecule in a host cell.
  • Recombinant expression vectors can include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence such as e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell.
  • the regulatory element is a promoter described herein.
  • the regulatory element is a terminator provided herein.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein such as, e.g., snRNAs, RNA-targeting nucleic acid molecules, CRISPR transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.
  • an expression vector, viral vector, or non-viral vector provided herein includes without limitation, an expression control element.
  • An “expression control element” as used herein refers to any sequence that regulates the expression of a coding sequence, such as a gene.
  • Exemplary expression control elements include but are not limited to promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements may be constitutive, inducible, repressible, or tissuespecific, for example.
  • a “promoter” is a control sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled.
  • Exemplary promoters include, without limitation, constitution and tissue specific promoters. Further exemplary promoters include viral promoters such as a CMV promoter, SV40 promoter, CAG promoter, or EFS promoter, which are encoded by SEQ ID NOS: 17-20, respectively. Selection of a suitable promoter is within the skill of persons of ordinary skill in the art. Vectors may also contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. An “enhancer” is a region of DNA that can be bound by activating proteins to increase the likelihood or frequency of transcription.
  • Vectors may also contain genetic elements for post-transcriptional regulation of genes, for example the woodchuck hepatitis posttranscriptional regulatory element (WPRE), polyadenylation sequences, untranslated regions isolated or derived from transcribed RNAs (5’ and 3’ UTRs) and the like.
  • WPRE woodchuck hepatitis posttranscriptional regulatory element
  • polyadenylation sequences untranslated regions isolated or derived from transcribed RNAs (5’ and 3’ UTRs) and the like.
  • an expression vector, viral vector or non-viral vector includes without limitation, vector elements such as a buffer sequence derived human genomic sequences downstream from an snRNA and as such have the capability of encoding multiple snRNAs from a single construct.
  • a vector of the disclosure is a non-viral vector.
  • the vector comprises or consists of a nanoparticle, a micelle, a liposome or lipoplex, a polymersome, a polyplex or a dendrimer.
  • the vector is an expression vector or recombinant expression system.
  • the term “recombinant expression system” refers to a genetic construct for the expression of certain genetic material formed by recombination.
  • the vector is a viral vector.
  • the vector is an adenoviral vector, an adeno-associated viral (AAV) vector, or a lentiviral vector.
  • the vector is a retroviral vector, an adenoviral/retroviral chimera vector, a herpes simplex viral I or II vector, a parvoviral vector, a reticuloendotheliosis viral vector, a polioviral vector, a papillomaviral vector, a vaccinia viral vector, or any hybrid or chimeric vector incorporating favorable aspects of two or more viral vectors.
  • viral vectors include but are not limited to adenoviral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.
  • AAV adeno-associated virus
  • the vector further comprises one or more expression control elements operably linked to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the AAV vector has low toxicity. In some embodiments, the AAV vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis. In some embodiments, the AAV vector can encode a range of total polynucleotides from 4.5 kb to 4.75 kb.
  • Exemplary AAV vectors that may be used in any of the herein described compositions, systems, methods, and kits can include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV2-Tyr mutant vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAVrh8 vector, an AAV9 vector, an AAV.rhlO vector, a modified AAV.rhlO vector, a MyoAAV vector, a modified MyoAAV vector, a MyoAAVl vector, a MyoAAV4 vector, an AAVrh.74 vector, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.r
  • the vector comprises a lentiviral vector.
  • the lentiviral vector is an integrase-competent lentiviral vector (ICLV).
  • the lentiviral vector can refer to the transgene plasmid vector as well as the transgene plasmid vector in conjunction with related plasmids (e.g., a packaging plasmid, a rev expressing plasmid, an envelope plasmid) as well as a lentiviral-based particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • the viral vector comprises a sequence isolated or derived from a retrovirus.
  • the viral vector comprises a sequence isolated or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is replication incompetent. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.
  • the vector further comprises one or more expression control elements operably linked to the polynucleotide comprising or encoding the snRNA described herein. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the vector has low toxicity. In some embodiments, the vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis.
  • rAAV vector or “rAAV expression vector” as used herein refers to a vector comprising, consisting essentially of, or consisting of one or more polynucleotide elements described herein (sometimes referred to as an expression vector, transgene or similar) and one or more AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • Such AAV vectors can be replicated and packaged into infectious viral particles, utilizing RepCap plasmids of the disclosure and comprising AAV capsid proteins of the disclosure, when present in a host cell that provides the functionality of rep and cap gene products; for example, by transfection of the host cell.
  • AAV vectors contain a promoter, at least one nucleic acid that may encode at least one protein or RNA, and/or an enhancer and/or a terminator within the flanking ITRs that is packaged into the infectious AAV particle.
  • the encapsidated nucleic acid portion may be referred to as the AAV vector genome.
  • Plasmids containing rAAV vectors may also contain elements for manufacturing purposes, e.g., antibiotic resistance genes, origin of replication sequences, etc., but these are not encapsidated and thus do not form part of the AAV particle.
  • the rAAV expression vector comprises one or more nucleotide elements for delivery to cell, tissue organ or subject.
  • nucleotide elements include transgenes, for example transgenes encoding proteins or RNA sequences.
  • the protein comprises a therapeutic protein.
  • therapeutic proteins include, without limitation, antibodies, fusion proteins, cytokines, chemokines, receptor fusion proteins, CRISPR/Cas proteins (Cas9, Cpfl and the like) and PUF proteins.
  • the RNA sequence comprises a non-coding RNA.
  • Exemplary non-coding RNAs include small nuclear RNA sequences, short hairpin RNAs (shRNA), small interfering RNAs (siRNA), microRNAs (miRNA) and guide RNAs for a CRISPR/Cas proteins.
  • the rAAV expression vector further comprises one or more expression control elements as described herein operably linked to the polynucleotide element or transgene. In some embodiments, the vector further comprises one or more selectable markers. In some embodiments, the vector has low toxicity. In some embodiments, the vector does not incorporate into the host genome, thereby having a low probability of causing insertional mutagenesis.
  • an rAAV vector can comprise at least two promoter sequences operably linked to nucleic acid elements such as a noncoding RNA or transgene.
  • an rAAV vector can comprise at least one AAV inverted terminal (ITR) sequence.
  • an rAAV vector can comprise at least one promoter sequence.
  • an rAAV vector can comprise at least one enhancer sequence.
  • an rAAV vector can comprise at least one polyadenylation (poly(A)) sequence.
  • an rAAV vector can comprise at least one reporter protein.
  • an rAAV vector can comprise more than one nucleic acid element, such as a transgene nucleic acid molecule, or more than one noncoding RNA molecule.
  • an rAAV vector can comprise at least two transgene nucleic acid molecules or at least two noncoding RNA molecules, such that the rAAV vector comprises a first transgene nucleic acid molecule or noncoding RNA molecule and an at least a second transgene nucleic acid molecule or noncoding RNA molecule.
  • the first and the at least second transgene nucleic acid molecule or noncoding RNA molecule can comprise the same nucleic acid sequence.
  • the first and the at least second transgene nucleic acid molecules or noncoding RNA molecules can comprise different nucleic acid sequences.
  • the rAAV vector comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or more nucleic acid elements (including noncoding RNA or transgenes), wherein each nucleic acid element is operably linked to a promoter.
  • the promoter operably linked to each nucleic acid element is unique and/or has low sequence homology relative to other promoters within the vector.
  • an rAAV vector can comprise more than one promoter sequences.
  • an rAAV vector can comprise at least two promoter sequences, such that the rAAV vector comprises a first promoter sequence and at least a second promoter sequence.
  • the first and the at least second promoter sequences can comprise the same sequence.
  • the first and the at least second promoter sequences can comprise different sequences.
  • the first and the at least second promoter sequences can be adjacent to each other.
  • an rAAV vector also comprises a first transgene nucleic acid molecule (for example, noncoding RNA molecule) and an at least second transgene nucleic acid molecule (for example a second noncoding RNA molecule), and the first promoter can be located upstream (5’) of the first transgene nucleic acid molecule and the at least second promoter can be located between the first transgene nucleic acid molecule o and the at least second transgene nucleic acid molecule, such that the at least second promoter is downstream (3’) of the first transgene nucleic acid molecule and upstream (5’) of the at least second transgene nucleic acid molecule.
  • a first transgene nucleic acid molecule for example, noncoding RNA molecule
  • an at least second transgene nucleic acid molecule for example a second noncoding RNA molecule
  • any of the preceding rAAV vectors can further comprise at least one enhancer.
  • the at least one enhancer can be located anywhere in the rAAV vector. In some aspects, the at least one enhancer can be located immediately upstream (5’) of a promoter. Enhancers can be tissue specific (for example, central nervous system, liver or muscle specific), or can be constitutive (for example, certain viral promoters). Selection of a suitable enhancer is within the skill of a person of ordinary skill in the art.
  • vectors comprising a nucleic acid encoding at least one RNA sequence or protein.
  • the RNA sequence is a noncoding RNA sequence.
  • the noncoding RNA is an RNA binding noncoding RNA.
  • the noncoding RNA is a small-nuclear RNA (snRNA) molecule, a single guide RNA molecule (sgRNA), a microRNA, a short hairpin RNA (shRNA), an enhancer RNA (eRNA), a small nucleolar RNA (snoRNA), or a long noncoding RNA (IncRNA).
  • the sgRNA is used in conjunction with CRISPR/Cas systems (e.g., any suitable Class 2 Type II, V or VI CRISPR/Cas system) to target, bind, and/or cleave nucleic acids including DNA and RNA sequences.
  • the noncoding RNA is an snRNA molecule.
  • Short nuclear RNA molecules of the present disclosure can be non-natural, modified, and/or engineered snRNA molecules.
  • the snRNA molecules of the present disclosure bind and target RNA molecules.
  • the protein is any protein known in the art. In some aspects, the protein is a therapeutic protein. In some aspects, the protein is a synthetic or non-naturally occurring protein. In some aspects, the protein is a fusion protein. In some aspects, the protein is a DNA or RNA binding protein. In some aspects, the RNA binding protein is a Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas protein. In some aspects, the RNA binding protein is a Pumilio and FBF (PUF) or Pumilio-based assembly (PUMBY) protein.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • RNA binding protein is a Pumilio and FBF (PUF) or Pumilio-based assembly (PUMBY) protein.
  • vectors of the present disclosure comprise one or more RNA molecules. In some embodiments, vectors of the present disclosure comprise one or more snRNA molecules. In some aspects, vectors encoding snRNA are capable of targeting multiple (z.e., two or more) RNAs of interest. In some aspects the RNA of interest is an mRNA or pre-mRNA sequence. In some embodiments, the two or more RNAs of interest can target the same mRNA or pre-mRNA molecule but different sequences within the mRNA or pre-mRNA molecule.
  • AAV Adeno-associated Virus
  • a vector described herein is an adeno-associated virus (AAV) viral vector.
  • AAV adeno-associated virus
  • An “AAV expression vector” or “AAV vector” as used herein is in reference to a vector comprising, consisting essentially of, or consisting of one or more transgene sequences (also referred to herein as nucleic acid elements) and one or more AAV inverted terminal repeat sequences (ITRs).
  • the AAV expression vector can be packaged into an AAV viral vector (a viral vector particle) using the methods described herein.
  • Adeno-associated virus refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae.
  • Adeno-associated virus is a single-stranded DNA virus that grows in cells in which certain functions are provided by a co-infecting helper virus.
  • General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).
  • the degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs).
  • ITRs inverted terminal repeat sequences
  • the similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types.
  • AAV is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two 145-nucleotide inverted terminal repeat (ITRs). ITR sequences are known in the art, and exemplary ITR sequences are provided herein as SEQ ID NOS:24 and 35.
  • the rAAV vector comprises ITRs isolated or derived from AAV2.
  • AAV serotypes of AAV There are multiple serotypes of AAV.
  • the nucleotide sequences of the genomes of the AAV serotypes are known.
  • the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077;
  • the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983);
  • the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829;
  • the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829;
  • the AAV-5 genome is provided in GenBank Accession No. AF085716;
  • the complete genome of AAV-6 is provided in GenBank Accession No.
  • AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).
  • the sequence of the AAV rh.74 genome is provided in U.S. Patent 9,434,928.
  • U.S. Patent No. 9,434,928 also provides the sequences of the capsid proteins and a self-complementary genome.
  • an AAV genome is a self-complementary genome.
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging, and host cell chromosome integration are contained within AAV ITRs.
  • Three AAV promoters (named p5, pl 9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and pl 9) coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and encodes the three capsid proteins, VP1, VP2, and VP3.
  • Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. More specifically, after the single mRNA from which each of the VP1, VP2 and VP3 proteins are translated is transcribed, it can be spliced in two different manners: either a longer or shorter intron can be excised, resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool.
  • the longer intron is often preferred and thus the 2.3-kb-long mRNA can be called the major splice variant.
  • This form lacks the first AUG codon, from which the synthesis of VP1 protein starts, resulting in a reduced overall level of VP1 protein synthesis.
  • the first AUG codon that remains in the major splice variant is the initiation codon for the VP3 protein.
  • upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak (translation initiation) context.
  • Each VP1 protein contains a VP1 portion, a VP2 portion and a VP3 portion.
  • the VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein.
  • the VP2 portion is the amino acid sequence present within the VP1 protein that is also found in the N-terminal portion of the VP2 protein.
  • the VP3 portion and the VP3 protein have the same sequence.
  • the VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 and VP2 proteins.
  • the VP3 protein can be further divided into discrete variable surface regions I-IX (VR-I-IX).
  • Each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that either alone or in combination with the specific amino acid sequences of each of the other VRs can confer unique infection phenotypes (e.g., decreased antigenicity, improved transduction, and/or tissue-specific tropism relative to other AAV serotypes) to a particular serotype as described in DiMatta et al., “Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9” J. Virol., Vol. 86 (12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy.
  • AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells, allowing the possibility of targeting many different tissues in vivo.
  • the AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible.
  • the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, RepCap) may be replaced with foreign DNA to generate AAV vectors (i.e., the rAAV expression vectors and nucleic acid elements of same described herein).
  • the Rep and Cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
  • the present disclosure provides single-stranded AAV (ssAAV) vectors.
  • the disclosure provides self-complementary AAV (scAAV).
  • the single-stranded nature of the parvoviral genome requires the use of cellular mechanisms to provide a complementary-strand for gene expression. This cellular recruitment activity is considered a rate-limiting factor in the efficiency of transduction and gene expression in parvoviruses and parvoviral particles.
  • the use of an scAAV versus an ssAAV remedies this well-known issue by packaging both strands as a single duplex DNA molecule (or inverted repeat genome) that can fold into dsDNA as a result of a self-complementary viral genome sequence. In this regard, the requirement for DNA synthesis or base-pairing between multiple viral genomes is eliminated.
  • an rAAV vector of the present disclosure comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NO: 23, or 52-56.
  • the present disclosure comprises, consists essentially of, or consists of a nucleic acid sequence set forth in any one of SEQ ID NO: 23, or 52-56, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the one or more additional viral gene is comprised within a plasmid that is transfected into an AAV producing cell alongside other plasmids encoding sequences necessary for AAV production including, but not limited to, AAV Rep and Cap genes, additional AAV helper genes, and an rAAV vector genome.
  • an AAV producing cell is modified to stably express the one or more additional viral gene.
  • the present disclosure provides methods of producing rAAV viral vectors comprising the use of baculovirus-mediated production.
  • one or more additional viral genes of the disclosure are encoded into a baculovirus for Sf9 cell-based AAV production.
  • the one or more viral gene is isolated or derived from any virus.
  • the virus is a human virus.
  • the virus is an adenovirus.
  • the one or more viral gene is isolated or derived from a virus that supports production of adenoviral vectors.
  • the one or more viral gene each encode a protein or polypeptide.
  • the one or more viral gene is isolated or derived from Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), Human Papillomavirus Type 16 (HPV-16), and Human Bocavirus 1 (HBoVl).
  • HSV-1 Herpes simplex virus 1
  • HSV-2 Herpes simplex virus 2
  • HCMV human cytomegalovirus
  • HPV-16 Human Papillomavirus Type 16
  • HBVl Human Bocavirus 1
  • the one or more viral gene is isolated or derived from HSV-1.
  • the one or more viral gene is isolated or derived from HSV-2.
  • the one or more viral gene is isolated or derived from HCMV.
  • the one or more viral gene is isolated or derived from HPV-16.
  • the one or more viral gene is isolated or derived from HBoVl .
  • the viral gene comprises ICP8, El, E2, UL97, ICP0, or E6, or a combination thereof.
  • ICP8 encodes herpes simplex virus type-1 single-strand DNA-binding protein, one of seven proteins encoded in the HSV-1 viral genome that is required for HSV-1 DNA replication. ICP8 protein has been demonstrated to anneal to single-stranded DNA (ssDNA) and can additionally melt small double-stranded DNA (dsDNA) fragments. ICP8 serves to destabilize duplex DNA during initiation of replication. Unlike helicases, it is ATP and Mg 2+ independent.
  • the nucleic acid sequence encoding ICP8 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 3.
  • the nucleic acid sequence encoding ICP8 comprises, consists essentially of, or consists of the sequence set forth in SEQ ID NO: 3, or a sequence having 1,
  • an ICP8 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 4.
  • the IPC8 amino acid sequences retains all, or substantially of the native ICP8 protein function.
  • an ICP8 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 4, or a sequence having 1, 2,
  • HPV-16 gene El encodes a helicase that is essential for the initiation of viral DNA replication.
  • the El helicase unwinds the viral origin and recruits host cellular factors to replicate the HPV-16 viral genome.
  • the nucleic acid sequence encoding El comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 5.
  • the nucleic acid sequence encoding El comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 5, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • an El amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 6, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the El amino acid sequences retains all, or substantially of the native El protein function.
  • HPV-16 gene E2 is a transcriptional regulator protein that helps recruit El helicase to the viral origin. E2 further plays a role in genome partitioning.
  • the nucleic acid sequence encoding E2 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 7.
  • the nucleic acid sequence encoding E2 comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 7, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • an E2 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 8.
  • an E2 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 8, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the E2 amino acid sequences retains all, or substantially of the native E2 protein function.
  • HCMV gene UL97 encodes a serine/threonine kinase that phosphorylates viral and cellular protein and impacts viral replication at a number of levels.
  • viral DNA synthesis has been shown to be inefficient, and structural proteins are observed to be sequestered in nuclear aggresomes. Mature viral capsids that do form fail to egress the nucleus as the nuclear lamina is not dispersed by the kinase.
  • the nucleic acid sequence encoding UL97 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 1.
  • the nucleic acid sequence encoding UL97 comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 1, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • a UL97 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 2.
  • a UL97 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 2, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the UL97 amino acid sequences retains all, or substantially of the native UL97 protein function.
  • the viral gene encodes a kinase.
  • the kinase is a serine/threonine kinase.
  • the serine/threonine kinase has structural and/or sequence homology to HCMV UL97 kinase.
  • HSV-1 gene ICP0 encodes a viral transactivator of HSV-1 promoters. ICP0 is capable of transactivating promoters of all three kinetic classes of HSV-1 genes.
  • the transactivator function of ICP0 is derived from an N-terminal RING-finger and a C-terminal nuclear domain 10 (ND 10) localization signal, a dimer/multimer motif, and a binding site for ubiquitin-specific protease 7 (USP7).
  • the nucleic acid sequence encoding ICP0 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 9.
  • the nucleic acid sequence encoding ICP0 comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 9, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • an ICP0 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 10.
  • an ICP0 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 10, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the IPC0 amino acid sequences retains all, or substantially of the native ICP0 protein function.
  • the viral gene is HPV E6.
  • the nucleic acid sequence encoding E6 comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 11.
  • the nucleic acid sequence encoding E6 comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 11, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • an E6 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 12.
  • an E6 amino acid sequence comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 12, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the E6 amino acid sequences retains all, or substantially of the native E6 protein function.
  • a nucleic acid sequence encoding the one or more viral gene is included within an AAV RepCap plasmid.
  • the placement of the one or more viral gene can be located 5’ to both the rep and cap sequences.
  • the placement of the one or more viral gene can be located 5’ to both the rep and cap sequences.
  • the one or more viral genes can be located 5’ to the rep sequence and 3’ to the cap sequence.
  • the one or more viral genes can be located 5’ to the cap sequence and 3’ to the rep sequence.
  • the one or more viral genes can be located between the rep sequence and the cap sequence.
  • promoter is a regulatory sequence that is a region of a polynucleotide sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors.
  • Any suitable promoter may be used to drive expression of one or more viral genes, wherein the viral gene is operably linked to the promoter.
  • the promoter is a ubiquitous promoter.
  • ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B 1-CBX3).
  • the promoter is a viral promoter. In some aspects, the promoter is a non-viral promoter.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • the promoter is a simian vacuolating virus 40 (SV40) promoter.
  • the promoter is a CAG promoter.
  • the CAG promoter is synthetic promoter comprised of the cytomegalovirus (CMV) early enhancer element, the first intron of the chicken beta-actin gene, and the splice acceptor of the rabbit beta-globin gene.
  • the promoter is an elongation factor la short (EFS) promoter.
  • the CMV promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 17.
  • the SV40 promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 18.
  • the CAG promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 19.
  • the EFS promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 20.
  • the EF-1 a promoter comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 21.
  • the CMV promoter comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 17, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the SV40 promoter comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 18, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the CAG promoter comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 19, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the EFS promoter comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 20, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the EF-1 a promoter comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 21, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • RepCap plasmids of the present disclosure comprise at least one promoter operably linked to the at least one viral gene. In some embodiments, RepCap plasmids of the present disclosure comprise at least one promoter operably linked to sequences encoding the replicase, the capsid and the at least one viral gene.
  • the disclosure provides RepCap plasmids comprising wild-type Rep genes encoding wild-type replicase proteins.
  • the wild-type replicase can be of any serotype.
  • the replicase is an AAV2 replicase.
  • the disclosure provides modified replicase proteins.
  • the disclosure provides RepCap plasmids comprising a modified Rep gene.
  • the modified Rep gene encodes a mutant or modified replicase protein.
  • the disclosure provides modified replicase from any AAV serotype.
  • Replicase derived from AAV2 is the standard replicase used in AAV production. It has been previously shown that the N-terminal DNA-binding and endonuclease domain of AAV2 replicase is distinct from the AAV1 or AAV8 replicase.
  • wild-type AAV2 replicase comprises, consists essentially of, or consists of an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 16.
  • wild-type AAV2 replicase comprises, consists essentially of, or consists of a nucleic acid sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 15.
  • Modified AAV2 replicase variants can be mutated at any position of the replicase sequence and the wild-type amino acid can be substituted with any natural or non-natural amino acid. Further, AAV2 replicase variants can have deletions or insertions of any length.
  • wild-type AAV2 replicase comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 16.
  • wild-type AAV2 replicase comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 15, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • AAV2 replicase variants of the disclosure comprise one or more mutations at amino acid positions: L45, T65, E66, M91, El 14, 1117, R119 and/or Q288., with the positions indicated relative to SEQ ID NO: 16.
  • AAV2 replicase variants of the disclosure comprise one or more of the following mutations: L45, T65, E66, M91, El 14, 1117, and R119.
  • AAV2 replicase variants of the disclosure comprise the following mutations: L45, T65, E66, M91, El 14, 1117, R119, and Q288.
  • AAV2 replicase variants of the disclosure comprise all of L45, T65, E66, M91, El 14, 1117, and R119. In some cases, AAV2 replicase variants of the disclosure comprise all of L45, T65, E66, M91, El 14, 1117, R119, and Q288.
  • the leucine at position 45 is mutated to arginine.
  • the threonine at position 65 is mutated to valine.
  • the glutamate at position 66 is mutated to glutamine.
  • the methionine at position 91 is mutated to leucine.
  • the glutamate at position 114 is mutated to arginine.
  • the isoleucine at position 117 is mutated to valine.
  • the arginine at position 119 is mutated to threonine.
  • the glutamine at position 288 is mutated to lysine.
  • AAV2 replicase variants of the disclosure comprise the following mutations: L45R, T65V, E66Q, M91L, El 14D, Il 17V, and R119T. In some aspects, AAV2 replicase variants of the disclosure comprise the following mutations: L45R, T65V, E66Q, M91L, El 14D, Il 17V, R119T, and Q288K. In some aspects, a modified AAV2 replicase comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 14.
  • a modified AAV2 replicase comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • a modified AAV2 replicase comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 13.
  • a modified AAV2 replicase comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 13, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto. Codon usage modification of nucleic acid sequences encoding capsid proteins
  • the disclosure provides nucleic acid sequences encoding Cap genes that have been codon “deoptimized” such that less frequently occurring codons are used in place of the naturally occurring codons.
  • Codon-optimization is a technique well known in the art. Codon optimization refers to the fact that different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence to match with the relative abundance of corresponding tRNAs, it is possible to increase expression - “codon optimization”. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in a particular cell type - “codon deoptimization”. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms. Based on the genetic code, nucleic acid sequences with desired codons corresponding to a given amino acid sequence can be generated.
  • such a sequence is optimized for expression in a host or target cell, such as a host or producer cell used to express a capsid or a cell in which the disclosed methods are practiced (such as in an AAV producing cell, e.g., a HEK293T cell or insect cell).
  • Codon preferences and codon usage tables for a particular species can be used to engineer isolated nucleic acid molecules encoding a protein (e.g. a capsid protein) that takes advantage of the codon usage preferences of that particular species.
  • an isolated nucleic acid molecule (which can be part of a vector) includes at least one coding sequence that is codon deoptimized for expression in a eukaryotic cell, or at least one coding sequence codon deoptimized for expression in a human cell. In some embodiments, an isolated nucleic acid molecule (which can be part of a vector) includes at least one coding sequence that is codon deoptimized for expression in an insect cell.
  • such a codon deoptimized coding sequence has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating sequence.
  • such a codon deoptimized coding sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in its corresponding wild-type or originating sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • a codon deoptimized nucleic acid sequence encodes a capsid protein having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to its corresponding wild-type or originating sequence.
  • a codon deoptimized nucleic acid sequence encodes a capsid protein comprising, consisting essentially of, or consisting of an amino acid sequence as set forth in its corresponding wildtype or originating sequence, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • a variety of clones containing functionally equivalent nucleic acids may be routinely generated, such as nucleic acids which differ in sequence but which encode the same sequence.
  • Silent mutations in the coding sequence result from the degeneracy (z.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue.
  • leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; isoleucine can be encoded by ATT, ATC, or ATA; phenylalanine can be encoded by TTT or TTC; valine can be encoded by GTT, GTC, GTA, or GTG; proline can be CCT, CCC, CCA, or CCG; threonine can be ACT ACC, ACA, or ACG; histidine can be encoded CAT
  • the codon deoptimized sequence exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% reduced transcription or translation in a human subject relative to a wild-type or non-codon optimized nucleic acid sequence.
  • a codon deoptimized nucleic acid sequence can have a GC content that differs from the GC content of the non-codon deoptimized nucleic acid sequence. In some aspects the GC content of a codon deoptimized nucleic acid sequence is less evenly distributed across the entire nucleic acid sequence, as compared to the non-codon deoptimized nucleic acid sequence. [0181] Without wishing to be bound by theory, by less evenly distributing the GC content across the entire nucleic acid sequence, the codon deoptimized nucleic acid sequence exhibits a less uniform melting temperature (“Tm”) across the length of the transcript.
  • Tm uniform melting temperature
  • the lack of uniformity of melting temperature results unexpectedly in decreased expression of the codon deoptimized nucleic acid in a human subject, as transcription and/or translation of the nucleic acid sequence occurs with more stalling of the polymerase and/or ribosome.
  • a codon deoptimized nucleic acid sequence can have more repressive microRNA target binding sites as compared to the non-codon deoptimized nucleic acid sequence. Without wishing to be bound by theory, by having more repressive microRNA target binding sites, the codon deoptimized nucleic acid sequence unexpectedly exhibits decreased expression in a human subject.
  • Nucleic acid sequences encoding cap genes or capsid proteins can be codon deoptimized at any position or length of the sequence. Accordingly, any individual codon or sequence of codons can be exchanged to use a less frequently occurring codon. In some aspects, native codons are changed to rare human codons.
  • the capsid encoded by the codon deoptimized nucleic acid sequence can be any serotype described herein or known in the art.
  • the capsid is of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrhlO, Anc80, PHP.eB, or MyoAAV.
  • the capsid is a modified capsid protein.
  • Modified capsid proteins of the disclosure can comprise any modification including mutation, deletion, or insertion of amino acids along the naturally occurring sequence.
  • the capsid is of serotype AAV9. In some aspects, the capsid is of serotype AAVrhlO. In some aspects, the capsid is of serotype AAVrh74. In some aspects, the capsid is of serotype MyoAAV.
  • the nucleic acid sequence encoding the AAV capsid protein is codon deoptimized in a portion of the nucleic acid sequence that encodes a VP1, VP2, or VP3 capsid protein. In some aspects, the nucleic acid sequence encoding the AAV capsid protein is codon deoptimized in a portion of the nucleic acid sequence that encodes a VP3 capsid protein. In some aspects, the nucleic acid sequence encoding the AAV capsid protein is codon deoptimized in a C-terminal region after the AAP reading frame.
  • a non-codon deoptimized AAV9 nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 22.
  • a non-codon deoptimized AAV9 nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 22, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • a non-codon deoptimized AAVrh74 nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 37.
  • a non-codon deoptimized AAVrh74 nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 37, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • a non-codon deoptimized AAVrhlO nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 36.
  • a non-codon deoptimized AAVrhlO nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 36, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • a non-codon deoptimized MyoAAV nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 38.
  • a non-codon deoptimized MyoAAV nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 38, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 39.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 39, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 40.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 40, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 41.
  • an AAV9 codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 41, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • an AAVrhlO codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 50.
  • an AAVrhlO codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 50, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • an MyoAAV codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 51.
  • an MyoAAV codon deoptimized nucleic acid sequence comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in SEQ ID NO: 51, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions relative thereto.
  • Translation efficiency is a measure of how readily the nucleic acid sequence is expressed as a protein. In some aspects, translation efficiency therefore is a measure of the amount of protein produced per a given quantity of mRNA encoding said protein. Translation efficiency can be expressed as a translation efficiency score. A score of 1 indicates maximum translation and reflects usage of the most frequently occurring codon for each amino acid in a protein sequence. A score of 0 is indicative of very low translation efficiency. In some aspects, the translation efficiency can be calculated as described in Sharp et al. Nucleic Acids Research, Volume 15, Issue 3, 11 February 1987, Pages 1281-1295 which is incorporated herein in its entirety by reference.
  • the codon deoptimized nucleic acid sequence has a translation efficiency score lower than the translation efficiency score of a non-codon deoptimized or wild-type nucleic acid sequence encoding a capsid protein.
  • the translation efficiency score is lower than about 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, or 0.2.
  • the translation efficiency score of the codon deoptimized nucleic acid sequence cannot be too low because expression of the sequence encoding the capsid protein may fall to a level where not enough capsid protein is produced. In some aspects, the translation efficiency score is higher than about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.6. [0207] The translation efficiency score was calculated for the native or wild-type capsid sequence for a variety of capsid serotypes and is reflected in Table 1.
  • Table 1 Translation efficiency scores for native capsid encoding nucleic acid sequences.
  • capsid expression levels from codon deoptimized nucleic acid sequences are at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or 1000% lower than expression of a capsid from a capsid encoding sequence that has not been codon deoptimized.
  • the disclosure provides RepCap plasmids comprising codon deoptimized nucleic acid sequences encoding capsid proteins.
  • rAAV viral vectors produced utilizing RepCap plasmids comprising the codon deoptimized capsid-encoding nucleic acid sequences have a higher percentage of full capsids relative to rAAV viral vectors produced using non-codon deoptimized capsidencoding nucleic acid sequences.
  • rAAV viral vectors produced utilizing a RepCap plasmid comprising a codon deoptimized capsid-encoding nucleic acid sequence are at least 40%, 50%, 60%, 70%, 80%, 90% or 100% full. In some aspects, rAAV viral vectors produced utilizing a RepCap plasmid comprising a codon deoptimized capsid-encoding nucleic acid sequence are at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or 100% more full than rAAV viral vectors produced using non-codon deoptimized capsid-encoding nucleic acid sequences.
  • a significant challenge for AAV viral vector production is purification. Separating full capsids from empty capsids is challenging and adds complexity to the manufacturing process. Enhancing the level of full capsids while also controlling the overall amount of capsid expressed can make the manufacture and purification process more efficient leading to a purer final AAV viral vector pharmaceutical composition. Accordingly, codon deoptimized nucleic acid sequences encoding capsid sequences and RepCap plasmids incorporating said sequences can simplify the manufacture of AAV viral vectors due to the controlled expression of capsid proteins.
  • manufacture of AAV viral vectors using codon deoptimized nucleic acid sequences of the disclosure or RepCap plasmids of the disclosure can eliminate downstream purification steps such as chromatographic separation (e.g. anion exchange chromatography (AEX)).
  • chromatographic separation e.g. anion exchange chromatography (AEX)
  • sequences provided herein can be used to provide the expression product as well as substantially identical sequences that encode an RNA or express and produce a protein that has the same biological properties.
  • biologically equivalent or “biologically active” or “equivalent” polypeptides are encoded by equivalent polynucleotides as described herein. They may possess at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98%, identical primary amino acid sequence to the reference polypeptide when compared using sequence identity methods run under default conditions.
  • polypeptide sequences are provided as examples of particular embodiments. Modifications to the sequences to amino acids with alternate amino acids that have similar charge.
  • an equivalent polynucleotide is one that hybridizes under stringent conditions to the reference polynucleotide or its complement or in reference to a polypeptide, a polypeptide encoded by a polynucleotide that hybridizes to the reference encoding polynucleotide under stringent conditions or its complementary strand.
  • an equivalent polypeptide or protein is one that is expressed from an equivalent polynucleotide.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi -stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • Examples of stringent hybridization conditions include: incubation temperatures of about 25°C to about 37°C; hybridization buffer concentrations of about 6x SSC to about lOx SSC; formamide concentrations of about 0% to about 25%; and wash solutions from about 4x SSC to about 8x SSC.
  • Examples of moderate hybridization conditions include: incubation temperatures of about 40°C to about 50°C; buffer concentrations of about 9x SSC to about 2x SSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5x SSC to about 2x SSC.
  • Examples of high stringency conditions include: incubation temperatures of about 55°C to about 68°C; buffer concentrations of about lx SSC to about O.
  • lx SSC formamide concentrations of about 55% to about 75%
  • wash solutions of about lx SSC, O.lx SSC, or deionized water.
  • hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes.
  • SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.
  • sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST.
  • the terms “about” and “approximately” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, z.e., the limitations of the measurement system.
  • “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art.
  • “about” or “approximately” can mean a range of up to 10% (z.e., ⁇ 10%) or more depending on the limitations of the measurement system.
  • about 5 mg can include any number between 4.5 mg and 5.5 mg.
  • the terms can mean up to an order of magnitude or up to 5-fold of a value.
  • the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.
  • the ranges and/or subranges can include the endpoints of the ranges and/or subranges.
  • operably linked and “operably joined” or related terms as used herein refers to the juxtaposition of components.
  • the components can be linked together covalently.
  • two nucleic acids can be linked together via a phosphodiester linkage.
  • a first component that confers a function on a second component without being directly physically linked can be considered to be operably linked.
  • rAAV viral vectors
  • AAV vectors (the expression vectors) of the disclosure can be packaged as an rAAV viral vector (the viral particle).
  • An “rAAV viral vector” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector (sometimes referred to herein as the “rAAV expression vector”). Thus, production of an rAAV viral vector necessarily includes production of an AAV vector.
  • the term "viral capsid” or “capsid” refers to the proteinaceous shell or coat of a viral particle. Capsids function to encapsidate, protect, transport, and release into the host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of protein ("capsid proteins"). As used herein, the term “encapsidated” means enclosed within a viral capsid.
  • the viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3.
  • AAV viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology.
  • AAV viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction.
  • RepCap plasmids of the disclosure are used to produce rAAV viral vectors.
  • rAAV viral vectors of the disclosure can be used to mediate gene transfer of a transgene to a cell of a subject.
  • the terms “gene transfer” or “gene delivery” refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g. episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Recombinant viral vectors may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses, including RepCap plasmids of the disclosure.
  • virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • AAV packaging cells include HEK293 cells, HEK293T cells, A549 cells, WEHI cells, 3T3 cells, 10T1/2 cells, BHK cells, MDCK cells, COS 1 cells, COS 7 cells, BSC 1 cells, BSC 40 cells, VERO cells, WI38 cells, HeLa cells and HepG2 cells.
  • the rAAV viral vector is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrh.10, MyoAAV or any other serotypes of AAV known in the art that can infect humans, monkeys or other species.
  • the disclosure provides rAAV expression vectors comprising one or more ITRs flanking a transgene (or nucleic acid element), and AAV viral vector particles comprising same.
  • inverted terminal repeats or “ITRs” is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus.
  • AAV ITRs together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
  • an "AAV ITR” does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
  • the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, ie., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.
  • an AAV inverted terminal repeat sequence can comprise any AAV ITR sequence known in the art.
  • an AAV ITR sequence can comprise or consist of an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV3 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAVrhlO ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, or an AAVrh74 ITR sequence.
  • the ITR sequence can comprise a modified AAV ITR sequence.
  • an AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 24 or SEQ ID NO: 35.
  • an AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 35, or a sequence having 1, 2, 3, 4, or 5 substitutions, insertions, or deletions relative thereto.
  • an AAV vector provided herein comprises a first and a second AAV ITR sequence.
  • a first AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 24 or SEQ ID NO: 35 and a second AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 24 or SEQ ID NO: 35.
  • a first AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 35, or a sequence having 1, 2, 3, 4, or 5 substitutions, insertions, or deletions relative thereto; and a second AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence as set forth in SEQ ID NO: 24 or SEQ ID NO: 35, or a sequence having 1, 2, 3, 4, or 5 substitutions, insertions, or deletions relative thereto.
  • the first AAV ITR sequence is positioned at the 5’ of an AAV vector. In some aspects the second AAV ITR sequence is positioned at the 3’ of an AAV vector.
  • the viral vector comprises a sequence isolated or derived from an adeno-associated virus (AAV).
  • AAV adeno-associated virus
  • RepCap plasmids suitable for the production of rAAV viral vectors have been modified for the improved manufacture of rAAV viral vectors relative to standard RepCap plasmids.
  • RepCap proteins of the disclosure comprise one or more of: a nucleic acid sequence encoding a modified replicase protein; a codon deoptimized nucleic acid sequence encoding a capsid protein; a nucleic acid sequence encoding a promoter; and/or a nucleic acid sequence encoding at least one viral gene.
  • the at least one viral gene is isolated or derived from Herpes simplex virus 1 (HSV-1), Herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), Human Papillomavirus Type 16 (HPV-16), and Human Bocavirus 1 (HBoVl).
  • HSV-1 Herpes simplex virus 1
  • HSV-2 Herpes simplex virus 2
  • HCMV human cytomegalovirus
  • HPV-16 Human Papillomavirus Type 16
  • HBVl Human Bocavirus 1
  • RepCap plasmids of the disclosure comprise at least one viral gene operably linked to a promoter.
  • the RepCap plasmid comprising the at least one viral gene comprises wild-type Rep and Cap sequences.
  • the RepCap plasmid comprising the at least one viral gene comprises a nucleic sequence encoding a modified Rep sequence that encodes a modified replicase.
  • the RepCap plasmid comprising the at least one viral gene comprises a nucleic sequence encoding a codon deoptimized nucleic acid sequence that encodes a capsid protein.
  • the modified RepCap plasmid comprising the at least one viral gene comprises a nucleic sequence encoding a modified Rep sequence that encodes a modified replicase and a nucleic sequence encoding a codon deoptimized nucleic acid sequence that encodes a capsid protein.
  • the RepCap plasmid comprises a nucleic sequence encoding a modified Rep sequence that encodes a modified replicase.
  • the modified RepCap plasmid comprises a nucleic sequence encoding a codon deoptimized nucleic acid sequence that encodes a capsid protein.
  • the RepCap plasmid comprises a nucleic sequence encoding a modified Rep sequence that encodes a modified replicase and a nucleic sequence encoding a codon deoptimized nucleic acid sequence that encodes a capsid protein.
  • a RepCap plasmid of the disclosure comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NO: 42 - 49.
  • a RepCap plasmid of the disclosure comprises, consists essentially of, or consists of a nucleic acid sequence as set forth in any one of SEQ ID NO: 42 - 49, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, insertions, or deletions relative thereto.
  • the disclosure provides methods of producing AAV viral vectors comprising the use of RepCap plasmids of the disclosure.
  • the disclosure provides a method of producing an AAV viral vector comprising contacting (e.g., transfecting or transducing) cells with i) a RepCap plasmid of the disclosure; ii) an adenovirus helper plasmid; and iii) a plasmid comprising an rAAV expression vector.
  • the method comprises culturing the cells under conditions sufficient to produce the AAV viral vector.
  • the method comprises purifying the AAV viral vector from the cells.
  • AAV viral vectors produced according to methods of the disclosure can be produced in any cell line amenable to the production of AAV viral vectors.
  • the cells are adherent cells. Alternatively, the cells can be in a suspension culture.
  • the cells are HEK293 cells.
  • the cells are HEK293T cells.
  • the cells are HEK293 cells, HEK293T cells, A549 cells, WEHI cells, 3T3 cells, 10T1/2 cells, BHK cells, MDCK cells, COS 1 cells, COS 7 cells, BSC 1 cells, BSC 40 cells, VERO cells, WI38 cells, HeLa cells or HepG2 cells.
  • Methods of producing AAV viral vectors comprising the use of RepCap plasmids of the disclosure yield higher AAV viral vector yield than conventional production methods.
  • conventional production methods comprise the use of standard RepCap plasmids.
  • methods of the disclosure yield about at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000% greater AAV viral vector yield relative to production using a standard RepCap plasmid, wherein the standard RepCap plasmid comprises an unmodified replicase sequence, a standard capsid encoding sequence, and does not comprise a further viral gene.
  • AAV viral vectors produced utilizing modified RepCap plasmids of the disclosure have greater genome integrity than AAV vectors produced according to conventional methods.
  • the term “genome integrity” expresses the percentage of AAV genomes that are the full length intended rAAV vector.
  • the genome integrity of a given rAAV vector is assessed by performing full molecule long read sequencing and assessing read lengths of all evaluated nucleic acids. The integrity or packaging score is then calculated from the mean read length over intended genome size as well as the coefficient of variation of the read lengths.
  • the genome integrity is at least 10%, at least 20%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 750%, at least 1000% greater for AAV viral vectors produced using modified RepCap plasmids relative to production using a standard RepCap plasmid.
  • the host cell for producing the viral vectors of the disclosure itself may be selected from any suitable biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells.
  • prokaryotic e.g., bacterial
  • eukaryotic cells including, insect cells, yeast cells and mammalian cells.
  • the host cells comprise mammalian cells.
  • the mammalian cells comprise human cells.
  • Particularly desirable host cells are selected from among any mammalian species, including, without limitation, cells such as HEK293 cells (which express functional adenoviral El), HEK293T cells, A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, and HepG2.
  • HEK293 cells which express functional adenoviral El
  • HEK293T cells A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, and HepG2.
  • the selection of the mammalian species providing the cells is not a limitation of the present disclosure; nor is the type of mammalian cell, z.e., fibroblast, hepatocyte, tumor cell, etc.
  • the requirements for the cell used is that it not carry any adenovirus gene other than El, E2a and/or E4; it not contain any other virus gene which could result in homologous recombination of a contaminating virus during the production of rAAV; and it is capable of infection or transfection of DNA and expression of the transfected DNA.
  • the host cell is one that has rep and cap stably transfected in the cell.
  • One host cell useful in the present disclosure is a host cell stably transformed with the sequences encoding rep and/or cap.
  • Stable rep and/or cap expressing cell lines such as B-50 (International Patent Application Publication No. WO 99/15685), or those described in U.S. Pat. No. 5,658,785, may also be similarly employed.
  • Yet other cell lines can be constructed using the AAV plasmid sequences of the present disclosure.
  • the preparation of a host cell according to this invention involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., use of overlapping oligonucleotide sequences of the adenovirus and AAV genomes, combined with polymerase chain reaction, synthetic methods, and any other suitable methods which provide the desired nucleotide sequence.
  • Introduction of the molecules (as plasmids or viruses) into the host cell may also be accomplished using techniques known to the skilled artisan and as discussed throughout the specification.
  • standard transfection techniques are used, e.g., CaPCh transfection or electroporation, and/or infection by hybrid adenovirus/ AAV vectors into cell lines such as the human embryonic kidney cell line HEK 293 (a human kidney cell line containing functional adenovirus El genes which provides trans-acting El proteins).
  • Culturing of host cells under conditions suitable to produce the vectors of the disclosure can be carried out using any suitable methods known in the art, and are described, for example, in WO2022112218, the contents of which are incorporated by reference herein in their entirety.
  • Host cells can be cultured in suspension, as adherent cells, or a combination thereof in a suitable culture medium.
  • exemplary culture media include, but are not limited, to serum- free culture media, such as OPM-293 CD03 medium (Optima, 81070-001), LV- MAX production medium (Gibco, A35834-01), and Expi293 expression medium (Gibco, A14351-01), as well as serum-containing cell culture media.
  • serum- free culture media such as OPM-293 CD03 medium (Optima, 81070-001), LV- MAX production medium (Gibco, A35834-01), and Expi293 expression medium (Gibco, A14351-01)
  • Selection of a suitable cell culture medium and culture conditions suitable to produce the viral vectors are known in the art.
  • the viral vectors can be purified by any suitable method including, but not limited to, methods that include lysis, followed by clarification, and one or more concentration and purification steps, which are additionally described in W02023061409, the contents of which are incorporated by reference herein in their entirety.
  • any conventional methods suitable for purifying AAV vectors can be used in the embodiments described herein to purify the recombinant AAV vector.
  • the AAV vector can be isolated and purified from packaging cells by lysing and/or the supernatant of the packaging cells.
  • the AAV vector can be purified by separation method using a CsCl gradient.
  • US Patent Publication No. 20020136710 describes another non-limiting example of method for purifying AAV vectors, in which AAV vector was isolated and purified from a sample using a solid support that includes a matrix to which an artificial receptor or receptor-like molecule that mediates AAV attachment is immobilized. See, for example, U.S. Patent No. 9,527,904, the contents of which are incorporated by reference herein.
  • Clarification refers to the removal of host cells and host cell debris after collection of the supernatant.
  • Exemplary clarification comprises centrifugation, of filtration using a 45 pm porous membrane.
  • Clarified viral vector particles can be concentrated and permeabilized into a suitable buffer using tangential flow filtration (TFF), including the use of membranes with 1 to 100 nm pore size.
  • Viral vector products may also undergo chromatographic purification.
  • Anion- exchange chromatography (AEX) is an efficient tool for viral vector purification, based on the fact that viral vectors are typically positively charged at neutral pH. When the viral vector supernatant is passed through a column with a negatively charged matrix, the positively charged viral particles bind to the negatively charged matrix, and impurities flow directly through the column. Afterwards, the viral vectors can be exposed to a high salt environment (0.5-1M NaCl), and the bound particles are eluted from the anion exchange column.
  • Affinity chromatography can also be used to purify the viral vectors disclosed herein.
  • Affinity chromatography is a purification technique used to separate biomolecules based on the specific interaction between target molecules and ligands attached to a chromatography column.
  • Affinity chromatography can be divided into several categories according to the interaction, such as affinity chromatography based on hydrogen bonding, electrostatic interaction, van der Waals force, or antibody-ligand, etc.
  • Antibody-ligand interactions are the most selective, and then affinity chromatography using antibodies that bind to the envelope protein has not been seen for viral vector purification.
  • Heparin is a relatively inexpensive affinity ligand that interacts with a variety of virus types. There is an interaction between positively charged molecules on the surface of virus particles and negatively charged heparin between the carrier and heparin, so NaCl is usually used to elute the carrier from the heparin column, optionally followed by a TFF step.
  • SEC size exclusion chromatography
  • gel filtration chromatography is used for viral vector purification based on the difference between the large volume of virus particles and the small volume of impurities. All impurities, smaller than the pore size, pass through the column and only bulky virus particles remain.
  • the disadvantage of SEC is that it is difficult to scale up, and the loading volume is only 10% of the column volume.
  • SEC purification requires linear low flow rates and long operating times.
  • cesium chloride density gradient centrifugation can also be used.
  • an iodixanol density gradient centrifugation can be used first, and folllwed by heparin affinity chromatography.
  • Yet another method contemplated within the scope of the disclosure is to first lyse the cells with deoxycholic acid, centrifuge to obtain the supernatant, heat at 56°C for 45 minutes, centrifuge to remove the precipitate after protein denaturation, and then perform heparin affinity chromatography.
  • compositions comprising AAV viral vectors produced by the methods of the disclosure, and a pharmaceutically acceptable carrier, diluent or excipient.
  • Excipients which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired.
  • Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, M D, 2006; incorporated herein by reference in its entirety).
  • kits comprising the RepCap plasmids of the disclosure.
  • the kit comprises a tube and/or carrier for the plasmid.
  • the kit comprises instructions for use.
  • the present disclosure provides an AAV RepCap plasmid comprising a nucleic acid sequence encoding a replicase protein, a nucleic acid sequence encoding a capsid protein, a nucleic acid sequence encoding a viral promoter, and a nucleic acid sequence encoding at least one viral gene.
  • the AAV RepCap plasmid comprises a nucleic acid encoding a replicase protein of an AAV2 replicase comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 13; a nucleic acid encoding a capsid protein comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 39; a nucleic acid encoding a viral promoter comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 17-20; and a nucleic acid encoding a viral gene UL97 comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 1.
  • the AAV RepCap plasmid comprises a nucleic acid encoding a replicase protein of an AAV2 replicase comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 13; a nucleic acid encoding a capsid protein comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 40; a nucleic acid encoding a viral promoter comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 17-20; and a nucleic acid encoding a viral gene UL97 comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 1.
  • the AAV RepCap plasmid comprises a nucleic acid encoding a replicase protein of an AAV2 replicase comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 15; a nucleic acid encoding a capsid protein comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 22, 37-41, or 50-51; a nucleic acid encoding a viral promoter comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 17-20; and a nucleic acid encoding a viral gene comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.
  • the AAV RepCap plasmid comprises a nucleic acid encoding a replicase protein of an AAV2 replicase comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 13; a nucleic acid encoding a capsid protein comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 39; a nucleic acid encoding a viral promoter comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 17-20; and a nucleic acid encoding a viral gene comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.
  • the AAV RepCap plasmid comprises a nucleic acid encoding a replicase protein of an AAV2 replicase comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 13; a nucleic acid encoding a capsid protein comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in SEQ ID NO: 40; a nucleic acid encoding a viral promoter comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 17-20; and a nucleic acid encoding a viral gene comprising, consisting essentially of, or consisting of a nucleic acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9, or 11.
  • the AAV RepCap plasmid comprises a nucleic acid sequence encoding a replicase protein, a nucleic acid sequence encoding a capsid protein, a nucleic acid sequence encoding a viral promoter, and a nucleic acid sequence encoding at least one viral gene as set forth in SEQ ID NO: 47.
  • the AAV RepCap plasmid comprises a nucleic acid sequence encoding a replicase protein, a nucleic acid sequence encoding a capsid protein, a nucleic acid sequence encoding a viral promoter, and a nucleic acid sequence encoding at least one viral gene as set forth in SEQ ID NO: 48.
  • Example 1 Improved vector yields from RepCap plasmids of the disclosure
  • HEK293 cells provide the adenoviral El gene, and E2A, E4, and VA are encoded on the Ad helper plasmid.
  • This set of Ad genes enables assembly and production of the rAAV virus and paved the way for the technological field of gene therapy.
  • current yields limit applications particularly when systemic administration is required, so improvements in rAAV manufacturing are desirable.
  • genes from HSV-1, HCMV, HPV-16, and HBoVl have been shown to support AAV replication. It was reasoned that some genes from these viruses could be used to supplement the Ad genes to increase rAAV production. Genes that act as single proteins rather than in multiprotein complexes, and whose function would be predicted to enhance rAAV production were specifically identified and evaluated. Six candidate genes from HPV-16 and HCMV were identified.
  • AAV viral vectors packaged by triple transfection have one plasmid encoding the AAV2 replicase gene and the capsid of the desired AAV serotype.
  • An additional expression cassette was inserted downstream of the capsid gene sequence to enable expression of candidate viral genes (FIG. 1A).
  • a small-scale adherent screen was performed testing the effectiveness of each viral gene to increase rAAV production measured by DNAse-protected vector genomes at harvest. This initial screen found that multiple genes from HP VI 6 trended towards greater rAAV yields.
  • AAV.RhlO was produced in adherent HEK293T using the RepCap plasmids from the design (FIG. IB). The harvest yield was assessed by qPCR titering of two different ssAAV genomes, AO 1383 encoding a ubiquitously expressed PUF -endonuclease fusion protein and A00676 encoding ubiquitously expressed GFP, and normalized to the RepCap plasmid without additional viral genes (Control).
  • AAV.RhlO was produced in adherent HEK293T using the RepCap plasmids from the design. The harvest yield was assessed by qPCR titering of two different ssAAV genomes and normalized to the RepCap plasmid without additional viral genes (Control). Expressing UL97 from the RepCap plasmid during AAV production in adherent HEK293T cells boosted yields of ssAAV between 1.7 to 5-fold (FIG. 4A and FIG. 4B).
  • AAV replicase engineering An additional strategy that has been previously demonstrated to boost rAAV production is through AAV replicase engineering. Standard AAV replicase used for rAAV production is derived from AAV2. Interestingly, the N-terminal DNA-binding and endonuclease domain of AAV2 is distinct from AAV1 or AAV8 replicase. AAV2 replicase variants with mutations in this region were produced and their productivity was evaluated when combined with UL97. CO 1831 contains the wild-type AAV2 replicase sequence and C02984 contains a mutated AAV2 replicase sequence (FIG. 5).
  • Single-stranded AAV9 was produced in adherent HEK293T using the CO 1831 and C02984. The harvest yield was assessed by qPCR titering. Modification of the replicase sequence doubled productivity (FIG. 6).
  • scAAV self-complementary AAV
  • scAAV use a non- naturally occurring AAV genome that is produced by mutating an ITR. Relative to ssAAV, scAAV can have reduced productivity and genome integrity, which is the packaging of full- length genomes. It was evaluated whether novel RepCap plasmids of the disclosure could improve the generation of scAAV encoding 2 or 4 snRNA expression cassettes. Such genomes can be challenging as they have repetitive and structured elements. It was found that the novel RepCap increased productivity 3.3-fold for the 2X snRNA and 5-fold for the 4X snRNA transgenes (FIG.
  • Nanopore long read sequencing was used to examine genome integrity. Intriguingly, in the 2X snRNA there was more uniform coverage across the intended AAV genome when the novel RepCap was used (FIG. 2B). The average mean read length was 2403 bp from the novel RepCap and 2080 bp from the control. This 15% increase is easily visualized when the overall distribution of read lengths was plotted, as there is a much greater number of short reads in the control (FIG. 2C). Shorter read lengths could be partial genomes or unintended ssAAV genomes in the scAAV preparation. A script called Nanoclip was created that assessed the percentage of ssAAV in scAAV production by analyzing soft clipping profiles.
  • scAAV9 was produced in adherent HEK293T using the C01779 and C02984 AAV2 replicase sequences. Two different AAV genomes constructs were used. One contained 4 snRNA expression cassettes, A05178, and one contained 2 snRNA expression cassettes, A04569. Two independent replicates were performed. The yield was assessed by ddPCR titering. A 5X enhancement in productivity was observed for C02984 production of A05178 (FIG. 7A). A 3X enhancement in productivity was observed for C02984 production of A04569 (FIG. 7B).
  • ssAAV9 was produced in two different suspension HEK293 -derived cell lines using the CO 1779 and C02984. The harvest yield was assessed by qPCR titering. C02984 increased productivity 5-fold in suspension HEK293T cells (FIG. 8A). C02984 increased productivity approximately 5-fold in a second HEK293 cell line for multiple ssAAV genomes (FIG. 8B). [0283] scAAV9 A04569 was produced in HEK293 (WuXi) cells in an AMBR250 bioreactor. Various ratios of RepCap:ALDX80:A04569 were used in the productions. The harvest vector genome yield was assessed by ddPCR titering.
  • the harvest capsid yield was assessed by AAV9 ELISA.
  • the percentage of the full particle was calculated by dividing the vector genome titer by the capsid titer.
  • C02984 increased both vector genome and capsid levels nearly 10-fold in the bioreactor production (FIG. 9).
  • the above data demonstrates that the addition of UL97 and modification of the AAV2 replicase sequence enhance AAV productivity across serotypes, genomes, and production platforms.
  • Example 3 AAV genome integrity from RepCap plasmids of the disclosure
  • the packaging integrity of the U7 vectors with the CO 1779 and C02984 was evaluated using an Agilent TapeStation.
  • the packaging integrity i.e. genomic integrity
  • Truncated genomes were detected in the CO 1779 but not the C02984 productions (FIG. 10). Two independent replicates were performed, and the genomic integrity profiles are consistent.
  • Example 4 Improved vector yields from RepCap plasmids of the disclosure
  • This plasmid (Cap9d375-736) was evaluated and a second less modified design (Cap9d375-600) in the bioreactor and observed harvest yields of 8.6E11 and 1.31E12 vg/ml with full percentages of 80% (FIG. 3E).
  • An optimized production for this genome with a standard RepCap has a range of harvest yield of 1 to 3E11 vg/ml with an approximately 40% full, so the novel RepCap plasmids increased productivity 2.8-fold and doubled the full percentage. This represents a significant advance as it enables purification without the need for AEX-based full enrichment both simplifying the process and increasing final yields.
  • the modified RepCap produces nearly 10X more vector genomes and 14X more capsid (FIG.
  • FIG. 14 shows a diagram of Rep and Cap gene open reading frames in the RepCap plasmid.
  • the Cap gene also encodes the viral assembly proteins MAAP and AAP in an alternate reading frame. Thus, only the C-terminal region downstream of the AAP is amenable to codon modification (FIG. 14).
  • FIG. 15 shows the modified RepCap plasmid and modified RepCap with codon modifications (codon deoptimized) (C05656).
  • the standard AAV9 VP3 Cap sequence in RepCap plasmid C04991 has a predicted translation efficiency of 0.74 on a 0-1 scale and the score of C05656 was 0.53. Slightly less of the sequence was codon altered in C05768 and C05769. These have predicted translation efficiency scores of 0.6 and 0.68 respectively (FIG. 15)
  • scAAV production in suspension flasks with C05656 showed slightly reduced vector genome compared to previous productions with the modified RepCap C04991, but was increased over the unmodified RepCap productions.
  • the amount of capsid produced by C05656 was significantly less than C04991, so there is a large increase in the percentage of full particles calculated by vector genome concentration divided by capsid concentration (FIG. 16)
  • scAAV was produced using C05656, C05768, and C05769 in a 250 ml bioreactor.
  • the harvest vector genome yield was assessed by ddPCR and the capsid yield by ELISA.
  • Use of C05656 and C05768 result in approximately 80% full capsid particles at harvest, which is a substantial increase over the non-codon altered RepCap plasmids (FIG. 17).

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Abstract

Sont divulgués des compositions et des procédés pour la production améliorée de vecteurs viraux AAV faisant intervenir l'utilisation de plasmides RepCap comprenant des protéines de réplicase modifiées, des gènes viraux supplémentaires et des séquences d'acides nucléiques présentant une utilisation de codon modifiée pour réguler la traduction de protéines.
PCT/US2025/014586 2024-02-06 2025-02-05 Compositions et procédés pour la production améliorée de vecteurs viraux aav Pending WO2025171001A1 (fr)

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