Method of AAV production

Abstract

The invention described herein provides an improved system and method for rAAV production. The system of the invention includes a recombinant DNA vector that comprises both the gene of interest (GO I) to be delivered to a target cell, as well as the Rep and Cap coding sequences for rAAV packaging.

Description

[0001] METHOD OF AAV PRODUCTION

[0002] REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to and the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 632,160, filed on April 10, 2024, the entire contents of which are incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] In the early days of rAAV production, cultured cells are first infected with adenovirus, then transfected with two additional DNA plasmids: one plasmid containing a pair of essential rAAV genes (e.g., Rep / Cap genes), and the other plasmid carrying a sequence of a gene of interest (GOI). This method produces a reasonable yield of rAAV particles, but there is a risk of adenovirus contaminating the preparation which can put patients treated with rAAV at risk.

[0006] In 1998, different research groups determined that the key genes of the adenovirus can be shifted into a third “helper” plasmid, and all three plasmids can be transfected into a preferred host cell at the same time to produce rAAV particles, circumventing the potential contamination of rAAV. Since then, this method known as the “triple transfection” (TT) technique has been widely used as the main method to produce recombinant adeno associated virus (rAAV), including commercial manufacturing of AAV viral stocks for gene therapy, and also may be referred herein as traditional triple transfection or conventional triple transfection.

[0007] However, a persistent problem of using AAV-based gene therapy is the demand for large quantities of suitable AAV viral particles e.g., in the range of about 1 * 1014to 1 * 1015vector genome) for efficacious treatment.

[0008] Today, rAAV is considered a key platform to deliver numerous types of genes for gene therapy, and the number of rAAV-based therapies in development continues to increase. Therefore, there is a need for further improving large scale rAAV production.

[0009] SUMMARY OF THE INVENTION

[0010] One aspect of the invention provides a vector system for production of a recombinant adeno-associated virus (AAV), said vector system comprises: (1) a first coding sequence for a gene-of-interest (GOI) flanked by AAV ITR sequences; (2) a second coding sequence for an AAV Rep and an AAV Cap; and, (3) optionally, a third coding sequence for AAV helper genes; wherein said AAV Rep is compatible with said AAV ITR sequences for AAV packaging; wherein expression of said AAV Rep, in a host cell comprising said AAV helper genes, is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the AAV ITR sequences into an AAV capsid comprising said AAV Cap, and, wherein the molar ratio of (1) : (2) : (3) is about 1 : about 2 : about 1.

[0011] In certain embodiments, the molar ratio of (1) : (2) : (3) is (0.95-1.05) : (1.90-2.10) : (0.95-1.05).

[0012] In certain embodiments, the vector system comprises: (a) a first vector comprising said first coding sequence and said second coding sequence; (b) a second vector comprising said second coding sequence; and, (c) optionally, a third vector comprising said third coding sequence.

[0013] In certain embodiments, the first coding sequence, the second coding sequence, and / or the third coding sequence is in a plasmid.

[0014] In certain embodiments, the first vector, the second vector, and / or the third vector is a plasmid.

[0015] In certain embodiments, the first vector is a first plasmid, and the second vector is a second plasmid.

[0016] In certain embodiments, the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter (such as a muscle-specific promoter (e.g., CK8 promoter), or a cardiac muscle-specific promoter e.g., MHCK7 promoter)).

[0017] In some embodiments, expression of the AAV Rep in a host cell comprising AAV helper genes is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the 5’- and 3-ITR sequences into an AAV capsid comprising the AAV Cap.

[0018] In certain embodiments, the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter.

[0019] In certain embodiments, the pro- AAV cassette further comprises: (1) an enhancer that promotes the transcription of the GOI from the promoter; (2) a 5’ UTR; (3) a Kozak sequence; (4) a heterologous intron that promotes transcription and / or translation of the GOI; (5) a 3’ UTR; (6) a WPRE sequence; and / or (7) a polyA (such as bGH polyA) signal sequence.

[0020] In certain embodiments, the coding sequence for the AAV Rep and the coding sequence for the AAV Cap are within a RepCap cassette comprising an operably-linked RepCap promoter (such as the AAV P5 promoter, e.g, a wild-type P5 promoter).

[0021] In certain embodiments, the RepCap cassette and the pro-AAV cassette, when present on the same vector: i) are immediately adjacent to each other (e.g, with substantially no intervening polynucleotide sequence); ii) are not immediately adjacent to each other; iii) have the same transcription direction; iv) have opposite transcription direction.

[0022] In certain embodiments, the first vector, the second vector, and / or the third vector each independently further comprises a bacterial replication Ori gene, a selection marker (such as an antibiotic resistance gene, e.g., KanR / NeoR, or AmpR) under the transcriptional control of a selection marker promoter (such as a bacterial promoter).

[0023] In certain embodiments, the GOI encodes a functional calsequestrin 2 (CASQ2) protein, a functional BCL-2-associated athanogene 3 (BAG3) protein, a functional cardiac troponin T (TNNT2) protein, a functional RNA binding motif protein 20 (RBM20) protein, a functional frataxin (FXN) protein, or a functional equivalent of dystrophin (e.g, a dystrophin minigene encoding a functional micro-dystrophin protein).

[0024] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0025] In certain embodiments, the GOI includes a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limbgirdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIEB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0026] In certain embodiments, the GOI is a microdystrophin gene (e.g., one described in US7,906,l l l; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854, or US 10, 166,272, each and every one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0027] In certain embodiments, the microdystrophin gene comprises a coding sequence for R16 and R17 spectrin-like repeats for the full length dystrophin protein (such as one described in US7,892,824).

[0028] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as the microdystrophin gene described in PCT / US2016 / 013733).

[0029] In certain embodiments, the 5’ and 3’ AAV ITR sequences flanking said GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0030] In certain embodiments, the 5’ and 3’ AAV ITR sequences flanking said GOI are both from AAV2.

[0031] In certain embodiments, the tropism of the AAV include skeletal muscle, cardiac muscle, or neuromuscular system (such as that of SLB-101, AAV1, AAV6, AAV7, AAV8, AAVrh74, or AAV9); optionally, said AAV has a capsid of AAVrh74 or a derivative thereof.

[0032] In certain embodiments, the AAV ITR, said AAV Rep, and said AAV Cap are from the same or different AAVs.

[0033] In certain embodiments, the AAV ITR is AAV2 ITR, said AAV Rep is Rep2 from AAV2, and the AAV Cap is SLB-101, AAV8, AAV9, AAVrh74, or a derivative thereof.

[0034] In certain embodiments, the coding sequence for AAV Rep and Cap proteins is under the transcriptional control of a promoter, such as an AAV p5 promoter, an upstream HSV promoter, a modified p5 promoter lacking RBE (Rep-Binding Element), an HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (such as CMV promoter, EFla promoter, CAG promoter, CB promoter etc).

[0035] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter. In some embodiments, the modified P5 promoter comprises a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site. In some embodiments, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

[0036] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site, and wherein said modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

[0037] In certain embodiments, the AAV helper genes comprise adenoviral, herpesviral, or papillomaviral genes useful for AAV packaging (such as El A, E1B, E2A, E4 and VA RNA), optionally operably linked to a promoter as one transcriptional unit.

[0038] In certain embodiments, the first coding sequence encodes BAG3, and the second coding sequence encodes AAVrh74.

[0039] Another aspect of the invention provides a composition or a kit, comprising the vector system of the invention, optionally including the third coding sequence in a helper plasmid.

[0040] In certain embodiments, the helper plasmid comprises a helper virus gene that sufficiently supports AAV packaging.

[0041] In certain embodiments, the helper virus gene comprises: (i) an adenovirus gene, optionally an Adenovirus 5 or Adenovirus 2 gene; and / or (ii) a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein.

[0042] Another aspect of the invention provides a host cell comprising the vector system of the invention, or the composition of the invention.

[0043] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell), a VP2 cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0044] Another aspect of the invention provides a method of propagating / amplifying / producing a recombinant replication-defective AAV viral particle encapsidating the GOI of the invention, the method comprising: introducing the vector system of the invention into a host cell, prior to, concurrently with, or subsequent to introducing said AAV helper genes of the invention to said host cell, thereby propagating / amplifying / producing the recombinant replication-defective AAV viral particle.

[0045] In certain embodiments, the method further comprises harvesting the recombinant replication-defective AAV viral particle of the invention from the host cell.

[0046] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell), a VP2 cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0047] In certain embodiments, the recombinant DNA vector is introduced to the host cell by transient transfection.

[0048] It should be understood that any one embodiment of the invention, including those only described in the Examples, claims, or one of the subsections, can be combined with any other one or more embodiments, unless improper or expressly disclaimed.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 A shows a schematic (not to scale) drawing of an enlarged section of an exemplary dual transfection GOI / RepCap vector to produce AAV-SLB101 (right) viral particles, wherein the gene-of interest (GOI) is a variant of microdystrophin (represented herein as “SGT003”) as a non-limiting example of the GOI, and the Cap encodes SLB101 Cap. The Pro- AAV cassette (ITR-GOI-ITR) is positioned upstream / 5’ to the RepCap cassette, the transcription of which is driven by the p5 promoter in the RepCap cassette.*: start codon for Rep.

[0051] FIG. IB shows a schematic (not to scale) drawing of an enlarged section of an exemplary dual transfection GOI / RepCap vector (top) and a plasmid containing said vector (bottom) to produce AAV-rh74 viral particles, wherein BAG3 is a non-limiting example of the GOI, and the Cap encodes rh74 Cap. The Pro- AAV cassette (ITR-GOI-ITR) is positioned upstream / 5’ to the RepCap cassette. *: start codon for Rep. This vector / plasmid is referred to as DT-BAG3-rh74-Vl .

[0052] FIG. 1C shows a schematic (not to scale) drawing of an enlarged section of an exemplary dual transfection GOI / RepCap vector (top) and a plasmid containing said vector (bottom) to produce AAV-rh74 viral particles, wherein BAG3 is a non-limiting example of the GOI, and the Cap encodes rh74 Cap. The Rep cassette has a 3kb intron, and the RepCap cassette is between the Pro- AAV cassette (ITR-GOI-ITR) and the p5 promoter. *: start codon for Rep. This vector / plasmid is referred to as DT-BAG3-rh74-V2.

[0053] FIG. ID shows a schematic (not to scale) drawing of an enlarged section of an exemplary dual transfection GOI / RepCap vector (top) and a plasmid containing said vector (bottom) to produce AAV-rh74 viral particles, wherein BAG3 is a non-limiting example of the GOI, and the Cap encodes rh74 Cap. The Rep cassette has a 3kb intron, and the Pro-AAV cassette (ITR-GOI-ITR) is between the RepCap cassette and the p5 promoter. *: start codon for Rep. This vector / plasmid is referred to as DT-BAG3-rh74-V3. FIGs. 2A and 2B show schematic of plasmids comprising the GOI (FIG. 2A) and the rh74 RepCap (FIG. 2B) used in triple transfection method. BAG3 is a non-limiting example of the GOI.

[0054] FIG. 3 shows GOI titre yield (viral genome (vg / ml) and percent full capsid using triple transfection method at varying pHelp:pRepCap:pGOI ratio.

[0055] FIG. 4 shows GOI titre yield (top left), percent full capsid at harvest (top right) and percent mis-packaging rates measured by the presence of KanRgene sequence (bottom left) using VPC 2.0 cells in shake flask method using traditional triple transfection (Triple) plasmids at pGOI:pRepCap:pHelp ratio of 1 :2: 1, dual transfection (Dual) plasmids at pDuakpHelp ratio of 1 : 1, and dual transfection with additional pRepCap (pRC) plasmid (Dual+RepCap) to match the triple transfection’s pGOI:pRepCap:pHelp ratio of 1 :2: 1. (z.e., pDual:pRepCap:pHelp ration of 1 : 1 : 1).

[0056] FIG. 5 shows GOI titre yield (left) and percent full capsid at harvest (right) by shake flask or AMBR®15 methods using traditional triple transfection (Triple) plasmids at pGOI:pRepCap:pHelp ratio of 1 :2: 1 and dual transfection with additional pRepCap plasmid (Dual+RepCap) to match the triple transfection’s pGOI:pRepCap:pHelp ratio of 1 :2: 1. (pRC: pRepCap).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] 1. Overview

[0059] Conventional AAV production system utilize three plasmid vectors (the so-called triple transfection vector system): one with a gene of interest (GOI) flanked by AAV ITR sequences, for packaging into the AAV viral particles; another with an expression cassette encoding the AAV Rep and Cap proteins useful for AAV packaging; and yet another provides the useful helper genes from other viruses (such as adenovirus, herpesvirus, or papillomavirus) for productive AAV life cycle.

[0060] A recombinant dual transfection vectors / system first disclosed in PCT / US2023 / 034596 improve the conventional system by inserting both the GOI cassette and the rep-cap expression cassette into a single plasmid, to create a dual transfection vector (e.g., plasmid) system that can be used in double transfection with the helper plasmid (e.g., the same helper plasmid used in the traditional triple transfection method).

[0061] At suitable (e.g., identical / equal molar) ratios of the Rep, Cap, and helper genes, the dual transfection system produced rAAV viral particles at about 200-300% level compared to the traditional triple transfection system that has been used in commercial production of rAAV viral particles for decades.

[0062] Further, in some embodiments, AAV production using the dual vector / plasmid system results in reduced frequency of mispackaging.

[0063] It was surprisingly found that, in both the triple and double transfection systems, when the molar ratio of the Rep / Cap coding sequence is increases (e.g., roughly doubled) with respect to that of the helper genes and that of the GOI, the overall yield of the AAV viral particle produced is significantly increases (e.g., by >100%, >150%, >200%, >250%, >300% or more).

[0064] Thus, one aspect of the invention provides vector system for production of a recombinant adeno-associated virus (AAV), said vector system comprises: (1) a first coding sequence for a gene-of-interest (GOI) flanked by AAV ITR sequences; (2) a second coding sequence for an AAV Rep and an AAV Cap; and, (3) optionally, a third coding sequence for AAV helper genes; wherein said AAV Rep is compatible with said AAV ITR sequences for AAV packaging; wherein expression of said AAV Rep, in a host cell comprising said AAV helper genes, is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the AAV ITR sequences into an AAV capsid comprising said AAV Cap, and, wherein the molar ratio of (1) : (2) : (3) is about 1 : about 2 : about 1.

[0065] In certain embodiments, the vector system does not comprise the optional third coding sequence, which may be provided / present in a suitable packaging host cell (such as HEK293 or a derivative thereof). In other embodiments, the vector system of the invention comprises the optional third coding sequence.

[0066] As used herein, the term “about,” especially when used to refer to the molar ratio of the invention, generally refers to a range of ±5%, ±10%, ±15%, or ±20% of the value that follows. Thus, for example, “about 1” may include a range of 0.95-1.05 when the range is ±5%; may include a range of 0.90-1.10 when the range is ±10%; may include a range of 0.85- 1.15 when the range is ±15%; and may include a range of 0.80-1.20 when the range is ±20%; etc. In certain embodiments, the term “about” when used to refer to the molar ratio of the invention refers to a range of ±5% of the value that follows. In certain embodiments, the term “about” when used to refer to the molar ratio of the invention refers to a range of ±10% of the value that follows. In certain embodiments, the term “about” when used to refer to the molar ratio of the invention refers to a range of ±15% of the value that follows. In certain embodiments, the term “about” when used to refer to the molar ratio of the invention refers to a range of ±20% of the value that follows.

[0067] In certain embodiments, the molar ratio of (1) : (2) : (3) is (0.95-1.05) : (1.90-2.10) : (0.95-1.05).

[0068] In certain embodiments, the vector system comprises: (a) a first vector comprising said first coding sequence and said second coding sequence; (b) a second vector comprising said second coding sequence; and, (c) optionally, a third vector comprising said third coding sequence. In certain embodiments, the vector system does not comprise the third vector. In certain embodiments, the vector system comprises the third vector. In certain embodiments, the molar ratio of (a) : (b) : (c) is about 1 (e.g., 0.95-1.05) : about 1 (e.g., 0.95-1.05) : about 1 (e.g., 0.95-1.05) (when present).

[0069] In certain embodiments, the first coding sequence, the second coding sequence, and / or said third coding sequence is in a plasmid. In certain embodiments, the first coding sequence, the second coding sequence, and the third coding sequence are each in a separate plasmid. In certain embodiments, the first coding sequence and the second coding sequence are on the same plasmid, and a further second coding sequence is in a different plasmid. In certain embodiments, the third coding sequence is not in a plasmid (e.g., it may be present in a host / packaging cell as an extrachromosomal replicable DNA element, or be integrated into the genome of the host / packaging cell). In certain embodiments, at least one of the coding sequences is not in a plasmid. In certain embodiments, the first and the second coding sequences are in a non-plasmid vector, such as an HSV vector, and a further second coding sequence is in a plasmid. In certain embodiments, at least one of the coding sequences is a in baculoviral vector. In certain embodiments, the first and / or the second coding sequences is / are in a baculoviral vector.

[0070] In certain embodiments, the first vector, the second vector, and / or the third vector is a plasmid.

[0071] As used herein, the term “plasmid” includes a nucleic acid molecule that can replicate independently of a cell chromosome. The term “plasmid” is intended to include circular nucleic acid molecules and linear nucleic acid molecules. Furthermore, the term “plasmid” is intended to include bacterial plasmids, cosmids, minicircles (Nehlsen etal., Gene Ther. Mol. Biol., 10: 233-244, 2006; and Kay et al., Nature Biotechnology, 28: 1287-1289, 2010) and ministrings (Nafissi et al., Mol Ther Nucleic Acids, 3:el65, 2014). In certain embodiments, the plasmid is a circular nucleic acid (DNA) molecule. In certain embodiments, the plasmid is a nucleic acid molecule that is of bacterial origin. In certain embodiments, the first vector, the second vector, and the third vector are each a separate plasmid. In certain embodiments, the first vector and the second vector are different plasmids. In certain embodiments, the third vector is not plasmid (e.g., it may be present in a host / packaging cell as an extrachromosomal replicable DNA element, or be integrated into the genome of the host / packaging cell). In certain embodiments, at least one of the vectors is not a plasmid. In certain embodiments, the first vector is a non-plasmid vector, such as an HSV vector, and the second vector is a plasmid. In certain embodiments, at least one of the vectors is a baculoviral vector. In certain embodiments, the first and / or the second vectors is / are a baculoviral vector.

[0072] In certain embodiments, the vector is suitable for use in a mammalian (e.g., human or non-human mammalian) cell. Vectors suitable for use in mammalian cells are widely described and well-known in the art. Those skilled in the art would appreciate that vectors of the present invention may also contain various additional sequences and elements useful for the replication of the vector in prokaryotic and / or eukaryotic cells, selection of the vector and the expression of the sequences in a variety of host cells. For example, the vectors of the present disclosure may include a prokaryotic replicon (that is, a sequence having the ability to direct autonomous replication and maintenance of the vector extra chromosomally in a prokaryotic host cell, such as a bacterial host, cell. Such replicons are well known in the art. In some embodiments, the vectors may include a shuttle element that makes the vectors suitable for replication and integration in both prokaryotes and eukaryotes. In addition, vectors may also include a gene whose expression confers a detectable marker such as a drug resistance gene (such as KanR, NeoR, AmpRetc.) or other selection marker, under the transcriptional control of a suitable promoter therefor (such as a bacterial promoter), which allows for selection and maintenance of the host cells. Vectors may also have a reportable marker, such as gene encoding a fluorescent or other detectable protein. Each vector may further comprise a bacterial replication Ori gene and other plasmid backbone sequences.

[0073] The vectors of the invention may also include transcriptional enhancers, translational signals, and transcriptional and translational termination signals. Examples of transcriptional termination signals include, but are not limited to, polyadenylation signal sequences, such as bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (RBG) poly(A), thymidine kinase (TK) poly (A) sequences, and any variants thereof. In certain embodiments, the polyA signal sequence is the bGH polyA signal sequence.

[0074] In certain embodiments, the dual transfection recombinant DNA vector of the invention (the dual transfection (DT) vector) comprises a gene-of-interest (GO I) flanked by a 5’ and 3’ adeno-associated virus (AAV, such as AAV2) ITR, a coding sequence for an AAV Rep (such as AAV2 Rep) and a coding sequence for an AAV Cap (such as AAV9 or derivative thereof) compatible with the AAV ITRs. In a host cell comprising AAV helper genes (which can be introduced into the host cell by any art-recognized means, such as infection of the host cell by adenovirus, or transfection of a helper plasmid comprising the requisite genes for AAV packaging), the expression of the AAV Rep and Cap proteins from the dual transfection vector is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the 5’ and 3’ AAV ITRs.

[0075] In certain embodiments, at least one of the vectors (e.g., all vectors) in the subject vector system is / are a plasmid(s), which may be suitable for transfection into an AAV packaging cell line, such as Expi, HEK293 or a derivative thereof (such as HEK293T cells), or other commonly used AAV packaging cell lines.

[0076] In some embodiments, the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter. In certain embodiments, the promoter is a muscle-specific promoter (e.g., CK8 promoter), or a cardiac muscle-specific promoter (e.g., MHCK7 promoter).

[0077] In certain embodiments, the rDNA vector comprises two or more copies of the pro- AAV cassette. In certain embodiments, all copies of the pro-AAV cassette comprise the same GOI. In certain embodiments, at least two of the pro-AAV cassettes comprise different GOI. The latter embodiment can be useful, for example, if AAV vectors are used to deliver different parts of the same functional assembly, such as a CRISPR / Cas effector enzyme and the coding sequence for one or more guide RNAs.

[0078] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that promotes the transcription of the GOI from the promoter; (2) a 5’ UTR; (3) a Kozak sequence; (4) a heterologous intron that promotes transcription and / or translation of the GOI; (5) a 3’ UTR; (6) a WPRE sequence; and / or (7) a polyA (such as bGH polyA) signal sequence.

[0079] In some embodiments, the coding sequence for the AAV Rep and the coding sequence for the AAV Cap are within a RepCap cassette comprising an operably-linked RepCap promoter. In certain embodiments, the operably-linked RepCap promoter comprises the AAV P5 promoter (e.g., a wild-type P5 promoter).

[0080] In some embodiments, the RepCap cassette and the pro-AAV cassette, when present on the same vector, are immediately adjacent to each other (e.g., with substantially no intervening polynucleotide sequence).

[0081] In some other embodiments, the RepCap cassette and the pro-AAV cassette, when present on the same vector, are not immediately adjacent to each other.

[0082] In some embodiments, the RepCap cassette and the pro-AAV cassette, when present on the same vector, have the same transcription direction.

[0083] In some other embodiments, the RepCap cassette and the pro-AAV cassette, when present on the same vector, have opposite transcription directions.

[0084] In some embodiments, the pro-AAV cassette is upstream of the RepCap cassette.

[0085] In some other embodiments, the pro-AAV cassette is downstream of the RepCap cassette.

[0086] In some embodiments, the GOI is in front of the coding sequence for the AAV Rep. In some embodiments, the GOI is in front of the coding sequence for the AAV Cap. In some embodiments, the GOI is in front of the coding sequences for both the AAV Rep and AAV Cap.

[0087] In certain embodiments, the coding sequence for the AAV Rep is under the transcriptional control of an AAV P5 promoter positioned downstream or 3’ (e.g., immediately downstream or 3’) to the GOI or pro-AAV cassette.

[0088] In certain embodiments, the coding sequence for the AAV Rep is under the transcriptional control of an AAV P5 promoter positioned downstream or 3’ (e.g., immediately downstream or 3’) to the RepCap cassette (e.g., downstream or 3’ to the Cap coding sequence). While not wishing to be bound by any particular theory, it is believed that Rep expression driven by the P5 promoter placed at the 3’ end of the RepCap cassette (or the Cap coding sequence) results in reduced level of Rep protein being produced and increased packaging efficiency of full length genomes with concurrent greatly reduced levels of backbone packaging.

[0089] In certain embodiments, the coding sequence for the AAV Rep is under the transcriptional control of an AAV P5 promoter positioned downstream or 3’ (e.g., immediately downstream or 3’) to the GOI or pro-AAV cassette.

[0090] In some embodiments, the expression of the AAV Rep in a host cell comprising AAV helper genes is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the 5’- and 3-ITR sequences into an AAV capsid comprising the AAV Cap. In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0091] In certain embodiments, the GOI includes a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limbgirdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0092] In certain embodiments, the GOI is a microdystrophin gene (e.g., one described in US7,906,l l l; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854, or US 10, 166,272, each and every one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0093] In certain embodiments, the microdystrophin gene comprises a coding sequence for R16 and R17 spectrin-like repeats for the full length dystrophin protein (such as one described in US7,892,824).

[0094] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as the microdystrophin gene described in PCT / US2016 / 013733).

[0095] In certain embodiments, the GOI encodes a functional calsequestrin 2 (CASQ2) protein, a functional BCL-2-associated athanogene 3 (BAG3) protein, a functional cardiac troponin T (TNNT2) protein, a functional RNA binding motif protein 20 (RBM20) protein, or a functional frataxin (FXN) protein.

[0096] In certain embodiments, the 5’ and 3’ AAV ITR sequences flanking said GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0097] In certain embodiments, the tropism of the AAV include skeletal muscle, cardiac muscle, or neuromuscular system (such as that of SLB-101, AAV1, AAV6, AAV7, AAV8, AAVrh74, or AAV9).

[0098] In certain embodiments, the AAV has a capsid of AAVrh74 or a derivative thereof.

[0099] In certain embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from the same or different AAVs.

[0100] In certain embodiments, the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is SLB-101, AAV8, AAV9, AAVrh74, or a derivative thereof.

[0101] In certain embodiments, the coding sequence for AAV Rep and Cap proteins is under the transcriptional control of a promoter, such as an AAV p5 promoter, a modified p5 promoter lacking RBE (Rep-Binding Element), an HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (such as CMV promoter, EFla promoter, CAG promoter, CB promoter etc).

[0102] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter. In some embodiments, the modified P5 promoter comprises a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site. In some embodiments, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

[0103] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site, and wherein said modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

[0104] In certain embodiments, the AAV helper genes comprise adenoviral, herpesviral, or papillomaviral genes useful for AAV packaging (such as El A, E1B, E2A, E4 and VA RNA), optionally operably linked to a promoter as one transcriptional unit.

[0105] In certain embodiments, the first coding sequence encodes BAG3, and wherein said second coding sequence encodes AAVrh74.

[0106] Another aspect of the invention provides a composition or a kit, comprising the recombinant DNA vector of the invention, and a helper plasmid.

[0107] In certain embodiments, the helper plasmid comprises a helper virus gene that sufficiently supports AAV packaging. In certain embodiments, the helper virus gene comprises: (i) an adenovirus gene, optionally an Adenovirus 5 or Adenovirus 2 gene; and / or (ii) a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein.

[0108] Another aspect of the invention provides a host cell comprising the recombinant DNA vector of the invention, or the composition of the invention.

[0109] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell), a VP2 cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0110] In another aspect, the invention provides a method of propagating, amplifying, or producing a recombinant replication-defective AAV viral particle encapsidating the GO I, comprising introducing the recombinant DNA vector (dual transfection vector) e.g., in a plasmid) of the invention into a host cell prior to, concurrently with, or subsequent to introducing the AAV helper genes (e.g., in a plasmid) into the host cells.

[0111] In some embodiments, the dual vector and the AAV helper genes in two separate plasmids are co-transfected into a host cell, thereby producing a recombinant replicationdefective AAV viral particle encapsidating the GOI.

[0112] In certain embodiments, the method further comprises harvesting the recombinant replication-defective AAV viral particle from the host cell.

[0113] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell), a VP2 cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0114] In certain embodiments, the recombinant DNA vector is introduced to the host cell by transient transfection.

[0115] With the general principles of the invention set forth herein, the sections below provides further detailed description for various aspects of the invention. It should be understood that any embodiment of the invention can be combined with any one or more additional embodiments of the invention, including those embodiments described in different sections of the application, and those described only in the examples, drawings, or claims.

[0116] Furthermore, all disclosures pertaining to “recombinant DNA vector” are also applicable to a “vector” and a “plasmid” described herein. 2. Rep and Cap Genes

[0117] In certain embodiments, the coding sequence for the AAV Rep and Cap proteins, and the gene of interest (GO I) flanked by AAV ITR sequences, are integrated into a single dual transfection (DT) vector of the invention, e.g., a DT plasmid, or the “first vector” as used herein. To maintain the roughly 1 :2: 1 ratio of the invention for the first coding sequence (for the GO I), the second coding sequence for the AAV Rep / Cap, and the third coding sequence for the helper genes, the “second vector” comprises a coding sequence for the same AAV Rep / Cap.

[0118] The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid, which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins (Rep78, Rep68, Rep52, and Rep40) from a single open reading frame and the generation of three capsid proteins (VP; VP1 / VP2 / VP3) from a single open reading frame. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1 : 1 : 10 of VP1 :VP2:VP3. As used herein, an “AAV serotype” is defined primarily by the AAV capsid. In some instances, the ITRs are also specifically described by the AAV serotype.

[0119] In certain embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from the same or different AAV serotypes.

[0120] In some embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from AAVPHP.B (PHP.B), AAVPHP.A (PHP. A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B- EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B- TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9 K449R, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2- 15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4-8 / r 11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5- 3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16.12 / hu.l l, AAV29.3 / bb. l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu. l6, AAV52 / hu.l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l l, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.l 8, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC 12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Civ 1-7, AAV Civ 1-8, AAV Civ 1-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAV SLB-101 (SEQ ID NO: 14 of WO 2021 / 072197, incorporated herein by reference), and / or AAVF9 / HSC9 and variants thereof.

[0121] In certain embodiments, the AAV Cap is AAVrh.74.

[0122] In certain embodiments, the AAV Cap is a derivative of wild-type AAV9. In certain embodiments, the derivative comprises an insertion of a short peptide (e.g., 3, 4, 5, 6, 7, 8, or 9 residues) in-between residues 588 and 589 of the wild-type AAV9 capsid VP1. In certain embodiments, the insertion comprises, consists essentially of, or consists of RGDLGLS into residues 588 and 589 of wild-type AAV9 Capsid VP1.

[0123] In certain embodiments, the rDNA vector / plasmid of the invention comprises a cap gene promoter. The cap gene promoter may be operably linked to a cap gene. In certain embodiments, the cap gene promoter is a native cap gene promoter.

[0124] The native cap gene (z.e., the cap gene of a wild type AAV) is operably linked to a p40 promoter, a p5 promoter and a pl9 promoter. Thus in one embodiment, the rDNA vector / plasmid of the invention comprises a cap gene promoter such as an AAV p40 promoter, a p5 promoter, and / or a pl9 promoter.

[0125] The native p40 promoter is contained within the native rep gene. In some embodiments, the p40 promoter has a sequence of or at least 95%, at least 98%, or 99% identical to that of AAV2. In some embodiments, the at least one cap gene promoter is comprised in a promoter region comprising a p40 promoter, a p5 promoter and a pl9 promoter.

[0126] The native p5 promoter is upstream of the native rep gene. In some embodiments, the p5 promoter has a sequence of or at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0127] In certain embodiments, the wild-type or native p5 promoter is modified to create a modified p5 promoter. In some embodiments, the modified p5 promoter further reduces mispackaging. In certain embodiments, the modified p5 promoter comprises a REP binding site and a transcription start site-localized Ying- Yang 1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site with an exogenous spacer sequence inserted between the REP binding site and YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

[0128] Without being bound to a particular theory, insertion of the spacer between the P5 REP binding sequence and the YY1 box at the transcription start site of the P5 promoter may cause or contribute to reduced or eliminated mis-packaging of vector / plasmid DNA upstream of the P5 promoter, while maintaining recombinant AAV titers.

[0129] In certain embodiments, the spacer includes an exogenous spacer sequence inserted between the REP binding site and YY1 binding site. In certain embodiments, excluding the exogenous spacer, the modified P5 promoter that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to wild-type P5 promoter, or a length within any range delimited by any pair of the foregoing values, such as between 80% and 99%, between 90% and 99% or between 95% and 99%, for example. In certain embodiments, the modified P5 promoter, into which the exogenous spacer is inserted, has a sequence of nucleotides corresponding to at least about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides, or a length within any range delimited by any pair of the foregoing values, such as between 40 and 50, between 50 and 160 or between 80 and 130, for example, adjacent (or upstream of) the transcription start site of the large REP proteins (i.e., REP78 and REP68) in an AAV genome, or a sequence having, or having at least, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or more, including ranges thereof, sequence identity thereto, wherein the promoter drives transcription of the large REP proteins, i.e., REP78 and REP68.

[0130] In some examples, the modified P5 promoter has a sequence derived from the sequence of the region adjacent to the transcription start site of REP78 and REP68 in AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 6 (AAV6}, AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or any of the following AAV serotype: AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant.

[0131] In certain embodiments, the modified P5 promoters comprises or consists of a sequence corresponding to the approximately 160 nucleotides adjacent to the transcription start site of REP78 and REP68 in an AAV genome, such as an AAV1, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, or AAVrh8R R533 A mutant genome.

[0132] As used herein, “REP binding sequence,” “REP binding site,” or “REP binding element” are used interchangeably herein to refer to an element of the P5 promoter composed of two to four imperfect GAGC repeats that are located just downstream, i.e., 3 of the TATA box and upstream of the transcription start site of the P5 promoter. Without being bound to a particular theory, Rep-mediated repression is thought to be the result of Rep binding to the REP binding element. In certain embodiments, the P5 REP binding sequence is or comprises GCCCGAGTGAGCACGC.

[0133] As used herein, the “Ying-Yang 1 binding site,” “YY1 binding site,” or “YY1 box” refers to a YY1 recognition sequence, which when bound by YY1, supports initiation from the transcription start site. The +1 YY1 binding of the P5 promoter is located at the transcription start site and has the consensus sequence CCAT.

[0134] As used herein, the term “exogenous spacer sequence” refers to a sequence that is not native to the P5 promoter.

[0135] The exogenous spacer sequence may be inserted anywhere between the REP binding site and YY1 binding site as described herein. In some embodiments, the exogenous spacer sequence is inserted into or replaces one or more of the seven nucleotides located between the REP binding site and YY1 binding site. By way of illustration, the exogenous spacer sequence may be inserted into or replace one or more nucleotides of the sequence AGGGTCT of the AAV8 P5 promoter.

[0136] In some embodiments, the total length of the exogenous spacer is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more nucleotides, or a length within any range delimited by any pair of the foregoing values, such as between 5 and 120, between 10 and 100 or between 5 and 100, for example. Ideally, the exogenous spacer is 5 to 100 nucleotides in length. As would be understood by those skilled in the art, the sequence of the spacer is not critical. The sequence of the spacer may be a random, artificial sequence, a sequence derived from AAV or a sequence derived from another source.

[0137] The pl9 promoter is contained within the native rep gene. In some embodiments, the pl9 promoter has a sequence of or at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0138] In some embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from the same AAV. In some other embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from different AAVs. For example, the AAV Rep may be from AAV2, and the AAV Cap may be from AAV9 or a derivative thereof such as the aforementioned wt AAV9 derivatives, and including SLB-101 (see SEQ ID NO: 14 and FIG. 6 of WO2021 / 072197, incorporated herein by reference).

[0139] In certain embodiments, the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0140] In certain embodiments, the coding sequence for AAV Rep protein encodes a wild type Rep 40, Rep 52, Rep 68 and / or Rep 78 of an AAV, such as AAV2. In certain embodiments, these Rep proteins are transcribed from one or more of the rep promoters p5, pl9 and / or p40.

[0141] The rep gene encodes at least one functional Rep proteins (Rep 78, Rep 68, Rep 52 and Rep 40). The gene region is under the control of the p5 and pl9 promoters. When the p5 promoter is used, a sequence that encodes Rep 78 and Rep 68 is transcribed. Rep 78 and Rep 68 are two alternative splice variants (Rep 78 comprises an intron that is excised in Rep 68). Similarly, when the pl9 promoter is used, a sequence that encodes Rep 52 and Rep 40 is transcribed. Rep 52 and Rep 40 are alternative splice variants (Rep 52 comprises an intron that is excised in Rep 40). The four Rep proteins are known to be involved in replication and packaging of the viral genome, and are thus useful in rAAV production.

[0142] In certain embodiments, it is not necessary for all four Rep proteins to be present. In certain embodiments, at least one encoded large Rep protein (Rep 78 or Rep 68) and one encoded small Rep protein (Rep 52 or Rep 40) are present. Rep 78 can be toxic to cells, and Rep 78 does not need to be present in order for AAV replication to take place. Thus, in some embodiments, Rep78 is absent. In some embodiments, the rep gene does not encode or transcribe Rep 78. In certain embodiments, the rep gene encodes / transcribes Rep 68.

[0143] In certain embodiments, the rDNA vector / plasmid of the invention comprises at least one rep gene encoding: (a) a functional Rep 52 protein; (b) a functional Rep 40 protein; and / or (c) a functional Rep 68 protein.

[0144] As used herein, a “functional” Rep protein is one which allows for production of AAV particles. In particular, Rep 78 or Rep 68 (the large Rep proteins) are believed to be involved in replication of the AAV genome, and Rep 52 and Rep 40 (the small Rep proteins) are believed to be involved in packaging of the AAV genome into a capsid. One of skill in the art can readily determine whether a given Rep protein is functional, such as by determining, e.g., whether the Rep protein supports AAV production using an AAV production assay as described above.

[0145] In some embodiments, the at least one rep gene encodes a “functional” Rep protein if the Rep protein supports rAAV production at a level of or at least 25%, of or at least 40%, of or at least 50%, of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95%, or a level within any range delimited by any pair of the foregoing values, such as between 25% and 95%, between 40% and 90% or between 25% and 70%, for example, of the level supported by the wild type Rep protein.

[0146] In certain embodiments, the coding sequence for Rep comprises a homologous intron, such as a human collagen intron into the Rep2 gene. It is believed that this reduces the ability to form a replication competent AAV during packaging. The incorporation of a 3071 bp human collagen intron places a size constraint on the ability to package the RepCap cassette into a viral particle, and would require at least three crossover events to produce a replication competent AAV virus.

[0147] In certain embodiments, the Rep protein is compatible with the ITR(s) surrounding the GOI. Some Rep proteins may only be able to package genomic material (such as an expression cassette) when it is flanked by ITR(s) of the same serotype as the Rep protein. Other Rep proteins are cross compatible, and can package genomic material that is flanked by ITR(s) of a different serotype.

[0148] In certain embodiments, the rDNA vector / plasmid of the invention comprises two or more rep genes that are separated on the vector / plasmid. For example, one of the separate rep genes could encode Rep 68 (for example using the p5 promoter or a different promoter situated near the normal position of the p5 promoter in the rep gene), another encoding Rep 40 (for example using the pl9 promoter or a different promoter situated near the normal position of the pl9 promoter). The one rep gene encoding Rep 40 may also encode Rep 52 since Rep 52 and Rep 40 are alternative splice variants.

[0149] In certain embodiments, if two genes encode a Rep 40 protein, one of the two genes that encodes a functional Rep 40 protein may comprise an intron. In one embodiment, both genes that encode a functional Rep 40 protein comprise an intron. In another embodiment, only one of the genes that encodes a functional Rep 40 protein comprises an intron.

[0150] For example, to avoid the at least one rep gene encoding Rep 78, the rep gene may be split through partial duplication into two genes. One gene could comprise nucleotides corresponding to the full length native rep gene with the sequence corresponding to the intron removed. Such a gene would encode Rep 68 and Rep 40, but would not encode either Rep 78 or Rep 52, as a portion of each of the Rep 78 and Rep 52 proteins is encoded by the sequence which acts as an intron in the context of rep 40. The second gene could comprise nucleotides corresponding to the region of the native rep gene downstream of the pl9 promoter, which would encode Rep 52 (intron spliced in) and Rep 40 (intron spliced out).

[0151] In some embodiments, the at least one rep gene does not comprise a functional internal p40 promoter. The native rep / cap gene comprises a p40 promoter (Pereira and Muzyczka, J. Virol. 71 : 1747-1756, 1997). The p40 promoter drives expression of the cap gene, but is not required for expression of rep genes. A functional p40 promoter is one that is capable to drive expression of the cap gene.

[0152] In certain embodiments, the coding sequence for AAV Rep and Cap proteins is under the transcriptional control of a promoter, such as an AAV p5, pl 9, or p40 promoter, a modified p5 promoter lacking RBE (Rep-Binding Element), or a ubiquitous promoter (such as CMV promoter, EFla promoter, CAG promoter, CB promoter etc).

[0153] In any of the above embodiments, either the rep gene or the cap gene or both may be replaced by a cloning site (e.g., a multi cloning site or MCS), in the rDNA vector of the invention, such that a suitable rep and / or cap gene can be cloned / inserted into the respective cloning sites. This will facilitate the swapping of any desired rep or cap genes into the rDNA vector / plasmid of the invention.

[0154] 3. Gene of Interest (GOI) in rAA V and Treatable Diseases

[0155] The system and method of the invention can be used to produce recombinant AAV vectors carrying a gene of interest (GOI) flanked by AAV ITR sequences.

[0156] In certain embodiments, the rDNA vector (plasmid) of the invention comprises ITR sequences derived from AAV1, AAV2, AAV4 and / or AAV6. In certain embodiments, the ITR sequences are AAV2 ITR sequences.

[0157] As used herein, “gene of interest” or GOI or goi generally refers to a nucleic acid or polynucleotide sequence, such as a gene, an open reading frame (ORF), or a coding sequence for protein or RNA, such as non-coding RNA that includes siRNA, miRNA, shRNA, antisense RNA or a precursor thereof. However, in certain circumstances or context, the term GOI also loosely refers to a protein (encoded by the GOI), or a disease or indication that can be remedied by the GOI, or a disease or indication can be (but is not necessarily) caused by loss of function of the GOI.

[0158] For example, and merely to illustrate, the gene GALGT2 encodes the protein GalNAc transferase (P-l,4-N-acetylgalactosamine galactosyltransferase), which is an enzyme that transfers a complex sugar molecule onto a few specific proteins, including dystroglycan. Under normal circumstances, GalNAc transferase is found only at the neuromuscular junction (NMJ), where some components of the dystroglycan-associated protein complex are different than elsewhere in muscle. Importantly, at the NMJ, utrophin is present instead of dystrophin. In the mdx mouse model of muscular dystrophy, viral gene transfer of GALGT2 results in expression of GalNAc transferase across the entire muscle membrane, instead of just at the normal expression domain of the NMJ, as well as upregulation of utrophin across the entire muscle fiber. In the mdx mouse, this expression can correct muscle functional deficits to the same degree as does microdystrophin gene expression. Furthermore, overexpression of GALGT2 corrects muscle pathology in mouse models of other muscular dystrophies, including LGMD2A and congenital muscular dystrophy (MDC1 A). Thus GALGT2 is a GOI for treating muscular dystrophy such as DMD, BMD, LGMD2A and MDC1 A, even though GALGT2 is not necessarily defective per se in the patient in need of treatment.

[0159] In another example, Sarcolipin (SLN) inhibits the sarco / endoplasmic reticulum (SR) Ca2+ATPase (SERCA), and is abnormally elevated in the muscle of DMD patients and animal models such as the mdx mouse model of DMD. Reducing SLN levels by AAV9- mediated RNA interference ameliorates dystrophic pathology in the severe dystrophin / utrophin double mutant (mdx:utr ) mouse model of DMD, including attenuation of muscle pathology and improvement of diaphragm, skeletal muscle and cardiac function. Thus the coding sequence for SLN RNAi is a GOI that remedies DMD.

[0160] Thus the GOI can be a gene (or protein) that, when expressed, replaces a mutated, damaged, or inactive gene or protein. The GOI can be a gene (or protein) that, when expressed, assists an already functioning process that can benefit from further modification for therapy in a disease, disorder, or dysfunction. The GOI can be a gene (or protein) that, when expressed, assists a dysfunctional process that can benefit from further modification for therapy in a disease, disorder, or dysfunction. A GOI nucleic acid sequence can be DNA, RNA, or synthetic nucleic acid molecule. The GOI can be a protein, an enzyme, a structural protein, a functional protein, or an adaptable protein based on cell function(s). The GOI can provide therapeutic benefit or a treatment modality for a disease, disorder, or dysfunction.

[0161] In certain embodiments, the recombinant DNA vector of the invention comprises more than one copy of the GOI. In some embodiments, the plasmid of the invention comprises more than one copy of the GOI. In certain embodiments, the multiple GOI copies are the same. In some other embodiments, the multiple GOI are different.

[0162] In certain other embodiments, the GOI may be a CRISPR / Cas effector enzyme, such as a Class 2, Type II, IV, V, or VI effector enzyme, including CRISPR-Cas9, Cas 12, Cas 13, etc. In certain embodiments, the GOI may be a TALEN, or other genetic based gene editing protein that functions upon intracellular delivery for their intended activity, such as gene editing or gene knockout in a target cell, tissue, or organism / individual.

[0163] In certain embodiments, CRISPR / Cas effector enzyme lacks endonuclease activity (dCas, such as dCas9).

[0164] In certain embodiments, the Cas or dCas is further fused to a base editor, such as a cytosine base editor (CBE, e.g., APOBEC, BE1, BE2, BE3, Target-AID base editor, SaBE3, BE3 PAM variants, BE3 editing window variants, AID, CDA1, AP0BEC3G, HF-BE3, BE4, BE4max, and AncBE4max), an adenine base editor (ABE, e.g., ABE7.10, ABE 6.3, ABE7.8, ABE7.9, ABEmax, ABE8e(TadA-8e V106W), ABE8 and variants thereof), or a dual base editor (SPACE, A&C-BEmax).

[0165] Any and all GOIs as used herein may benefit from codon optimization for enhanced expression and activity via known computer-based algorithms or other codon optimization method, such as manual optimization.

[0166] In certain embodiments, the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter.

[0167] In some embodiments, the promoter operably linked to the GOI is an ubiquitous promoter which drives or promotes expression of the GOI in most tissues. In some embodiments, the promoter is elongation factor la-subunit (EFla) promoter, cytomegalovirus (CMV) immediate-early enhancer promoter, chicken P-actin (CBA) and its derivative CAG promoter, P glucuronidase (GUSB) promoter, or ubiquitin C (UBC) promoter.

[0168] In some other embodiments, the promoter operably linked to the GOI is a tissuespecific promoter which can be used to restrict expression to certain cell types such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0169] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, and mammalian skeletal alpha-actin (ASKA) promoter.

[0170] In some embodiments, the pro-AAV cassette may further comprise an enhancer that promotes the transcription of the GOI from the promoter, a 5’ UTR, a Kozak sequence, a heterologous intron that promotes transcription and / or translation of the GOI, a 3’ untranslated region (UTR), a WPRE sequence, and / or a polyA signal sequence.

[0171] In certain embodiments, the pro-AAV cassette comprises a transcription regulatory element comprising the promoter element and / or enhancer element from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and / or LSP. These transcription regulatory elements are described in more detail in the following references: HLP2: WO16 / 075473; HLP1 : McIntosh et al., Blood 121(17):3335-44, 2013; LP1 : Nathwani et al., Blood 107(7): 2653-2661, 2006; HCR-hAAT: Miao et al., Mol Ther. 1 :522-532, 2000; ApoE-hAAT: Okuyama etal., Human Gene Therapy 7:637-645, 1996; and LSP: Wang et al., Proc Natl Acad Sci U S A. 96(7): 3906-3910, 1999 (all incorporated herein by reference). Each of these transcription regulatory elements may comprise a promoter, an enhancer, and / or optionally other nucleotides.

[0172] In certain embodiments, the plasmid of the invention comprises a promoter region comprising one or more promoters, and the promoter region does not comprise dispensable translation initiation codons (e.g., ATG or GTG codons) to prevent unintended / undesirable translation initiation. In certain embodiments, the promoter region comprises p5, pl9 and / or p40 promoters, and wherein ATG or GTG codons at one or more positions within these promoters are absent or mutated.

[0173] The GOI can be any gene or coding sequence within the packaging capacity of the AAV, e.g., about 4-5 kb, or about 4.7 kb including the ITR sequences, or about 4.4 kb without accounting for the ITR sequences.

[0174] The AAV ITR sequences flanking the GOI can be from any AAV ITR, and can be from the same or different AAV serotypes. In certain embodiments, the AAV ITR sequences flanking the GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0175] In some embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from the same AAV. In some other embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from different AAVs.

[0176] In certain embodiments, the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0177] In certain embodiments, the tropism of the AAV include skeletal muscle (such as AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9, or a derivative thereof, such as SLB-101 supray).

[0178] In certain aspects, the rAAV that may be produced by using the DT vectors (e.g., DT plasmids) of the invention and the suitable host cells (which can supply any useful helper functions for rAAV production in trans), may encode a gene of interest (GOI) useful for, e.g., gene therapy to treat a disease or condition.

[0179] Representative (non-limiting) gene of interest (GOI) may include: a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0180] In certain embodiments, the GOI is a microdystrophin gene.

[0181] In certain embodiments, the microdystrophin gene is any such one described in the following patents: US7,906,l l l; US7,001,761; US7,510,867; US6, 869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854, or US 10, 166,272, each and every one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference). In certain embodiments, the microdystrophin gene is capable of being packaged into a rAAV virion, e.g., no more than about 4.7 kb in size.

[0182] In certain embodiments, the microdystrophin gene contains within its coding sequence spectrin-like repeats R16 and R17 that are capable of restoring nitric oxide synthase (nNOS) activity to the sarcolemma (such as those described in US7,892,824).

[0183] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats (i.e., SRI, SR16, SR17, SR23, and SR24, respectively) of the full-length dystrophin protein, such as one described in PCT / US2016 / 013733 (incorporated herein by reference). In certain embodiments, the microdystrophin gene does not encode any other spectrin repeats of the full-length dystrophin protein, other than SRI, SR16, SR17, SR23, and SR24.

[0184] Diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention include: Huntington’s disease, X-linked myotubular myopathy (XLMTM), Acid maltase deficiency (e.g., Pompe disease), Spinal Muscular Atrophy (SMA), Myasthenia Gravis (MG), Amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Mitochondrial myopathy, Muscular dystrophies (Duchenne’s muscular dystrophy, Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery- Dreifuss muscular dystrophy (EDMD), Mucopolysaccharidoses (MPS), Metachromatic leukodystrophy (MLD), Batten Disease, Rett Syndrome, Krabbe Disease, Canavan disease, X-Linked Retinoschisis, Achromatopsia (CNGB3 and CNGA3), X-Linked Retinitis Pigmentosa, Age-Related Macular Degeneration, neovascularized macular degeneration, Pompe, Fabry’s disease, MPS I, II, IIIA, IIIB, Gaucher’s disease, Dannon Disease, AlAt Deficiency, Friedreich ataxia, Wilson’s Disease, Batten Disease (CLN1, CLN3, CLN6, CLN8), Wolman Disease, Tay-Sachs, Niemann-Lick Type C, CDKL5 deficiency Disorder, B-thalassemia, Sickle cell disease.

[0185] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include: Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Bethlem CMD, Fukuyama CMD, Muscle-eye-brain diseases (MEBs), Rigid spine syndromes, Ullrich CMD, Walker-Warburg syndromes (WWS), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Motor neuron diseases including ALS (amyotrophic lateral sclerosis), Spinal -bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA).

[0186] In certain embodiments, the GOI is a coding sequence for BAG3 (BCL-2-associated athanogene 3). BAG3 has been implicated in selected macroautophagy (aggrephagy), wherein aggregated proteins are degraded. Under stress conditions and during normal cellular aging, BAG3 acts with other molecular chaperones HSP70 and HSPB8, along with ubiquitin receptor p62 / SQSTMl to target aggregated proteins for autophagic degradation. Loss of function of BAG3 can disrupt cellular clearing of protein aggregates which may lead to physiological complications and dysfunction. BAG3 mediated clearance is involved in many cellular processes which require the clearance of aggregate or aggregate prone proteins, and may be associated with age-related neurodegenerative disorders, like Alzheimer’s disease (marked by tau- protein), Huntington’s disease (involving mutated huntingtin / polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). Additionally, BAG3 has been shown to play a role in a variety of other disease states, including cancer and myopathies. BAG3 mutations in cardiomyopathy may significantly increase burdens associated with heart disease and increase severe cardiac events.

[0187] In certain embodiments, the coding sequence for BAG3 is a human coding sequence for BAG3. In some embodiments, the human BAG3 coding sequence is codon-optimized for expression in human cells, such as human cardiac muscle cells. In some embodiments, the coding sequence for BAG3 is CpG depleted. In certain embodiments, the coding sequence for BAG3 is any one of the BAG3 constructs in WO2023108159A1 (incorporated herein by reference).

[0188] In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to one or a combination of the amino acid sequences set forth as SEQ ID NOs: 23-26, 101, or 83-85 of WO2023108159A1 (incorporated herein by reference). In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NOs: 23-26 of WO2023108159A1 (incorporated herein by reference) arranged in sequence. In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NO: 101 of WO2023108159A1 (incorporated herein by reference). In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NOs: 83-85 of WO2023108159A1 (incorporated herein by reference) arranged in sequence.

[0189] In certain embodiments, the coding sequence for BAG3 is operatively linked to a promoter and / or an enhancer element, such as a CMV promoter / enhancer, or a cardiac muscle-specific promoter / enhancer, such as a MHCK7 promoter / enhancer. In certain embodiments, the promoter is CBA (Chicken P-Actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is CK8. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CMV, mini-CMV, HSV, TK, RSV, SV40, MMTV, Ad E1A, CBA, CK8, MHCK7, Desmin (optionally mDES), and combinations thereof.

[0190] In certain embodiments, the coding sequence for BAG3 is used / encompassed in AAV viral particles for treating dilated cardiomyopathy.

[0191] In certain embodiments, the GOI is a coding sequence for cardiac troponin T (TNNT2). TNNT2 is a component of the troponin complex within the thin filament of the sarcomere which allows actomyosin interaction and contraction to occur in response to Ca2+. Mutations in or perturbations in the function of TNNT2 are causative of hypertrophic (HCM) and dilated cardiomyopathy (DCM).

[0192] In certain embodiments, the coding sequence for TNNT2 is a human coding sequence for TNNT2. In some embodiments, the human TNNT2 coding sequence is codon-optimized for expression in human cells, such as human cardiac muscle cells. In some embodiments, the coding sequence for TNNT2 is CpG depleted. In certain embodiments, the coding sequence for TNNT2 is any one of the TNNT2 constructs in WO2023178339A2 (incorporated herein by reference).

[0193] In certain embodiments, the amino acid sequence of TNNT2 encoded by the coding sequence for TNNT2 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 32-46 of WO2023178339A2 (incorporated herein by reference) arranged in sequence. In certain embodiments, the amino acid sequence of the TNNT2 encoded by the coding sequence for TNNT2 is any one of the synthetic TNNT2 sequences in Table 1 of WO2023178339A2 (incorporated herein by reference).

[0194] In certain embodiments, the coding sequence for TNNT2 is operatively linked to a promoter and / or an enhancer element. In certain embodiments, the promoter is a cardiac specific promoter. In some embodiments, the promoter is TNNT2. In some embodiments, the promoter is MHCK9. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CBA (Chicken P- Actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is a Desmin promoter.

[0195] In certain embodiments, the coding sequence for TNNT2 is used / encompassed in AAV viral particles for treating dilated cardiomyopathy or hypertrophic cardiomyopathy.

[0196] In certain embodiments, the GOI is a coding sequence for frataxin (FXN), which mutation is linked to Friedreich’s ataxia. More specifically, Friedreich’s ataxia is caused by a mutation in the FXN gene which encodes frataxin. Individuals who inherit two defective copies of the gene will develop this disease. Although rare, Friedreich’s ataxia is the most common form of hereditary ataxia in the United States, affecting about 1 in every 50,000 people. There is currently no approved cure for Friedreich’s ataxia.

[0197] In certain embodiments, the coding sequence for frataxin / FXN is a human coding sequence for frataxin / FXN. In some embodiments, the human frataxin / FXN coding sequence is codon-optimized for expression in human cells, such as human cardiac and / or CNS tissues / cells. In some embodiments, the coding sequence for frataxin / FXN is CpG depleted. In certain embodiments, the coding sequence for frataxin / FXN is any one of the frataxin / FXN constructs in WO2022147575A1 (incorporated herein by reference).

[0198] In certain embodiments, the coding sequence for frataxin / FXN has at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the coding sequence for frataxin / FXN encodes human frataxin comprising the amino acid sequence of SEQ ID NO: 3 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the coding sequence for frataxin / FXN exhibits enhanced expression of human frataxin as compared to a polynucleotide encoding frataxin but not having the same sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 of WO2022147575A1. In certain embodiments, the coding sequence for frataxin / FXN further comprises an untranslated region (UTR) that imparts regulatory control on expression of the human frataxin encoded by the polynucleotide. The UTR region may comprise a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 6 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the amino acid sequence of the frataxin / FXN, and / or the coding sequence for frataxin / FXN is any one disclosed in WO2022147575A1 (incorporated herein by reference).

[0199] In certain embodiments, the coding sequence for frataxin / FXN further comprises a promoter driving expression of the frataxin. In certain embodiments, the promoter is a cardiac-specific promoter. For example, promoter is a cardiac-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). In some embodiments, the promoter is a muscle-specific promoter, such as desmin. In some embodiments, the promoter is a CNS-specific promoter, such as the synapsin (SYN) promoter. In certain embodiments, the promoter comprises a cytomegalovirus (CMV) enhancer element functionally coupled to a chicken beta actin (CBA) promoter. In certain embodiments, the CMV enhancer element comprises a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 4 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the CBA promoter comprises a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 5 of WO2022147575A1 (incorporated herein by reference).

[0200] In certain embodiments, the coding sequence for frataxin / FXN is used / encompassed in AAV (e.g., AAV9 or AAVrh74) viral particles for treating Friedreich’s ataxia. In certain embodiments, the treatment may further comprise administering an immunomodulatory regimen comprising an antibody directed against a cancer marker (such as CD20) and an additional agent (such as an mTOR pathway inhibitor). In certain embodiments, the GOI is a coding sequence for RNA binding motif protein 20 (RBM20). Mutations in or perturbations in the function of RBM20 are known to be causative of DCM (Dilated Cardiomyopathy). RBM20 is a major regulator of heart- specific alternative splicing of the TTN gene, which is found to be most frequently mutated in patients with idiopathic DCM (approximately 20- 25%). The TTN gene has the largest number of exons (364 in humans) and titin, a sarcomeric protein encoded by the TTN gene, is the largest known protein in mammals. In an RBM20 mutant rat strain lacking nearly all the RBM20 exons, the shortest cardiac titin isoform N2B is not expressed. Therefore, RBM20 is a key regulator of TTN pre-mRNA processing in the heart and may cause DCM phenotypes through altered splicing of the RBM20-regulated genes. Missense mutations in a highly conserved RSRSP stretch, within an arginine / serine (RS)-rich region and not in the RNA binding domains are the most frequent disease alleles.

[0201] In certain embodiments, the coding sequence for RBM20 is a human coding sequence for RBM20. In some embodiments, the human RBM20 coding sequence is codon-optimized for expression in human cells, such as human cardiac and / or CNS tissues / cells. In some embodiments, the coding sequence for RBM20 is CpG depleted. In certain embodiments, the coding sequence for RBM20 is any one of the RBM20 constructs in WO2023178337A2 (incorporated herein by reference).

[0202] In certain embodiments, the coding sequence for RBM20 is at least about 85% sequence identity to SEQ ID NO: 5 of WO2023178337A2 (incorporated herein by reference).

[0203] In certain embodiments, the coding sequence for RBM20 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter, such as a TNNT2 promoter, a MHCK9 promoter, a CB A (Chicken beta- Actin) promoter or a truncated chicken beta-actin (smCB A) promoter, and combinations thereof. In certain embodiments, the promoter is a CMV or mini-CMV promoter. In some embodiments, the promoter is a Desmin promoter. In some embodiments, the promoter is a muscle creatine kinase (MCK) promoter. In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the promoter is a MHCK9 promoter. In certain embodiments, the promoter is a constitutive viral promoter, which may include the Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the Simian Virus 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Ad El A promoter and the cytomegalovirus (CMV) promoters. In certain embodiments, the promoter comprises a nucleic acid sequence having at least about 85% sequence identity (e.g., identical) to the sequence of SEQ ID NO: 2 or 16 of WO2023178337A2 (incorporated herein by reference).

[0204] In certain embodiments, the coding sequence for RBM20 is used / encompassed in AAV (e.g., AAV9, AAVrh74, or AAVrhlO) viral particles for treating dilated cardiomyopathy. In certain embodiments, the treatment may further comprise administering an immunomodulatory regimen comprising an antibody directed against a cancer marker (such as CD20) and an additional agent (such as an mTOR pathway inhibitor). In certain embodiments, the coding sequence for RBM20 is in an expression cassette that further comprises a silencing element, wherein the coding sequence for RBM20 and the silencing element are each operably linked to a promoter and optionally an enhancer element. The silencing elements can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In some embodiments, the silencing element is a short hairpin RNA (shRNA). In some embodiments, the silencing element is an siRNA. In a nonlimiting example, epigenetic modulation of gene expression by siRNA silencing elements can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression.

[0205] In certain embodiments, the GOI is a coding sequence for MYBPC3. The MYBPC3 gene provides instructions for making cardiac myosin binding protein C (cardiac MyBP-C). Cardiac MyBP-C is found in cardiac muscle cells, where it plays a role in sarcomere contraction. Muscle contraction depends heavily on the activity of sarcomeres resident in myocytes. Mutations in MYBPC3 are common causes of familial hypertrophic cardiomyopathy, accounting for up to 30% of all cases. Though some individuals have no obvious health effects, all affected individuals possess an increased risk of heart failure and sudden death. MYBPC3 mutations generally present phenotypically shorter or otherwise altered MyBP-C proteins. Reduced MyBPC in the sarcomere disrupts myosin conformations, which may contribute to various cardiac disease states.

[0206] In certain embodiments, the coding sequence for MYBPC3 is a human coding sequence for MYBPC3. In some embodiments, the human MYBPC3 coding sequence is codon-optimized for expression in human cells, such as human cardiac and / or CNS tissues / cells. In some embodiments, the coding sequence for MYBPC3 is CpG depleted. In certain embodiments, the coding sequence for MYBPC3 is any one of the MYBPC3 constructs in WO2023108157A1 (incorporated herein by reference).

[0207] In certain embodiments, the coding sequence for MYBPC3 is at least about 85% sequence identity to the sequence of SEQ ID NO: 9, 29 or 43 of WO2023108157A1 (incorporated herein by reference).

[0208] In certain embodiments, the coding sequence for MYBPC3 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter. In certain embodiments, the promoter is selected from the group consisting of: CMV, mini-CMV, CBA, HSV, TK, RSV, SV40, MMTV, Ad El A, and combinations thereof, and wherein the cardiac specific enhancer or regulatory element comprises an alphaMHC enhancer. In certain embodiments, the MYBPC3 promoter sequence has at least about 85% sequence identity to the sequence of SEQ ID NO: 5, 24, or 39 of WO2023108157A1 (incorporated herein by reference).

[0209] In certain embodiments, the coding sequence for MYBPC3 is used / encompassed in AAV (e.g., AAV9, AAVrh74, or AAVrhlO) viral particles for treating hypertrophic cardiomyopathy.

[0210] In certain embodiments, the GOI is a coding sequence for calsequestrin 2 (CASQ2), the mutation of which is responsible for the primary inherited arrhythmia syndrome CPVT (catecholaminergic polymorphic ventricular tachycardia). CASQ2 is a calcium-binding protein which, through its role in Ca2+regulation, is integral to excitation-contraction coupling in the heart and in regulating the rate of heart beats. CPVT is a rare, serious and life-threatening disease which primarily manifests in children in the first and second decades of life, with the mean onset of CPVT symptoms being between seven and twelve years. CPVT is an inherited cardiac arrhythmia syndrome characterized by adrenergically induced polymorphic arrythmias in the presence of a normal resting sinus rhythm and a structurally normal heart. It is estimated that the prevalence of CPVT is 1 per 10,000 persons. CPVT manifestations typically involve syncope, cardiac arrest and / or sudden cardiac death. The most common symptoms / signs include syncope (52-100%), cardiac arrest (8-48%), seizurelike events (40%), and hypoxic-ischemic encephalopathy (20%). CPVT is a significant cause of sudden death at a young age and mortality is high (up to 50%). Data from the recent Pediatric and Congenital Electrophysiology Society CPVT registry suggest that three of every four children with CPVT present with life-threatening symptoms, which often occur during resting wakeful activities highlighting the unpredictable nature of CPVT. CPVT may be caused by a gain-of-function mutation in the ryanodine receptor 2 (encoded by the RYR2 gene), which is referred to as CPVT-1, as well as a loss-of-function mutations in the calsequestrin 2, or CASQ2, gene, which is referred to as CPVT-2. In certain embodiments, the coding sequence for CASQ2 is a human coding sequence for CASQ2. In some embodiments, the human CASQ2 coding sequence is codon-optimized for expression in human cells, such as human cardiac cells. In some embodiments, the coding sequence for CASQ2 is CpG depleted. In certain embodiments, the coding sequence for CASQ2 is any one of the CASQ2 constructs in US20170360957A1 (incorporated herein by reference).

[0211] In certain embodiments, the coding sequence for CASQ2 encodes a protein that is at least about 85%, 90%, 95% or higher sequence identity to the sequence of SEQ ID NO: 2 of US20170360957A1 (incorporated herein by reference).

[0212] In certain embodiments, the coding sequence for CASQ2 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter, such as a TNNT2 promoter, a MHCK9 promoter, a CB A (Chicken beta- Actin) promoter or a truncated chicken beta-actin (smCB A) promoter, and combinations thereof. In certain embodiments, the promoter is a CMV or mini-CMV promoter. In some embodiments, the promoter is a Desmin promoter. In some embodiments, the promoter is a muscle creatine kinase (MCK) promoter. In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the promoter is a MHCK9 promoter. In certain embodiments, the promoter is a constitutive viral promoter, which may include the Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the Simian Virus 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Ad El A promoter and the cytomegalovirus (CMV) promoters. In certain embodiments, the promoter comprises a nucleic acid sequence having at least about 85% sequence identity (e.g., identical) to the sequence of SEQ ID NO: 2 or 16 of WO2023178337A2 (incorporated herein by reference).

[0213] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include ion channel diseases, which are typically marked by muscular weakness, absent muscle tone, or episodic muscle paralysis. They include Andersen-Tawil syndrome, Hyperkalemic periodic paralysis, Hypokalemic periodic paralysis, Myotonia congenita, Becker myotonia, Thomsen myotonia, Paramyotonia congenita, Potassium-aggravated myotonia.

[0214] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include mitochondrial diseases, which occur when structures that produce energy for a cell malfunction. Such diseases include: Friedreich’s ataxia (FA), Mitochondrial myopathies, Kearns-Sayre syndrome (KSS), Leigh syndrome (subacute necrotizing encephalomyopathy), Mitochondrial DNA depletion syndromes, Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Myoclonus epilepsy with ragged red fibers (MERRF), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Progressive external opthalmoplegia (PEO).

[0215] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include myopathies, which is a disease of muscle in which the muscle fibers do not function properly, resulting in muscular weakness. Myopathies include: Cap myopathies, Centronuclear myopathies, Congenital myopathies with fiber type disproportion, Core myopathies, Central core disease, Multiminicore myopathies, Myosin storage myopathies, Myotubular myopathy, Nemaline myopathies, Distal myopathies, GNE myopathy / Nonaka myopathy / hereditary inclusion-body myopathy (HIBM), Laing distal myopathy, Markesberg-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, Vocal cord and pharyngeal distal myopathy, Welander distal myopathy, Endocrine myopathies, Hyperthyroid myopathy, Hypothyroid myopathy, Inflammatory myopathies, Dermatomyositis, Inclusion-body myositis, Polymyositis, Metabolic myopathies, Acid maltase deficiency (AMD, Pompe disease), Carnitine deficiency, Carnitine palmityl transferase deficiency, Debrancher enzyme deficiency (Cori disease, Forbes disease), Lactate dehydrogenase deficiency, Myoadenylate deaminase deficiency, Phosphofructokinase deficiency (Tarui disease), Phosphoglycerate kinase deficiency, Phosphoglycerate mutase deficiency, Phosphorylase deficiency (McArdle disease), Myofibrillar myopathies (MFM), Scapuloperoneal myopathy.

[0216] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include neuromuscular junction diseases, which result from the destruction, malfunction or absence of one or more key proteins involved in the transmission of signals between muscles and nerves. Such diseases include: Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG).

[0217] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include peripheral nerve diseases, in which the motor and sensory nerves that connect the brain and spinal cord to the rest of the body are affected, causing impaired sensations, movement or other functions. Such diseases include: Charcot-Marie-Tooth disease (CMT), Giant axonal neuropathy (GAN), muscle wasting in cachexia and aging.

[0218] In certain embodiments, the GOI coding sequence comprises a polyA signal sequence or polyadenylation site.

[0219] In certain embodiments, the polyadenylation site is a bovine growth hormone (bGH) polyadenylation site.

[0220] In certain embodiments, the polyadenylation site or poly(A) signal sequence is from other suitable sources, e.g., synthetic sequences or sequences from other eukaryotic genes or viruses.

[0221] In certain embodiments, the GOI coding sequence is partially or fully codon- optimized for expression in a mammalian host cell. For example, the most 3’ 300-350 nucleotides of the coding sequence may be codon-optimized for expression in the mammalian host cell.

[0222] 4. Composition or Kits of Two-Vector (e.g., two-plasmid) System

[0223] One aspect of the invention provides a composition or a kit, comprising a two-vector (e.g., two-plasmid) system that comprises, consists essentially of, or consists of any one of the dual recombinant DNA vector (dual transfection vector or “DT vector”) of the invention, and a helper plasmid sufficient to support AAV packaging in the presence of the DT vector of the invention, further including another plasmid (or “the second vector” as described herein) for adjusting the molar ratio of the RepCap coding sequence.

[0224] The two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention is useful for producing rAAV. The two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention is suitable for use in producing rAAV. The two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention is for producing rAAV. The two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention is for producing rAAV suitable for use in gene therapy. The two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention is for producing rAAV for use in gene therapy.

[0225] The phrase “two-vector (two-plasmid) system supplemented by said another plasmid (or “the second vector” as described herein)” refers to a system that comprises two vectors (e.g., plasmids) plus said another plasmid (or “the second vector” as described herein), and can be used together, without the need for additional plasmids to produce rAAV (z.e., they in combination are sufficient for AAV production). In certain embodiments, the two-vector (plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) can be used to produce rAAV without the need for helper virus such as adenovirus. In certain embodiments, the two-vector (plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) can be used to produce rAAV without the need for genetic material originating from a host cell, optionally with the exception of a gene encoding El A / B. However, the vector system in the composition / kit of the invention may comprise additional non-plasmid components. Optionally, the composition or kit of the invention comprising the two-vector (plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) of the invention does not comprise a helper virus.

[0226] In certain embodiments, the two-vector (plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) of the invention comprises all the necessary genetic information for the production of rAAV. For example, the two-vector (plasmid) system supplemented by said another plasmid (or “the second vector” as described herein) of the invention may comprise at least one rep gene, at least one cap gene and at least one helper gene. In certain embodiments, the two-plasmid system supplemented by said another plasmid (or “the second vector” as described herein) of the invention comprises all the necessary genetic information useful for the production of rAAV suitable for use in gene therapy. For example, the two-plasmid system supplemented by said another plasmid (or “the second vector” as described herein) of the invention may comprise at least one rep gene, at least one cap gene, at least one helper gene and an expression cassette comprising a transgene operably linked to at least one regulatory control element.

[0227] One of the vectors / plasmids in the composition or kit of the invention is the vector / plasmid comprising the AAV Rep, Cap and GOI (flanked by AAV ITR sequences). The other vector / plasmid in the composition or kit of the invention is the helper plasmid / vector. Said another plasmid (or “the second vector” as described herein) in the composition or kit of the invention can be a plasmid having the RepCap (but no GOI) coding sequence.

[0228] In certain embodiments, the helper plasmid / vector may comprise at least one helper virus gene. AAV is only able to propagate in the presence of a helper virus. Examples of helper viruses include adenoviruses, and herpes viruses.

[0229] In certain embodiments, the helper plasmids of the invention comprise sufficient helper genes to allow for AAV replication and packaging. In certain embodiments, the helper plasmid or vector that can be used with the rDNA vector of the invention provides all the helper genes / functions for AAV packaging (except for the ITR sequences flanking the GOI).

[0230] For example, for host cells expressing the adenoviral E1A / B genes, such as the HEK293T cells, the remaining adenoviral helper genes encode E4, E2A and VA RNA I and II. These helper genes can be included in the helper plasmid or vector in any order or orientation, so long as all these genes can be transcribed and expressed to facilitate AAV packaging.

[0231] Whether a helper plasmid comprises sufficient helper genes to facilitate AAV production can be assessed using any art recognized AAV production assay to show sufficient titer of AAV so produced.

[0232] In one embodiment, the helper gene products will be considered (sufficient) to facilitate AAV production if they support rAAV production at a level of or at least 25%, of or at least 40%, of or at least 50%, of or at least 60%, of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95%, or a level within any range delimited by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, for example, of the level supported by adenoviral helper genes encoding E4, E2A and VA RNA I and II. In certain embodiments, if the yield of rAAV produced is or is at least 25%, or is at least 40%, or is at least 50%, or is at least 60%, or is at least 70%, or is at least 80%, or is at least 90%, or is at least 95%, or a level within any range delimited by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, or a level within any range delimited by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, for example, of the yield of rAAV produced using the reference two-plasmid system in which one is a helper plasmid comprising adenoviral helper genes encoding E4, E2A and VA RNA I and II.

[0233] Since the helper plasmids encode the genes that allow for efficient AAV production, the addition of a helper virus is not required (z.e., the composition or kit of the invention comprising the two vector / plasmid of the invention is sufficient to support AAV production and packaging). In certain embodiments, the at least one helper virus gene comprises an adenovirus gene. Adenovirus is a virus which is known to aid propagation of AAV (Xiao et al., J. Virol, 72:2224-2232, 1998). In certain embodiments, the at least one helper virus gene comprises an Adenovirus 5 gene or an Adenovirus 2 gene. In certain embodiments, the helper genes of adenoviruses encode E1A, E1B, E4, E2A and VA RNA I and II.

[0234] E1A is encoded by nucleotides 560-1545 of the Adenovirus 5 genome. The E1A may optionally lack the non-essential intron at nucleotides 1113-1228.

[0235] E1B is actually two proteins E1B 19K and E1B 55K, which work together to block apoptosis in adenovirus-infected cells. E1B is encoded by nucleotides 1714-2244 (E1B 19K), and by nucleotides 2019-3509 (E1B 55 K) of the Adenovirus 5 genome.

[0236] E4 is encoded by a number of different open reading frames (ORFs) of the Adenovirus 5 genome. E4 ORF 6 / 7 is encoded by nucleotides 32914-34077, which may optionally lack the intron between nucleotides 33193 and 33903. E4 34K is encoded by nucleotides 33193-34077 of the Adenovirus 5 genome. E4 ORF 4 is encoded by nucleotides 33998-34342 of the Adenovirus 5 genome. E4 ORF 3 is encoded by nucleotides 34353- 34703 of the Adenovirus 5 genome. E4 ORF B is encoded by nucleotides 34700 to 35092 of the Adenovirus 5 genome. E4 ORF 1 is encoded by nucleotides 35140-35526 of the Adenovirus 5 genome. A functional E4 protein may only comprise amino acids encoded by ORFs 6 and 7, as only the amino acids encoded by ORFs 6 and 7 are mandatory for activity. In certain embodiments, the functional E4 protein comprises a polypeptide sequence encoded by all or a significant portion of ORFs 6 and 7. Optionally, the functional E4 protein does not comprise polypeptide sequence encoded by all or a portion of ORFs 1-4 and 34K.

[0237] In certain embodiments, the functional E4 protein comprises amino acids encoded by ORFs 1-7, as the amino acids encoded by ORFs 1-3 and 34K of E4, though not required, do improve the activity of the E4 protein.

[0238] The E2 (E2A) gene is encoded by nucleotides 22443-24032 of the Adenovirus 5 genome.

[0239] The VA RNA I and II is encoded by nucleotides 10589-11044 of the Adenovirus 5 genome.

[0240] E1B and E4 are believed to enhance AAV mRNA accumulation, and E2A and VA RNA I and II are believed to enhance AAV mRNA splicing and translation. E1B, E4 and E2A are proteins encoded by genes present in the adenovirus genome, whereas the VA nucleic acid encodes two RNA transcripts known as VA RNA I and VA RNA II. The transcripts themselves are functional in the cell, and are never translated into amino acid sequences. It will be appreciated, therefore, that the VA nucleic acid does not encode a protein, but does “encode” or “correspond to” an RNA, z.e., whilst the term “encode” is used the VA nucleic acid is a non-translated nucleic acid sequence.

[0241] Of the five adenovirus genes, in some embodiment, it is possible not to include E1A or E1B in the helper plasmid, as some host cell lines (such as HEK293 cells) express one or more of E1A or E1B constitutively. In certain embodiments, the helper plasmid does not comprise a gene encoding a functional adenoviral E1A / B protein.

[0242] In one embodiment, the at least one helper virus gene comprises: (a) a VA (viral associated) nucleic acid encoding functional VA RNA I and II; (b) an E2A gene encoding a functional E2A protein; and / or (c) n E4 gene encoding a functional E4 protein.

[0243] In certain embodiments, the at least one helper virus gene comprises a VA nucleic acid (such as VA RNA I and / or II), an E2A gene and an E4 gene.

[0244] A “functional” VA RNA I and II, E2A protein or E4 protein is able to facilitate production of AAV.

[0245] One of skill in the art can determine whether a given VA RNA I and II, E2A protein or E4 protein is functional by determining, for example, whether the VA RNA I and II, E2A protein or E4 protein supports AAV production using an AAV production assay as described above. In some embodiments, the VA RNA I and II, E2A protein or E4 protein is considered to be functional if it supports rAAV production at a level of or at least 25%, of or at least 40%, of or at least 50%, of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95%, or a level within any range delimited by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, for example, of the level supported by the wild type (for example as found in native Adenovirus 5) VA RNA I and II, E2A protein or E4 protein. In certain embodiments, the E4 protein will be considered to be “functional” if it supports rAAV production at a level of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95% of the level supported by the wild type E4 protein.

[0246] In certain embodiments, the E4 gene is not located between the VA nucleic acid and the E2A gene, z.e., the sequence of the plasmid is such that E4 gene sequence does not appear in the plasmid between the VA nucleic acid sequence and the E2A gene sequence. In certain other embodiments, the E2A gene is located between the VA nucleic acid and the E4 gene.

[0247] Any of the genes on the helper plasmid can take any orientation without regard to the other genes.

[0248] In certain embodiments, the VA nucleic acid has an activity level which has or has at least 75%, has or has at least 80%, has or has at least 90%, has or has at least 95%, or between 95% and 100% of the activity of a wild type VA nucleic acid from Adenovirus 5. The activity level of the VA nucleic acid can be determined by, for example, measuring rAAV yield, for example using the AAV production assay described above.

[0249] Expression of the E4 gene is driven by a promoter (designated “'E4 promoter”). A fully active promoter comprises nucleotides corresponding to 35793-35848 of the Adenovirus 5 genome. In certain embodiments, the E4 gene is operably linked to an E4 promoter that has at least 50%, at least 70%, or at least 90% of the activity of a wild type promoter from Adenovirus 5. The activity of the E4 promoter may be determined by testing its ability to drive expression of a protein. In one embodiment, the E4 promoter has or has at least 25%, has or has at least 40%, has or has at least 50%, has or has at least 70%, has or has at least 90% of the activity of a wild type E4 promoter from Adenovirus 5 if it supports rAAV production at a level of or at least 25%, of or at least 40%, of or at least 50%, of or at least 60%, of or at least 70%, of or at least 90% of the level supported by a wild type E4 gene promoter, i.e., if the yield of rAAV produced is or is at least 50%, or is at least 70%, is or is at least 90% of the yield of rAAV produced using the reference two-plasmid system comprising wild type helper genes with wild-type promoters. Preferably, the E4 promoter will be considered to facilitate AAV production if it supports rAAV production at a level of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95% of the yield of rAAV produced using the reference two-plasmid system comprising wild type helper genes with wild-type promoters.

[0250] 5. Host Cells

[0251] One aspect of the invention comprises a host cell comprising the dual recombinant DNA vector (dual transfection vector) supplemented by said another plasmid (or “the second vector” as described herein) of the invention.

[0252] In general, a host cell of the invention is capable or suitable for the production of rAAV. The host cell is typically derived from a eukaryotic cell line, such as a vertebrate cell line, including a mammalian cell line (e.g., a human cell line).

[0253] In certain embodiments, the host cell is a cell selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1.CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, and an A549 cell.

[0254] In certain embodiments, the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1.CR cell, a PerC6 cell, a C139 cell, and an EB66 cell.

[0255] In certain embodiments, the host cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, and a HEK293EBNA cell.

[0256] In certain embodiments, the host cell is a HEK293T cell.

[0257] In certain embodiments, the host cell is a cell that expresses a functional adenoviral E1A / B protein. For example, the host cell may comprise a chromosome comprising a gene encoding a functional adenoviral El A / B protein.

[0258] The host cell is considered suitable or capable of producing recombinant AAV if it supports AAV production at a level of or at least 30%, of or at least 40%, of or at least 50%, of or at least 70%, of or at least 80%, of or at least 90%, of or at least 95% of the level supported by HEK293T cells. In this case, the test two-vector / plasmid system of the invention can be introduced / transfected into the host cell whose suitability for the production of recombinant AAV is to be tested, and the reference two-vector / plasmid system is be introduced / transfected into HEK293T cells.

[0259] In certain embodiments, the dual transfection vector and / or the helper plasmid (or equivalent function DNA coding sequences) is stably integrated into the host cell genome.

[0260] In certain embodiments, the host cell is derived from a vertebrate, such as human, monkey, bovine, porcine, equine and other equids, canine, feline, ovine, goat, murine, rat, rabbit, mink, opossum, camel and other cameloids, chicken and other avian, armadillo, frog, or reptile, or derived from an insect cell.

[0261] In certain embodiments, the host cell is a cell line suitable for AAV packaging, such as Expi cells, HEK293 cells (or a derivative thereof such as HEK293T cells).

[0262] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell), a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0263] In certain embodiments, the host cell is HEK293 (human embryonic kidney), which can be grown using standard tissue culture media such as DMEM complemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin. In some embodiments, the HEK293 cells are grown on a solid support, including tissue culture plates, dishes, flasks, and bottles. For growing adherent HEK293 cells, the percentage of FBS can be reduced during rAAV production in order to limit contamination by animal-derived components.

[0264] In some embodiments, the HEK293 cells are adapted to grow in suspension.

[0265] In certain embodiments, the host cell is a Vero cell, such as a Vero75.4 or V75 cell described herein. Such cells may grow on a solid support, including tissue culture plates, dishes, flasks, bottles, and microcarrier that allows the adherent Vero cells to grow in suspension-like conditions.

[0266] In certain embodiments, the host cell is a BHK (baby hamster kidney) cell, such as BHK21 or sBHK27. In certain embodiments, the BHK cells are adapted to grow in serum- free suspension.

[0267] In certain embodiments, the host cell is a HEK293 cell. In certain embodiments, the HEK293 cell is adapted for growth in serum-free media (such as F 17 or Expi293 media) and in suspension, thus is amenable for large scale growth in a bioreactor. See, for example, Grieger et al. (Mol. Ther. 24:287-297, 2016, incorporated herein by reference).

[0268] In certain embodiments, the HEK293 cell is a HEK293T cell which expresses SV40 T antigen (the temperature sensitive allele tsA1609) and the neomycin / geneticin-resistance gene.

[0269] In certain embodiments, for production of rAAV particles, the host cell comprises helper virus proteins useful (e.g., required) for AAV packaging.

[0270] In some embodiments, the coding sequences of the helper virus proteins are introduced into the host cell in a plasmid through transfection. In some other embodiments, the coding sequences of the helper virus proteins are integrated into the host cell genome.

[0271] In certain embodiments, the host cell of the invention may be adapted for use in producing recombinant AAV vectors encoding a gene of interest (GO I), which may be used in gene therapy. See the section entitled “Recombinant AAV Production” below. In such embodiments, one or more rAAV production cell lines may be infected by the DT vectors of the invention, such as vectors or plasmids encoding AAV Rep and Cap proteins.

[0272] In certain embodiments, such producer / host cell line for rAAV production is a HeLa- or A549-derived cell line transfected with the DT vector / plasmid of the invention, optionally containing a drug selection marker. In certain embodiments, such producer cell line for rAAV production is a Vero cell.

[0273] In certain embodiments, such producer cell line for rAAV production is a BHK cell.

[0274] In certain embodiments, such producer cell line for rAAV production is a HEK293 cell.

[0275] In certain embodiments, such producer / host cell line for rAAV production comprises a DT vector / vector of the invention comprising the GOI flanked by the AAV ITR sequences. The GOI can be any one of the GOI described herein useful for gene therapy, such as a dystrophin minigene or a microdystrophin gene described in US7, 906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; W02020 / 086844; or US10,166,272, WO 2023 / 018854, or in PCT / US2016 / 013733 (all incorporated herein by reference).

[0276] For example, PCT / US2016 / 013733 (WO2016 / 115543 A2) provides a microdystrophin gene operatively connected to a regulatory cassette, wherein the micro-dystrophin gene encodes a protein comprising: an amino-terminal actin-binding domain; a P- dystroglycan binding domain; and a spectrin-like repeat domain, comprising at least four spectrin-like repeats, wherein two of the at least four spectrin-like repeats comprise a neuronal nitric oxide synthase binding domain. In certain embodiments, the at least four spectrin-like repeats include spectrin-like repeat 1 (SRI), spectrin-like repeat 16 (SRI 6), spectrin-like repeat 17 (SRI 7), and spectrin-like repeat 24 (SR24). In certain embodiments, the protein encoded by the micro-dystrophin gene further comprises at least a portion of a hinge domain, such as at least one of a Hinge 1 domain, a Hinge 2 domain, a Hinge 3 domain, a Hinge 4 domain, and a hinge-like domain. In certain embodiments, the microdystrophin gene comprises, in N- to C-terminal order: a Hinge 1 domain (Hl); a spectrin-like repeat 1 (SRI); a spectrin-like repeat 16 (SR16); a spectrin-like repeat 17 (SR17); a spectrinlike repeat 24 (SR24); and a Hinge 4 domain (H4). In certain embodiments, Hl is directly coupled to the SRI. In certain embodiments, SR 1 is directly coupled to SR16. In certain embodiments, SRI 6 is directly coupled to SRI 7. In certain embodiments, SR 17 is directly coupled to SR24. In certain embodiments, SR24 is directly coupled to the H4. In certain embodiments, the protein encoded by the micro-dystrophin gene further comprises between SRI and SRI 6, in N- to C-terminal order, a spectrin-like repeat 2 (SR2) and a spectrin-like repeat 3 (SR3). In certain embodiments, SRI is directly coupled to SR2 and SR2 is further coupled to SR3. In certain embodiments, Hl is directly coupled to SRI, SRI is directly coupled to SR16, SR16 is directly coupled to SR17, SR17 is directly coupled to SR23, SR23 is directly coupled to SR24, and SR24 is directly coupled to H4.

[0277] In certain embodiments, the regulatory cassette is selected from the group consisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter. In certain embodiments, the protein encoded by the micro-dystrophin gene has between five spectrin-like repeats and eight spectrin-like repeats. In certain embodiments, the protein encoded by the microdystrophin gene has at least 80% or 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 5 in WO2016 / 115543 A2 (incorporated herein by reference).

[0278] In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0279] In certain embodiments, the GOI is a coding sequence for any one of a functional calsequestrin 2 (CASQ2) protein, a functional BCL-2-associated athanogene 3 (BAG3) protein, a functional cardiac troponin T (TNNT2) protein, a functional RNA binding motif protein 20 (RBM20) protein, a functional frataxin (FXN) protein, or a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein). See, for example, WO2023108159A1 (BAG3), WO2023178339A2 (TNNT2); WO2022147575A1 (frataxin / FXN); WO2023178337A2 (RBM20); WO2023108157A1 (MYBPC3); or US20170360957A1 (CASQ2), all incorporated herein by reference.

[0280] 6. Recombinant AA V Production

[0281] The vector system of the present invention, as well as the production cell lines, can be used for large scale production of recombinant AAV vectors (rAAV) useful for gene therapy. In certain embodiments, the subject vector system and the associated method of use, can be used for propagating / amplifying / producing of AAV viral particles comprising the ITR- flanked GOI.

[0282] Recombinant replication-defective AAV vectors, which can be produced with the present vector system (including the dual transfection vectors) and production cell lines, typically comprise a gene of interest (GOI) and expression regulators (such as promoters for the GOI) in lieu of the wild-type AAV virus rep and cap open reading frames (ORFs). The AAV rep and cap ORFs, optionally their native promoters p5, pl 9, and p40 are supplied by the vector system of the invention, and other helper functions useful for AAV packaging are supplied in another construct or production cell line. The rep ORF encodes four nonstructural Rep proteins involved in the AAV viral life cycle, and the cap ORF encodes the three structural proteins (z.e., VP1, VP2, and VP3) that form the icosahedral AAV capsid. Typically, the only AAV viral sequences that are retained in the rAAV vector genome are the inverted terminal repeats (ITRs) - the minimal cv.s-acting elements for AAV DNA replication and packaging.

[0283] In certain aspects, the method of propagating / amplifying / producing a replicationdefective AAV particle encapsidating the GOI comprises introducing the vector system of the invention into a host cell, prior to, concurrently with, or subsequent to introducing the AAV helper genes for virus packaging.

[0284] In some embodiments, the vector system of the invention is introduced to the host cell by transient transfection.

[0285] The method for producing a replication-defective AAV particle comprises the steps of simultaneously co-transfecting mammalian cells, such as, but not limited to HEK293 cells, with the vector system of the invention which may include a construct comprising the AAV helper genes.

[0286] In some embodiments, the AAV helper genes are introduced into the host cell through transfection, e.g., transient transfection. In some other embodiments, the AAV helper genes are integrated into the host cell genome prior to introduction of the vector system of the invention to the host cell.

[0287] In certain embodiments, the host cell is derived from a vertebrate, such as human, monkey, bovine, porcine, equine and other equids, canine, feline, ovine, goat, murine, rat, rabbit, mink, opossum, camel and other cameloids, chicken and other avian, armadillo, frog, or reptile, or derived from an insect cell. Human cells include BHK cells, Vero cells, HEK293 cells, etc.

[0288] In certain embodiments, the host cell is a HEK293 cell (such as an Expi293F cell or VP2 cell), a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0289] In certain embodiments, the host cell is HEK293 (human embryonic kidney), which can be grown using standard tissue culture media such as DMEM complemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin.

[0290] In some embodiments, the HEK293 cells are grown on a solid support, including tissue culture plates, dishes, flasks, and bottles. For growing adherent HEK293 cells, the percentage of FBS can be reduced during rAAV production in order to limit contamination by animal -derived components. In some embodiments, the HEK293 cells are adapted to grow in suspension.

[0291] In certain embodiments, the HEK293 cell is adapted for growth in serum-free media (such as F 17 or Expi293 media) and in suspension, thus is amenable for large scale growth in a bioreactor. See, for example, Grieger et al. (Mol. Ther. 24:287-297, 2016, incorporated herein by reference).

[0292] In certain embodiments, the HEK293 cell is a HEK293T cell which expresses SV40 T antigen (the temperature sensitive allele tsA1609) and the neomycin / geneticin-resistance gene.

[0293] In certain embodiments, the host cell is a Vero cell, such as a Vero75.4 or V75 cell described herein. Such cells may grow on a solid support, including tissue culture plates, dishes, flasks, bottles, and microcarrier that allows the adherent Vero cells to grow in suspension-like conditions.

[0294] In certain embodiments, the host cell is a BHK (baby hamster kidney) cell, such as BHK21 or sBHK27. In certain embodiments, the BHK cells are adapted to grow in serum- free suspension.

[0295] In some embodiments, the method further comprises harvesting the replicationdefective AAV particle comprising the GOI. In some embodiments, the method comprises purifying the recombinant replication-defective AAV particle.

[0296] The gene of interest (GOI) may include genes useful for gene therapy in treating certain diseases or conditions. Representative (non-limiting) GOI may include a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0297] Suitable microdystrophin genes include those described in the following patents:

[0298] US7,906,l l l; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854; US10, 166,272; W02020 / 086844 (all incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0299] Diseases or conditions having a potential to benefit from the rAAV produced by the DT vector of the invention-based system include: Huntington’s disease, X-linked myotubular myopathy (XLMTM), Acid maltase deficiency (e.g., Pompe disease), Spinal Muscular Atrophy (SMA), Myasthenia Gravis (MG), Amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Mitochondrial myopathy, Muscular dystrophies (Duchenne’s muscular dystrophy, Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery- Dreifuss muscular dystrophy (EDMD), Mucopolysaccharidoses (MPS), Metachromatic leukodystrophy (MLD), Batten Disease, Rett Syndrome, Krabbe Disease, Canavan disease, X-Linked Retinoschisis, Achromatopsia (CNGB3 and CNGA3), X-Linked Retinitis Pigmentosa, Age-Related Macular Degeneration, neovascularized macular degeneration, Pompe, Fabry’s disease, MPS I, II, IIIA, IIIB, Gaucher’s disease, Dannon Disease, AlAt Deficiency, Friedreich ataxia, Wilson’s Disease, Batten Disease (CLN1, CLN3, CLN6, CLN8), Wolman Disease, Tay-Sachs, Niemann-Lick Type C, CDKL5 deficiency Disorder, B-thalassemia, Sickle cell disease, etc.

[0300] Additional GOI may encode BAG3 (BCL-2-associated athanogene 3) for treating dilated cardiomyopathy; cardiac troponin T (TNNT2) for treating dilated cardiomyopathy or hypertrophic cardiomyopathy; frataxin (FXN) for treating Friedreich’s ataxia; RNA binding motif protein 20 (RBM20) for treating dilated cardiomyopathy; MYBPC3 for treating hypertrophic cardiomyopathy; or calsequestrin 2 (CASQ2) for treating catecholaminergic polymorphic ventricular tachycardia (CPVT).

[0301] Being a naturally replication-defective human parvovirus, wild-type AAV integrates its genome site-specifically within the host cell chromosome in the absence of helper assistance for its replication, where it persists indefinitely unless rescued via cellular infection with a helper virus. The introduction of a helper virus into the host cell triggers AAV replication and the generation of progeny virions. In the case of rAAV virions useful for gene therapy, introduction of the helper virus function into a suitable host cell triggers the packaging of the GOI in the rAAV virions, when the requisite rep and cap coding sequences are also supplied in the same system.

[0302] In other words, production of recombinant AAV relies on (1) the presence of the AAV rep and cap coding sequences, and (2) the helper virus functions.

[0303] In certain embodiments, the tropism of the AAV include serotypes such as SLB-101, AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9, AAV10, AAV11, preferably AAV9, AAVrh74, AAVrhlO. In certain embodiments, AAV capsids may be genetically modified, or capsids may be synthetic, designer capsids that enhance tissue specific or physiologic compartments delivery of a GOI to a specific tissue such as muscle, skeletal muscle, cardiac muscle, smooth muscle, and the like, as described (see, e.g., Zinn and Grimm, High- Throughput Dissection of AAV-Host Interactions: The Fast and the Curious, JMB 430( 17):2626-2640, 2018; Kotterman and Schaffer, Engineering adeno-associated viruses for clinical gene therapy. Nature Reviews Genetics (2014) 4445-4451, both incorporated herein by reference) and / or detargets certain tissue. Tropism of AAV through pseudotyping, or the mixing of a capsid and genome from different viral serotypes may also be employed. These serotypes are denoted using a slash, so that AAV2 / 5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5. Use of these pseudotyped viruses can improve transduction efficiency, as well as alter tropism. For example, pseudotyped AAV2 / 5 targets myoblasts (Duan et al.. Enhancement of muscle gene delivery with pseudotyped adeno-associated virus type 5 correlated with myoblast differentiation. J Virol 75(16):7662- 7671, 2001). Other pseudotyped AAV includes AAV2 / 6. In certain embodiments, In-silico- derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina (Zinn et al.. In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector, Cell Reports 12(6): 1056-1068, 2015); Buning et al.. Engineering the AAV capsid to optimize vector-host-interactions, Current Opinion in Pharmacology, 24:94-104, 2015).

[0304] In certain embodiments, the tropism of the AAV includes skeletal muscle (such as SLB-101, AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9). In certain embodiments, the tropism of the AAV includes cardiac muscle (such as AA1, AAV5, AAV6 AAV8, AAV9, AAVrh74, AAV2-8, AAV2-9,). In certain embodiments, the AAV detargets certain tissue, such as the liver. In certain embodiments, the tropism of the AAV is targeted to certain tissue(s) and detargets certain tissue(s).

[0305] In certain embodiments, the gene of interest (GO I) includes a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IHB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0306] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0307] In certain embodiments, the GOI is a microdystrophin gene.

[0308] In certain embodiments, the microdystrophin gene is one described in US7, 906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854, or US10,166,272 (each and every one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0309] In certain embodiments, the microdystrophin gene comprises a coding sequence for R16 and R17 spectrin-like repeats for the full length dystrophin protein (such as one described in US7,892,824).

[0310] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as the microdystrophin gene described in PCT / US2016 / 013733).

[0311] In certain embodiments, the microdystrophin gene does not comprise coding sequence for and spectrin repeats of the full-length dystrophin protein other than the SRI, SRI 6, SRI 7, SR23, and SR24 repeats (e.g., in that order).

[0312] In certain embodiments, the rAAV vectors of the invention are produced in in vitro culture conditions, such as in bioreactors e.g., 0.5L, IL, 2L, 3L, 5L, 10L, 20L, 50L, 100L, 250L, 500L, or l,000L working volume bioreactors), such as a CelliGen Plus packed-bed bioreactor (New Brunswick Scientific) for fed-batch vector production for 3 days post infection.

[0313] In certain embodiments, the recombinant replication-defective AAV viral particles are produced as in vitro culture on adherence-dependent cell lines, such as Vero and Vero- derived cell lines or HEK293 cells and HEK293 -derived cells, that rely on a solid support. In certain embodiments, the solid support is a tissue culture surface, such as tissue culture dishes, plates, bottles, flasks, cell factory, etc. In certain embodiments, the solid support is a microcarrier, such as Cytodex 1 (GE Healthcare Life Sciences, Piscataway, NJ); a macrocarrier, such as FibraCel (New Brunswick Scientific, Edison, NJ), or a multilayered culture vessel, such as a CellCube (Corning Life Sciences, Lowell, MA) that permit medium perfusion.

[0314] In certain embodiments, the recombinant replication-defective AAV viral particles are produced as in vitro culture in eukaryote cells adapted to grow in suspension, such as a suspension culture of the BHK cell line or HEK293 cell line adapted for growth in suspension.

[0315] In certain embodiments, the culture supernatant yields in excess of 1 x IO10plaqueforming units (PFU) of rAAV, 1 x 1011plaque-forming units (PFU) of rAAV, 1 x 1012plaque-forming units (PFU) of rAAV, 1 x 1013plaque-forming units (PFU) of rAAV, 1 x 1014plaque-forming units (PFU) of rAAV, or a yield within any range delimited by any pair of the foregoing values, such as between 1 x io10PFU and 1 x 1014PFU, between 1 x io11PFU and 1 x io13PFU, or 1 x 1012PFU and 1 x 1014PFU, for example.

[0316] In certain embodiments, vector stock is produced by one or more post processing steps, such as filtration and / or concentration (e.g., depth filtration, dead-end filtration, tangential flow filtration (TFF), and diafiltration), multi-column chromatography purification, final concentration / buffer exchange, etc., to obtain vector stocks with sufficient purity for administration to animals, including human. In certain embodiments, the purification process and the purified vector stock satisfy GMP standard.

[0317] In certain embodiments, the titer of the rAAV vector stocks is about 1-2 x 107PFU / ml, about 1-2 x 108PFU / ml, about 1-2 x 109PFU / ml, about 1-2 x 1O10PFU / ml, about 1-2 x 1011PFU / ml, about 1-2 x 1012PFU / ml, or a titer within any range delimited by any pair of the foregoing values.

[0318] In certain embodiments, the total yield of the rAAV vector stock is about 1-25 x IQ14 total VG of purified rAAV, about 1-10 x 1014total VG of purified rAAV, about 1-5 x io14total VG of purified rAAV, about 2-4 x 1014total VG of purified rAAV, or a total yield within any range delimited by any pair of the foregoing values.

[0319] In certain embodiments, rAAV vectors so produced are further purified from crude cell lysates by ion-exchange chromatography and / or by iodixanol density gradient centrifugation to ensure high final product purity. In certain embodiments, rAAV vectors so produced qualify as a clinical-grade vector batch.

[0320] In certain embodiments, the AAV production method of the invention further comprises determining the titer, purity, and / or potency of the rAAV vectors so produced. This may include characterizing the purified rAAV stocks using, e.g., silver staining of SDS- PAGE separation of proteins to determine purity.

[0321] 7. Treatment of Muscular Dystrophy using AA V

[0322] The DT vector of the invention-based system can be used for large scale production of rAAV, which in turn can be used in gene therapy for treating various forms of muscular dystrophy, such as Duchenne’s muscular dystrophy (DMD), Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), etc. In certain embodiments, the muscular dystrophy is DMD or BMD.

[0323] Thus another aspect of the invention provides a method of treating muscular dystrophy (such as DMD and BMD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV) vector encoding a functional version of the gene defective in the muscular dystrophy, such as a microdystrophin gene, wherein the rAAV is produced by the method of the invention using the DT vector of the invention and complementary system.

[0324] In certain embodiments, the microdystrophin gene is one described in US7, 906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; W02020 / 086844; WO2016115543; WO 2023 / 018854, or US10,166,272 (all incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023 / 018854 (incorporated herein by reference).

[0325] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as one described in PCT / US2016 / 013733).

[0326] In certain embodiments, the method further comprises producing the rAAV by the method of the invention using the DT vector system of the invention, prior to administering to the subject the rAAV so produced.

[0327] 8. Treatment of Cardiomyopathy using AA V

[0328] The vector system of the invention can be used for large scale production of rAAV, which in turn can be used in gene therapy for treating various forms of cardiomyopathy. Cardiomyopathy represents a collection of diverse conditions of the heart muscle and is the second most common cause of heart disease in subjects and medical management of the secondary signs is the only therapeutic option. These diseases have many causes, symptoms, and treatments, and can affect people of all ages and races. When cardiomyopathy occurs, the normal muscle in the heart can thicken, stiffen, thin out, or fill with substances the body produces that do not belong in the heart muscle. As a result, the heart muscle's ability to pump blood is reduced, which can lead to irregular heartbeats, the backup of blood into the lungs or rest of the body, and heart failure. Cardiomyopathy can be acquired or inherited. The cause isn't always known but there is an increasing understanding of the genetic underpinnings of inherited forms of disease. Gene transfer strategies have been shown to ameliorate heart diseases.

[0329] Cardiomyopathy is a class of disease of heart muscle that adversely impacts the heart’s ability to circulate blood through the cardiovascular system. Various types of cardiomyopathies exist, including dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (AVC), left ventricular noncompaction cardiomyopathy (LVNC), and restrictive cardiomyopathy (RCM). There are a number of genetic forms of cardiomyopathies, including but not limited to DCM associated with Duchenne and Becker muscular dystrophies. In the case of certain forms of Becker muscular dystrophy, as well as in most cases of Duchenne muscular dystrophy, the cardiomyopathy can ultimately limit the patient’s survival. Accordingly, some aspects of the present invention provide AAV or rAAV vectors for delivering transgenes into the heart of a subject, whether directly, systemically or by both methods.

[0330] Thus another aspect of the invention provides a method of treating cardiomyopathy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV) vector encoding a functional version of the gene defective in the cardiomyopathy, genes such as a B AG3 gene, a MYBPC gene, a TMEM43 gene, a TNNT2 gene, an RBM20 gene, an RYR2 gene, a CASQ2 gene, wherein the rAAV is produced by the method of the invention using the vector system of the invention and complementary system.

[0331] Other genes related to various cardiomyopathies include the following:

[0332] Table 1

[0333]

[0334]

[0335] EXAMPLES

[0336] Example 1 AA V production using transfection system

[0337] The transfection experiments were carried out according to the methods described below. Similar experiments can be carried out using techniques known in the art.

[0338] Shake flask method

[0339] VP2 cells were seeded into shake flasks at 0.4E6 (0.4* 106) cells / mL and allowed to grow for 3 days. On day 3, the cells were transfected at target cell density of 3.0E6 cells / mL. The transfection reagent used was FectoVIR (PolyPlus, France), added at a ratio of 1 : 1 (FectoVIR: DNA) and the complex were allowed to proceed for 15 mins prior to addition to the bioreactors. Helper plasmid of pALD-HELP was selected for use in this experiment. Three days post transfection the vessels were harvested.

[0340] Microreactor method (AMBR®15)

[0341] Ambr® 15 Cell Culture is a commercial high throughput, automated bioreactor system for 24 or 48 parallel cultivations at the 10 -15 mL microbioreactor scale. This platform enables selection of the best clones, media and feeds under small-scale bioreactor conditions, ensuring a seamless scale-up.

[0342] Example 2 Identification of superior GOI, RepCap and Helper plasmid ratio for triple transfection method pGOI plasmid encoding BAG3 (FIG. 2A) and pRepCap plasmid encoding AAV rh74 capsid (FIG. 2B) were tested with helper plasmid pALD-HELP in triple transfection method at varying ratio.

[0343] Based on the results as shown in FIG. 3, 1 :2: 1 ratio of helper plasmid (pHelp): RepCap plasmid (pRepCap): GOI plasmid (pGOI) yielded the highest GOI titre, while the percentage of full capsid was comparable to numerous other tested ratios. Example 3 Comparison between triple transfection, dual transfection and dual transfection with additional RepCap plasmid in shake flasks

[0344] This experiment was carried to compare outcomes of AAV production using triple transfection, dual transfection, and dual transfection with additional RepCap plasmid to adjust the ratio of the GOI and the RepCap / Helper coding sequences.

[0345] Dual plasmids (pDual) comprising BAG3 as GOI and rh74 Cap as depicted in FIG. 1C were used in dual transfection (DT) experiments. Shake flask transfection method as described in Example 1 was employed for this experiment with the parameters provided in Table 1

[0346] Table 1

[0347] At pDuakpHelp (w / w) ratio of 1 : 1, GOI titre and percentage full capsid were both lower compared to triple transfection with pGOI:pRepCap:pHelp (w / w) ratio of 1 :2: 1 (see FIG. 4).

[0348] With pDuakpHelp ratio of 1 : 1, the effective ratio of GOI:RepCap:Help is 1 : 1 : 1. To test whether production of AAV is dependent on GOI:RepCap:Help ratio in dual transfection method the same way it is dependent on this ratio in triple transfection method, additional RepCap plasmids were added to match the effective GOI:RepCap:Help 1 :2: 1 ratio of the triple transfection.

[0349] When additional RepCap plasmid was added to match the GOI:RepCap:Help 1 :2: 1 ratio, significantly higher yield GOI titres and percentage of full capsid was achieved compared to dual transfection alone (FIG. 4).

[0350] Although the percentage of full capsid was slightly lower compared to that of triple transfection, GOI titre yield of dual transfection with additional RepCap plasmid was surprisingly ~3 folds higher than triple transfection method (FIG. 4). It was also unexpected that Dual and additional RepCap plasmid transfection resulted in a much lower rate of DNA mispackaging as measured by mis-packaged KanRgene sequence (FIG. 4).

[0351] In summary, this experiment showed that adding RepCap plasmid to a dual transfection to match the superior 1 :2: 1 GOI:RepCap:Help ratio of triple transfection method can improve yield of dual transfection, and may even achieve unexpectedly better AAV production outcome (e.g., in terms of lower mis-packaging rate) compared to that of triple transfection.

[0352] Example 4 Production outcome of dual transfection is reproducible in other transfection platform

[0353] Comparison between triple transfection and dual transfection with additional RepCap plasmid was carried out using microbioreactor AMBR® 15 cell culture system.

[0354] Similar to the results using shake flask, dual transfection with additional RepCap plasmid to match GOI:RepCap:Help ratio 1 :2: 1 in AMBR® 15 cell culture bioreactor system yielded slightly lower full capsid percentage, but significantly higher GOI titre yield (~3 folds) compared to triple transfection method of the same ratio (see FIG. 5).

Claims

CLAIMS:

1. A vector system for production of a recombinant adeno-associated virus (AAV), said vector system comprises:(1) a first coding sequence for a gene-of-interest (GO I) flanked by AAV ITR sequences;(2) a second coding sequence for an AAV Rep and an AAV Cap; and,(3) optionally, a third coding sequence for AAV helper genes; wherein said AAV Rep is compatible with said AAV ITR sequences for AAV packaging; wherein expression of said AAV Rep, in a host cell comprising said AAV helper genes, is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the AAV ITR sequences into an AAV capsid comprising said AAV Cap, and, wherein the molar ratio of (1) : (2) : (3) is about 1 : about 2 : about 1.

2. The vector system of claim 1, wherein the molar ratio of (1) : (2) : (3) is (0.95-1.05) : (1.90-2.10) : (0.95-1.05).

3. The vector system of claim 1 or 2, comprising:(a) a first vector comprising said first coding sequence and said second coding sequence;(b) a second vector comprising said second coding sequence; and,(c) optionally, a third vector comprising said third coding sequence.

4. The vector system of any one of claims 1-3, wherein said first coding sequence, said second coding sequence, and / or said third coding sequence is in a plasmid.

5. The vector system of claim 3, wherein said first vector, said second vector, and / or said third vector is a plasmid.

6. The vector system of claim 5, wherein said first vector is a first plasmid, and said second vector is a second plasmid.

7. The vector system of any of claims 1-6, wherein the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter (such as a muscle-specific promoter (e.g., CK8 promoter), or a cardiac muscle-specific promoter (e.g., MHCK7 promoter)).

8. The vector system of claim 7, wherein the pro- AAV cassette further comprises:(1) an enhancer that promotes the transcription of the GOI from the promoter;(2) a 5’ UTR;(3) a Kozak sequence;(4) a heterologous intron that promotes transcription and / or translation of the GOI;(5) a 3’ UTR;(6) a WPRE sequence; and / or(7) a polyA (such as bGH polyA) signal sequence.

9. The vector system of any one of claims 1-8, wherein the coding sequence for the AAV Rep and the coding sequence for the AAV Cap are within a RepCap cassette comprising an operably-linked AAV P5 promoter (e.g., a wild-type P5 promoter).

10. The vector system of claim 9, wherein the RepCap cassette and the pro- AAV cassette, when present on the same vector: i) are immediately adjacent to each other (e.g., with substantially no intervening polynucleotide sequence); ii) are not immediately adjacent to each other; iii) have the same transcription direction; or, iv) have opposite transcription direction.

11. The vector system of any one of claims 3-10, wherein said first vector, said second vector, and / or said third vector each independently further comprises a bacterial replication Ori gene, a selection marker (such as an antibiotic resistance gene, e.g., KanR, NeoR, or AmpR) under the transcriptional control of a selection marker promoter (such as a bacterial promoter).

12. The vector system of any one of claims 1-11, wherein said GOI encodes a functional calsequestrin 2 (CASQ2) protein, a functional BCL-2-associated athanogene 3 (BAG3) protein, a functional cardiac troponin T (TNNT2) protein, a functional RNA binding motif protein 20 (RBM20) protein, a functional frataxin (FXN) protein, or a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

13. The vector system of any one of claims 1-12, wherein said GOI includes a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E),LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N- acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain- 3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

14. The vector system of any one of claims 1-13, wherein the GOI is a microdystrophin gene (e.g., one described in US7,906,l l l; US7,001,761; US7,510,867; US6, 869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854, or US 10, 166,272).

15. The vector system of claim 14, wherein the microdystrophin gene comprises a coding sequence for R16 and R17 spectrin-like repeats for the full length dystrophin protein (such as one described in US7,892,824).

16. The vector system of claim 15, wherein the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as the microdystrophin gene described in WO2016115543 or SEQ ID NO: 1 of WO 2023 / 018854).

17. The vector system of any one of claims 1-16, wherein said AAV ITR sequences flanking said GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

18. The vector system of claim 17, wherein the tropism of the AAV include skeletal muscle, cardiac muscle, or neuromuscular system (such as that of SLB-101, AAV1, AAV6, AAV7, AAV8, AAVrh74, or AAV9); optionally, said AAV has a capsid of AAVrh74 or a derivative thereof.

19. The vector system of any one of claims 1-18, wherein said AAV ITR sequences, said AAV Rep, and said AAV Cap are from the same or different AAVs.

20. The vector system of claim 19, wherein said AAV ITR sequences are AAV2 ITR sequences, said AAV Rep is Rep2 from AAV2, and said AAV Cap is SLB-101,AAV8, AAV9, AAVrh74, or a derivative thereof.

21. The vector system of any one of claims 1-20, wherein said coding sequence for AAV Rep and Cap proteins is under the transcriptional control of a promoter, such as an AAV P5 promoter, an upstream HSV promoter, a modified P5 promoter lacking RBE (Rep-Binding Element), an HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (such as CMV promoter, EFla promoter, CAG promoter, CB promoter etc).

22. The vector system of claim 21, wherein the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized Ying- Yang 1 (YY1) binding site, and wherein said modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nts).

23. The vector system of any one of claims 1-22, wherein said AAV helper genes comprise adenoviral, herpesviral, or papillomaviral genes for AAV packaging (such as El A, E1B, E2A, E4 and VA RNA), optionally operably linked to a promoter as one transcriptional unit.

24. The vector system of any one of claims 3-23, wherein said first coding sequence encodes BAG3, and wherein said second coding sequence encodes AAVrh74.

25. A composition or a kit, comprising the vector system of any one of claims 1-24, optionally including said third coding sequence in a helper plasmid.

26. The composition or kit of claim 25, wherein the helper plasmid comprises a helper virus gene that supports AAV packaging.

27. The composition or kit of claim 26, wherein the helper virus gene comprises:(i) an adenovirus gene, optionally an Adenovirus 5 or Adenovirus 2 gene; and / or(ii) a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein.

28. A host cell comprising the vector system of any one of claims 1-24, or the composition of any one of claims 25-27.

29. The host cell of claim 28, which is a HEK293 cell (such as an Expi293F cell), a VP2cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

30. A method of propagating / amplifying / producing a recombinant replication-defective AAV viral particle encapsi dating the GOI of any one of claims 1-24, the method comprising: introducing the vector system of any one of claims 1-24 into a host cell, prior to, concurrently with, or subsequent to introducing said AAV helper genes of any one of claims 1-24 to said host cell, thereby propagating / amplifying / producing the recombinant replication-defective AAV viral particle.

31. The method of claim 30, further comprising harvesting the recombinant replicationdefective AAV viral particle from the host cell.

32. The method of claim 30 or 31, wherein the host cell is a HEK293 cell (such as an Expi293F cell), a VP2 cell, a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

33. The method of any one of claims 30-32, wherein the recombinant DNA vector is introduced to the host cell by transient transfection.

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