Dual transfection vector

Abstract

The invention described herein provides improved systems and methods for rAAV production. The systems of the invention include a recombinant DNA vector containing a gene of interest (GOI) to be delivered to a target cell, as well as both Rep and Cap coding sequences for rAAV packaging.

Description

[Background technology]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Application No. 63 / 413,887, filed on October 6, 2022, the entire contents of which are incorporated herein by reference.

[0002] In early rAAV production, cultured cells are first infected with adenovirus and then transfected with two additional DNA plasmids: one containing a pair of essential rAAV genes (e.g., the Rep / Cap genes), and the other carrying the sequence of the gene of interest (GOI). While this method results in reasonable yields of rAAV particles, there is a risk that adenovirus will contaminate the preparation, which may put patients being treated with rAAV at risk.

[0003] In 1998, a different research group determined that essential genes of adenovirus could be transferred onto a third "helper" plasmid, and that all three plasmids could be simultaneously transfected into a preferred host cell to produce rAAV particles, avoiding potential contamination of the rAAV. Since then, this method, known as the "triple transfection" (TT) technique, has been widely used as the primary method for generating recombinant adeno-associated virus (rAAV), including for commercial production of AAV viral stocks for gene therapy, and may also be referred to herein as conventional triple transfection or traditional triple transfection.

[0004] However, a persistent problem with using AAV-based gene therapy is the lack of sufficient quantities of suitable AAV viral particles (e.g., approximately 1×10) for effective treatment. 14 ~1×10 15 The demand for vector genomes is

[0005] Today, rAAV is considered an important platform for delivering many types of genes for gene therapy, and the number of rAAV-based therapies in development continues to increase, necessitating further improvements in large-scale rAAV production. Summary of the Invention

[0006] One embodiment of the present invention is a recombinant DNA vector comprising: (a) a gene of interest (GOI) flanked by a 5' adeno-associated virus (AAV) ITR (e.g., AAV2 5' ITR) sequence and a 3' ITR sequence (e.g., AAV2 3' ITR); (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally wherein the coding sequence for AAV Rep is under transcriptional control of an AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap, wherein expression of the AAV Rep in a host cell containing AAV helper genes is facilitated by the AAV Provided is a recombinant DNA vector that is sufficient to package an AAV vector genome (vg) containing a GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid containing Cap, with the proviso that the recombinant DNA vector is not a herpes simplex virus (HSV) vector.

[0007] Another aspect of the present invention provides a vector comprising: (a) a gene of interest (GOI) flanked by adeno-associated virus (AAV) ITRs; (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) that is compatible with the ITRs, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3′ to the GOI; and (c) a coding sequence for AAV Cap, wherein the vector is not a herpes simplex virus (HSV) vector.

[0008] In certain embodiments, the recombinant DNA vector, or vectors, are plasmids.

[0009] In another aspect, the invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by (AAV) ITRs (e.g., AAV2 5' ITR sequence and AAV2 3' ITR sequence); (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) that is compatible with the (AAV) ITRs, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap.

[0010] In another aspect, the invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (e.g., AAV2 5' ITR) and 3' ITR sequences (e.g., AAV2 3' ITR); (b) a coding sequence for AAV Rep (e.g., AAV2 Rep or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap, wherein expression of the AAV Rep in a host cell containing 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.

[0011] In certain embodiments, the plasmids of the present invention do not include polynucleotide sequences encoding helper genes.

[0012] In yet another aspect, the present invention provides a two-plasmid system comprising a vector plasmid and a helper plasmid, wherein the vector plasmid comprises a GOI, a rep gene encoding a functional Rep protein, and a cap gene encoding a functional Cap protein.

[0013] In some embodiments, expression of AAV Rep in a host cell containing AAV helper genes is sufficient to package an AAV vector genome (vg) containing a GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid containing AAV Cap.

[0014] In certain embodiments, the GOI is in a pro-AAV cassette that comprises the GOI operably linked to a promoter.

[0015] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that facilitates transcription of the GOI from the promoter; (2) a 5'UTR; (3) a Kozak sequence; (4) a heterologous intron that facilitates transcription and / or translation of the GOI; (5) a 3'UTR; (6) a WPRE sequence; and / or (7) a polyA signal sequence.

[0016] In certain embodiments, the coding sequence for AAV Rep and the coding sequence for AAV Cap are in a RepCap cassette that comprises an operably linked RepCap promoter (e.g., the AAV P5 promoter).

[0017] In certain embodiments, the RepCap cassette and the pro-AAV cassette i) are immediately adjacent to each other (e.g., substantially free of intervening polynucleotide sequences); ii) are not immediately adjacent to each other; iii) have the same direction of transcription; or iv) have opposite directions of transcription.

[0018] In certain embodiments, the recombinant DNA vector comprises a bacterial replication Ori gene, a selectable marker (e.g., an antibiotic resistance gene, e.g., Kan) under the transcriptional control of a selectable marker promoter (e.g., a bacterial promoter).R or Amp R ) further includes.

[0019] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0020] In certain embodiments, the GOI includes a gene causing / 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 2I), or a gene encoding Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB). IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (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.

[0021] In some embodiments, the GOI is the microdystrophin gene (e.g., as described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO2023 / 018854, or US10,166,272 (each of which is incorporated herein by reference in its entirety). In some embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (incorporated herein by reference).

[0022] In certain embodiments, the microdystrophin gene comprises coding sequences for the R16 and R17 spectrin-like repeats (e.g., as described in US 7,892,824) for a full-length dystrophin protein.

[0023] In some embodiments, the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats of a full-length dystrophin protein (e.g., the microdystrophin gene described in PCT / US2016 / 013733).

[0024] In some embodiments, the 5' and 3' 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.

[0025] In certain embodiments, both the 5' and 3' AAV ITR sequences flanking the GOI are from AAV2.

[0026] In certain embodiments, the tropism of the AAV includes skeletal muscle (e.g., AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9).

[0027] In certain embodiments, the AAV ITRs, the AAV Rep, and the AAV Cap are from the same or different AAVs.

[0028] In certain embodiments, the AAV ITRs are AAV2 ITRs, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or a derivative thereof (e.g., a spectral or transcriptional derivative thereof).

[0029] In certain embodiments, the coding sequences for the AAV Rep and Cap proteins are under the transcriptional control of a promoter, such as the AAV p5 promoter, an upstream HSV promoter, a modified p5 promoter lacking the RBE (Rep binding element), the HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (e.g., a CMV promoter, an EF1a promoter, a CAG promoter, a CB promoter, etc.).

[0030] In some 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-Yang1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site. In some embodiments, the spacer is 5 nucleotides to 100 nucleotides (e.g., about 5 nt) in length.

[0031] In some embodiments, the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized Ying-Yang1 (YY1) binding site, wherein the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides (e.g., about 5 nt) in length.

[0032] In certain embodiments, the AAV helper genes comprise adenovirus, herpesvirus, or papillomavirus viral genes useful for AAV packaging (e.g., E1A, E1B, E2A, E4, and VA RNAs), optionally operably linked to a promoter as a single transcription unit.

[0033] Another aspect of the present invention provides a composition or kit comprising the recombinant DNA vector of the present invention and a helper plasmid.

[0034] In certain embodiments, the helper plasmid contains helper virus genes sufficient to support AAV packaging.

[0035] In certain embodiments, the helper virus genes include (i) adenovirus genes, optionally adenovirus 5 or adenovirus 2 genes; 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.

[0036] Another aspect of the present invention provides a host cell comprising a recombinant DNA vector of the present invention, or a composition of the present invention.

[0037] In certain embodiments, the host cell is a HEK293 cell (e.g., an Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

[0038] Another aspect of the present invention provides a method for propagating / amplifying / producing recombinant replication-deficient AAV viral particles harboring a GOI of the present invention, the method comprising introducing a recombinant DNA vector of the present invention into a host cell before, simultaneously with, or after introducing said AAV helper genes of the present invention into said host cell, thereby propagating / amplifying / producing recombinant replication-deficient AAV viral particles.

[0039] In certain embodiments, the method further comprises recovering the recombinant, replication-defective AAV viral particles of the invention from the host cell.

[0040] In certain embodiments, the host cell is a HEK293 cell (e.g., an Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

[0041] In certain embodiments, the recombinant DNA vector is introduced into the host cell by transient transfection.

[0042] It is to be understood that any one embodiment of the present invention, including those set forth solely in one of the examples, claims, or subsections, may be combined with any other one or more embodiments, unless inappropriate or expressly disclaimed. [Brief explanation of the drawings]

[0043] [Figure 1A] 1 shows a schematic diagram (not to scale) of an enlarged section of an exemplary vector of the invention, showing the Pro-AAV cassette and the RepCap cassette (which may or may not be directly adjacent to each other, as shown here, and may or may not be in the same transcriptional orientation). pZ-KanR (kanamycin resistance gene under transcriptional control of a bacterial promoter) is an optional feature as well. GOI: gene of interest. [Figure 1B] Figure 1 shows the plasmid maps of two exemplary plasmids containing the GOI / RepCap vector of the present invention for generating AAV9 (left) and AAV-SLB101 (right) viral particles. The GOI is a variant of microdystrophin, as a non-limiting example of a GOI. μDys: microdystrophin. In both constructs, the Pro-AAV cassette is located upstream / 5' to the RepCap cassette, and its transcription is driven by the p5 promoter in the RepCap cassette. As a result, in the circular plasmid vector, the KanR-encoding sequence is further upstream / 5' to the p5 promoter than the equivalent KanR-encoding sequence in the RepCap plasmid used in triple transfection (absent the Pro-AAV cassette). [Figure 2]Figure 1 shows the results of rAAV production using Expi cells by dual transfection of a GOI / RepCap plasmid of the invention and a suitable helper plasmid at exemplary GOI:helper (w / w) ratios of 1:1 and 1.5:1, respectively, and DNA amounts of 0.5 pg / cell and 0.75 pg / cell. [Figure 3A] We demonstrate surprisingly high titers (e.g., 200-300%) of rAAV viral particle yields for AAV9 and AAV-SLB101 viral particles produced in Expi cells using the double-transfection (DT) plasmid of the present invention (see Figure 1B) compared with conventional triple-transfection (TT) plasmids. Viral titers were determined by ddPCR at 24, 48, and 72 hours post-transfection by lysing infected Expi cells. [Figure 3B] This shows the superior yield of AAV produced by various methods using the DT plasmid of the present invention (3rd to 5th bars from the left) compared to the triple transfection method (1st bar from the left). [Figure 3C] 1 shows scalable, high-yield production of AAV in 2 L bioreactors using the DT plasmid of the present invention. [Figure 4] Figure 1 shows the reduced mispackaging rate of KanR using the DT plasmid of the present invention compared to the TT plasmid, based on ddPCR and KanR / GOI ratios using two commercially available transfection reagents: AAVMAX: Gibco AAV-MAX Transfection Kit (ThermoFisher Scientific); FectoVIR: FECTOVIR®-AAV (Polyplus, NY). [Figure 5] We show that the modified p5 promoter further reduces mispackaging rates (as measured by % p5-associated mispackaging / GOI) and increases GOI titers. DETAILED DESCRIPTION OF THE INVENTION

[0044] 1. Overview Conventional AAV production systems utilize three plasmid vectors (so-called triple transfection vector systems): one carries the gene of interest (GOI) flanked by AAV ITR sequences for packaging into AAV viral particles; another carries expression cassettes encoding the AAV Rep and Cap proteins useful for AAV packaging; and yet another provides useful helper genes from other viruses (e.g., adenovirus, herpesvirus, or papillomavirus) for the productive AAV life cycle.

[0045] The recombinant double transfection vectors of the invention described herein improve upon previous systems by inserting both a GOI cassette and a rep-cap expression cassette into a single plasmid to generate a double transfection vector (e.g., a plasmid) of the invention that can be used in double transfections with a helper plasmid (e.g., the same helper plasmid used in conventional triple transfection methods).

[0046] We surprisingly found that with suitable (e.g., identical / equimolar) ratios of Rep, Cap, and helper genes, the dual transfection system of the present invention produced rAAV viral particles at levels of approximately 200-300% compared to the conventional triple transfection system used in the commercial production of rAAV viral particles for decades.

[0047] Furthermore, in some embodiments, AAV production using the dual vector / plasmid system of the present invention results in a reduced frequency of mispackaging. Exemplary such embodiments for rDNA vectors of the present invention include the configuration shown in Figure 1A.

[0048] Thus, one aspect of the present invention provides a vector comprising: (a) a gene of interest (GOI) flanked by adeno-associated virus (AAV) ITRs; (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) that is compatible with the ITRs, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap, wherein the vector is not a herpes simplex virus (HSV) vector.

[0049] Another aspect of the invention is a recombinant DNA vector (rDNA) comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (e.g., AAV2 5' ITR) and 3' ITR sequences (e.g., AAV2 3' ITR); (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally wherein the coding sequence for AAV Rep is under transcriptional control of the AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap, wherein expression of AAV Rep in a host cell containing AAV helper genes is facilitated by the AAV A recombinant DNA vector (rDNA) is provided that is sufficient to package an AAV vector genome (vg) containing a GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid containing Cap, with the proviso that the recombinant DNA vector is not a herpes simplex virus (HSV) vector.

[0050] In certain embodiments, the vector or recombinant DNA vector is a plasmid.

[0051] In certain embodiments, the vector is suitable for use in mammalian cells. Vectors suitable for use in mammalian cells have been widely described and are well known in the art. Those skilled in the art will understand that the vectors of the present invention may also contain various additional sequences and elements useful for replicating the vector in prokaryotic and / or eukaryotic cells, selecting the vector, and expressing the sequence in various host cells. For example, vectors of the present disclosure may include a prokaryotic replicon (i.e., a sequence capable of directing autonomous replication and maintenance of the vector extrachromosomally in a prokaryotic host cell, e.g., a bacterial host cell). Such replicons are well known in the art. In some embodiments, the vector may include shuttle elements that generate a vector suitable for replication and integration in both prokaryotes and eukaryotes. The vector may also include a gene that confers a detectable marker, such as a drug resistance gene, whose expression allows for selection and maintenance of the host cell. The vector may also have a reportable marker, e.g., a gene encoding a fluorescent or other detectable protein.

[0052] The vectors of the present invention may also contain transcription enhancers, translation signals, and transcription and translation termination signals. Examples of transcription 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) sequence, and any variants thereof.

[0053] In certain embodiments, a double-transfection recombinant DNA vector of the invention (double-transfection (DT) vector) comprises a gene of interest (GOI) flanked by 5' and 3' adeno-associated virus (AAV, e.g., AAV2) ITRs, a coding sequence for AAV Rep (e.g., AAV2 Rep) compatible with the AAV ITRs, and a coding sequence for AAV Cap (e.g., AAV9 or a derivative thereof). In host cells containing AAV helper genes (which can be introduced into host cells by any art-recognized means, e.g., infection of host cells with adenovirus or transfection of a helper plasmid containing the necessary genes for AAV packaging), expression of AAV Rep and Cap proteins from the double-transfection vector is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the 5' and 3' AAV ITRs.

[0054] In certain embodiments, the recombinant DNA vector is a plasmid that may be suitable for transfection into an AAV packaging cell line, such as Expi, HEK293 (e.g., HEK293T cells), or other commonly used AAV packaging cell lines.

[0055] In a related aspect, the invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by (AAV) ITRs; (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) that is compatible with the (AAV) ITRs, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3′ to the GOI; and (c) a coding sequence for AAV Cap.

[0056] In another related aspect, the invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (e.g., AAV2 5' ITR) and 3' ITR sequences (e.g., AAV2 3' ITR); (b) a coding sequence for AAV Rep (e.g., AAV2 Rep, or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter located downstream or 3' to the GOI; and (c) a coding sequence for AAV Cap, wherein expression of the AAV Rep in a host cell containing 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.

[0057] In some embodiments, the plasmids of the present invention do not include polynucleotide sequences encoding helper genes.

[0058] In yet another aspect, the present invention provides a two-plasmid system comprising a vector plasmid and a helper plasmid, wherein the vector plasmid comprises a GOI, a rep gene encoding a functional Rep protein, and a cap gene encoding a functional Cap protein.

[0059] As used herein, the term "plasmid" includes a nucleic acid molecule that can replicate independently of a cellular chromosome. The term "plasmid" is intended to include circular and linear nucleic acid molecules. Additionally, the term "plasmid" is intended to include bacterial plasmids, cosmids, minicircles (Nehlsen et al., 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, a plasmid is a circular nucleic acid (DNA) molecule. In certain embodiments, a plasmid is a nucleic acid molecule that is of bacterial origin.

[0060] In some embodiments, the GOI is in a pro-AAV cassette that comprises the GOI operably linked to a promoter.

[0061] In some embodiments, the rDNA vector contains two or more copies of a pro-AAV cassette. In some embodiments, all copies of the pro-AAV cassette contain the same GOI. In some embodiments, at least two of the pro-AAV cassettes contain different GOIs. The latter embodiment can be useful, for example, when an AAV vector is used to deliver different parts of the same functional assembly, such as coding sequences for a CRISPR / Cas effector enzyme and one or more guide RNAs.

[0062] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that facilitates transcription of the GOI from the promoter; (2) a 5'UTR; (3) a Kozak sequence; (4) a heterologous intron that facilitates transcription and / or translation of the GOI; (5) a 3'UTR; (6) a WPRE sequence; and / or (7) a polyA signal sequence.

[0063] In some embodiments, the coding sequence for AAV Rep and the coding sequence for AAV Cap are in a RepCap cassette comprising an operably linked RepCap promoter, in certain embodiments, the operably linked RepCap promoter comprises the AAV P5 promoter.

[0064] In some embodiments, the RepCap cassette and the pro-AAV cassette are immediately adjacent to each other (e.g., substantially free of intervening polynucleotide sequences).

[0065] In some other embodiments, the RepCap cassette and the pro-AAV cassette are not immediately adjacent to each other.

[0066] In some embodiments, the RepCap cassette and the pro-AAV cassette have the same direction of transcription.

[0067] In some other embodiments, the RepCap cassette and the pro-AAV cassette have opposite transcriptional directions.

[0068] In some embodiments, the pro-AAV cassette is upstream of the RepCap cassette.

[0069] In some other embodiments, the pro-AAV cassette is downstream of the RepCap cassette.

[0070] In some embodiments, the GOI precedes the coding sequence for AAV Rep. In some embodiments, the GOI precedes the coding sequence for AAV Cap. In some embodiments, the GOI precedes the coding sequence for both AAV Rep and AAV Cap.

[0071] In certain embodiments, the coding sequence for AAV Rep is under the transcriptional control of the AAV P5 promoter, which is located downstream or 3' to the GOI.

[0072] In some embodiments, expression of AAV Rep in a host cell containing AAV helper genes is sufficient to package an AAV vector genome (vg) containing a GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid containing AAV Cap.

[0073] In some embodiments, the recombinant DNA vector (double transfection vector) further comprises a bacterial replication Ori gene, a selectable marker promoter, e.g., a selectable marker (e.g., an antibiotic resistance gene, e.g., KanR or AmpR) under the transcriptional control of a bacterial promoter.

[0074] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0075] In certain embodiments, the GOI includes a gene causing / 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 2I), or a gene encoding Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB). IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (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.

[0076] In some embodiments, the GOI is the microdystrophin gene (e.g., as described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO2023 / 018854, or US10,166,272 (each of which is incorporated herein by reference in its entirety). In some embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (incorporated herein by reference).

[0077] In certain embodiments, the microdystrophin gene comprises coding sequences for the R16 and R17 spectrin-like repeats (e.g., as described in US 7,892,824) for a full-length dystrophin protein.

[0078] In some embodiments, the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats of a full-length dystrophin protein (e.g., the microdystrophin gene described in PCT / US2016 / 013733).

[0079] In some embodiments, the 5' and 3' 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.

[0080] In certain embodiments, the tropism of the AAV includes skeletal muscle (e.g., AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9).

[0081] In certain embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from the same or different AAVs.

[0082] In certain embodiments, the AAV ITRs are AAV2 ITRs, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or a derivative thereof (e.g., a spectral or transcriptional derivative thereof).

[0083] In certain embodiments, the coding sequences for the AAV Rep and Cap proteins are under the transcriptional control of a promoter, such as the AAV p5 promoter, a modified p5 promoter lacking the RBE (Rep binding element), the HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (e.g., a CMV promoter, an EF1a promoter, a CAG promoter, a CB promoter, etc.).

[0084] In some 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-Yang1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site. In some embodiments, the spacer is 5 nucleotides to 100 nucleotides (e.g., about 5 nt) in length.

[0085] In some embodiments, the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized Ying-Yang1 (YY1) binding site, wherein the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides (e.g., about 5 nt) in length.

[0086] In certain embodiments, the AAV helper genes comprise adenovirus, herpesvirus, or papillomavirus viral genes useful for AAV packaging (e.g., E1A, E1B, E2A, E4, and VA RNAs), optionally operably linked to a promoter as a single transcription unit.

[0087] Another aspect of the present invention provides a composition or kit comprising the recombinant DNA vector of the present invention and a helper plasmid.

[0088] In certain embodiments, the helper plasmid contains helper virus genes sufficient to support AAV packaging.

[0089] In certain embodiments, the helper virus genes include (i) adenovirus genes, optionally adenovirus 5 or adenovirus 2 genes; 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.

[0090] Another aspect of the present invention provides a host cell comprising a recombinant DNA vector of the present invention, or a composition of the present invention.

[0091] In certain embodiments, the host cell is a HEK293 cell (e.g., an Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

[0092] In another aspect, the present invention provides a method for propagating, amplifying, or generating recombinant, replication-deficient AAV viral particles harboring a GOI, comprising introducing a recombinant DNA vector (e.g., in a plasmid) of the present invention (double transfection vector) into a host cell before, simultaneously with, or after introducing AAV helper genes (e.g., in a plasmid) into the host cell.

[0093] In some embodiments, the dual vector and AAV helper genes on two separate plasmids are co-transfected into host cells, thereby generating recombinant, replication-deficient AAV viral particles harboring the GOI.

[0094] In certain embodiments, the method further comprises recovering the recombinant, replication-defective AAV viral particles from the host cells.

[0095] In certain embodiments, the host cell is a HEK293 cell (e.g., an Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

[0096] In certain embodiments, the recombinant DNA vector is introduced into the host cell by transient transfection.

[0097] With the general principles of the invention set forth herein, the following sections provide further detailed descriptions for various aspects of the invention. It should be understood that any embodiment of the invention may be combined with any one or more additional embodiments of the invention, including those embodiments described in different sections of this application and those described only in the examples, drawings, or claims.

[0098] Furthermore, all disclosures regarding "recombinant DNA vectors" are also applicable to "vectors" and "plasmids" described herein.

[0099] 2. Rep and Cap genes In certain embodiments, the coding sequences for the AAV Rep and Cap proteins and the gene of interest (GOI) flanked by AAV ITR sequences are incorporated into a single double transfection (DT) vector of the invention, e.g., a DT plasmid.

[0100] While Rep proteins are important for replication and packaging, capsid proteins assemble to generate the protein shell of AAV, i.e., the AAV capsid, which forms the outer capsid shell that protects the viral genome and also actively participates in cell binding and internalization. Alternative splicing and alternative start 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. Without wishing to be bound by theory, AAV capsid proteins typically contain a 1:1:10 molar ratio of VP1:VP2:VP3. As used herein, "AAV serotype" is primarily defined by the AAV capsid. In some instances, the ITRs are also specifically represented by the AAV serotype.

[0101] In certain embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from the same or different AAV serotypes.

[0102] In some embodiments, AAV ITR, AAV Rep, and AAV Capは、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-G GT、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-QGT、AAVPHP.B-NQT、AAVPHP.B-QGT P.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、AAVG2A 15 / G2A3(G2A3)、AAVG2B4(G2B4)、AAVG2B5(G2B5)、PHP.S、AAV1、AAV2、AAV2G9、AAV3、AAV3a、AAV3 b、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、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-1 5、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、AA V223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.61 .9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10 AV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、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、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、A AV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAV3、AAV5、AAV2 rh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、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-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh. R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AA Vhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、AAVhu.2 AVhu.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.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、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、AA Vhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.1 8、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. rh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R. A586R mutation、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr r2.29、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-L K04、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-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM 10-2、AAVシャッフル100-1、AAVシャッフル100-3、AAVシャッフル100-7、AAVシャッフル10-6、AAVシャッフル10-8、AAV SM 10-1、AAV SM 10-8、AAV SM 100-3 100-10、BNP61 AAV、BNP62 AAV、BNP63 AAV、AAVrh.50、AAVrh.43、AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, 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 AAV10, Japanese AAV10 serotype, 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-E1、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 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-P1、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-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、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-F1、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 CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11. , AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, 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, and / or AAVF9 / HSC9 and variants thereof.

[0103] In some embodiments, the AAV Cap is a derivative of wild-type AAV9. In some embodiments, the derivative comprises an insertion of a short peptide (e.g., 3, 4, 5, 6, 7, 8, or 9 residues) between residues 588 and 589 of wild-type AAV9 capsid VP1. In some embodiments, the insertion comprises, consists essentially of, or consists of RGDLGLS into residues 588 and 589 of wild-type AAV9 capsid VP1.

[0104] In certain embodiments, the rDNA vectors / plasmids of the present invention comprise a cap gene promoter. The cap gene promoter can be operably linked to a cap gene. In certain embodiments, the cap gene promoter is a native cap gene promoter.

[0105] The native cap gene (i.e., the cap gene of wild-type AAV) is operably linked to the p40 promoter, p5 promoter, and p19 promoter. Thus, in one embodiment, an rDNA vector / plasmid of the invention comprises a cap gene promoter, e.g., an AAV p40 promoter, p5 promoter, and / or p19 promoter.

[0106] The native p40 promoter is contained in the native rep gene. In some embodiments, the p40 promoter has a sequence that is at least 95%, at least 98%, or 99% identical to that of AAV2. In some embodiments, at least one cap gene promoter is contained in a promoter region that includes the p40 promoter, the p5 promoter, and the p19 promoter.

[0107] The native p5 promoter is upstream of the native rep gene. In some embodiments, the p5 promoter has a sequence that is at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0108] In certain embodiments, a wild-type or native p5 promoter is modified to generate 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-Yang1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises a REP binding site and a transcription start site-localized Ying-Yang1 (YY1) binding site with an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides (e.g., about 5 nt) in length.

[0109] Without being bound by any particular theory, the insertion of a 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 the reduction or elimination of mispackaging of vector / plasmid DNA upstream of the P5 promoter while maintaining recombinant AAV titer.

[0110] In certain embodiments, the spacer comprises an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site. In certain embodiments, excluding the exogenous spacer, the modified P5 promoter has 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 the wild-type P5 promoter, or a length within any range delimited by any pair of the foregoing values, such as 80%-99%, 90%-99%, or 95%-99%, etc. In certain embodiments, the modified P5 promoter into which the exogenous spacer has been inserted comprises, for example, a sequence of nucleotides corresponding to at least about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 nucleotides adjacent to (upstream of) the transcription start site of the large REP proteins (i.e., REP78 and REP68) in the AAV genome, or any range of lengths delimited by any pair of the foregoing values, e.g., 40-50, 50-160, or 80-130, or The promoter has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or more sequence identity, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or more (inclusive), and the promoter directs transcription of the major REP proteins, i.e., REP78 and REP68.

[0111] In some examples, the modified P5 promoter is selected from AAV serotype 1 (AAVl), 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 the following AAV serotypes: AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, 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.38, AAVrh.39, AAVrh.40, AAVrh.41, AAVrh.42, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.63, AAVrh.64, AAVrh.65, AAVrh.66, AAVrh.67, rh.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, A The sequence is derived from the sequence of the region adjacent to the transcription start site of EP78 and REP68 in any of AVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, and AAVrh8R R533A mutant.

[0112] In certain embodiments, the modified P5 promoter is located within an AAV genome, e.g., AAVl, AAV2, AAV3, AAV4, AAV6, AAV7, AAV8, AAV9, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, 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, AAVr h.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.46, A AVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AA Vrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, A AVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R It comprises or consists of a sequence corresponding to approximately 160 nucleotides adjacent to the transcription start sites of REP78 and REP68 in the AAVrh8R A586R mutant or AAVrh8R R533A mutant genome.

[0113] As used herein, the terms "REP binding sequence," "REP binding site," or "REP binding element" are used interchangeably herein to refer to an element of the P5 promoter consisting of two to four imperfect GAGC repeats located immediately downstream of the TATA box, i.e., 3' 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 includes GCCCGAGTGAGCACGC.

[0114] As used herein, "Ying-Yang1 binding site," "YY1 binding site," or "YY1 box" refers to a YY1 recognition sequence that, when bound by YY1, supports initiation from the transcription start site. The +1 YY1 binding site to the P5 promoter is located at the transcription start site and has the consensus sequence CCAT.

[0115] As used herein, the term "exogenous spacer sequence" refers to a sequence that is not native to the P5 promoter.

[0116] The exogenous spacer sequence can be inserted anywhere between the REP binding site and the YY1 binding site described herein.In some embodiments, the exogenous spacer sequence is inserted into or replaces one or more of the 7 nucleotides located between the REP binding site and the YY1 binding site.For example, the exogenous spacer sequence can be inserted into or replace one or more nucleotides of the AGGGTCT sequence of the AAV8 P5 promoter.

[0117] 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 any range of lengths bounded by any pair of the foregoing values, such as 5-120, 10-100, or 5-100. Ideally, the exogenous spacer is 5-100 nucleotides in length. As will be understood by those skilled in the art, the sequence of the spacer is not critical. The sequence of the spacer can be random, artificial, derived from AAV, or derived from another source.

[0118] The p19 promoter is contained in the native rep gene. In some embodiments, the p19 promoter has a sequence that is at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0119] In some embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from the same AAV. In some other embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from different AAVs. For example, the AAV Rep can be from AAV2, and the AAV Cap can be from AAV9 or a derivative thereof, such as the wtAAV9 derivatives described above (including SLB-101) (see SEQ ID NO: 14 and FIG. 6 of WO2021 / 072197, which is incorporated herein by reference).

[0120] In certain embodiments, the AAV ITRs are AAV2 ITRs, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0121] In certain embodiments, the coding sequence for the AAV Rep proteins encodes wild-type Rep40, Rep52, Rep68, and / or Rep78 of AAV, e.g., AAV2. In certain embodiments, these Rep proteins are transcribed from one or more of the rep promoters p5, p19, and / or p40.

[0122] The rep gene encodes at least one functional Rep protein (Rep78, Rep68, Rep52, and Rep40). The gene region is under the control of the p5 and p19 promoters. When the p5 promoter is used, sequences encoding Rep78 and Rep68 are transcribed. Rep78 and Rep68 are two alternative splice variants (Rep78 contains an intron excised in Rep68). Similarly, when the p19 promoter is used, sequences encoding Rep52 and Rep40 are transcribed. Rep52 and Rep40 are alternative splice variants (Rep52 contains an intron excised in Rep40). The four Rep proteins are known to be involved in viral genome replication and packaging and are therefore useful in rAAV production.

[0123] In certain embodiments, all four Rep proteins need not be present. In certain embodiments, at least one encoded large Rep protein (Rep78 or Rep68) and one encoded small Rep protein (Rep52 or Rep40) are present. Rep78 can be toxic to cells, and Rep78 does not need to be present for AAV replication to occur. Thus, in some embodiments, Rep78 is absent. In some embodiments, the rep gene does not encode or transcribe Rep78. In certain embodiments, the rep gene encodes / transcribes Rep68.

[0124] In certain embodiments, the rDNA vectors / plasmids of the present invention comprise at least one rep gene encoding (a) a functional Rep52 protein; (b) a functional Rep40 protein; and / or (c) a functional Rep68 protein.

[0125] As used herein, a "functional" Rep protein is one that allows for the production of AAV particles. In particular, Rep78 or Rep68 (large Rep proteins) are thought to be involved in replication of the AAV genome, and Rep52 and Rep40 (small Rep proteins) are thought to be involved in packaging the AAV genome into capsids. One skilled in the art can readily determine whether a given Rep protein is functional by, for example, determining whether the Rep protein supports AAV production using an AAV production assay such as those described above.

[0126] In some embodiments, at least one rep gene encodes a "functional" Rep protein if the Rep protein supports rAAV production at, for example, 25% or at least 25%, 40% or at least 40%, 50% or at least 50%, 70% or at least 70%, 80% or at least 80%, 90% or at least 90%, 95% or at least 95% of the level supported by wild-type Rep protein, or at any level within a range delimited by any pair of the foregoing values, e.g., 25%-95%, 40%-90%, or 25%-70%.

[0127] In certain embodiments, the Rep proteins are compatible with the ITR(s) surrounding the GOI. Some Rep proteins may only be able to package genomic material (e.g., an expression cassette) if it is flanked by ITR(s) of the same serotype as the Rep protein. Other Rep proteins may be cross-compatible and package genomic material flanked by ITR(s) of a different serotype.

[0128] In certain embodiments, the rDNA vectors / plasmids of the invention contain two or more rep genes separated on the vector / plasmid. For example, one of the separate rep genes can encode Rep68 (e.g., using the p5 promoter or a different promoter located near the normal position of the p5 promoter in the rep gene), and another can encode Rep40 (e.g., using the p19 promoter or a different promoter located near the normal position of the p19 promoter). One rep gene that encodes Rep40 can also encode Rep52, since Rep52 and Rep40 are alternative splice variants.

[0129] In certain embodiments, when two genes encode Rep40 proteins, one of the two genes encoding the functional Rep40 protein may contain an intron. In one embodiment, both genes encoding the functional Rep40 protein contain an intron. In another embodiment, only one of the genes encoding the functional Rep40 protein contains an intron.

[0130] For example, to avoid at least one rep gene encoding Rep78, the rep gene could be split via partial duplication into two genes. One gene could contain nucleotides corresponding to the full-length native rep gene with sequences corresponding to the removed intron. Because portions of each of the Rep78 and Rep52 proteins are encoded by sequences that act as introns in the context of rep40, such a gene would encode Rep68 and Rep40 but not either Rep78 or Rep52. The second gene could contain nucleotides corresponding to the region of the native rep gene downstream of the p19 promoter that would encode Rep52 (intron spliced ​​in) and Rep40 (intron spliced ​​out).

[0131] In some embodiments, at least one rep gene does not contain a functional internal p40 promoter. The native rep / cap gene contains the 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 the rep gene. A functional p40 promoter is one that is capable of driving expression of the cap gene.

[0132] In certain embodiments, the coding sequences for the AAV Rep and Cap proteins are under the transcriptional control of a promoter, such as the AAV p5, p19, or p40 promoter, a modified p5 promoter lacking the RBE (Rep binding element), or a ubiquitous promoter (e.g., a CMV promoter, an EF1a promoter, a CAG promoter, a CB promoter, etc.).

[0133] In any of the above embodiments, either the rep gene or the cap gene, or both, may be replaced in the rDNA vectors of the present invention by a cloning site (e.g., a multiple cloning site or MCS), such that suitable rep and / or cap genes can be cloned / inserted into the respective cloning sites. This will facilitate the replacement of any desired rep or cap gene into the rDNA vectors / plasmids of the present invention.

[0134] 3. Genes of interest (GOI) and treatable diseases in rAAV The systems and methods of the present invention can be used to generate recombinant AAV vectors carrying a gene of interest (GOI) flanked by AAV ITR sequences.

[0135] In certain embodiments, the rDNA vectors (plasmids) of the invention comprise ITR sequences derived from AAV1, AAV2, AAV4, and / or AAV6, hi certain embodiments, the ITR sequences comprise AAV2 ITR sequences.

[0136] As used herein, "gene of interest" or GOI or goi typically refers to a nucleic acid or polynucleotide sequence, e.g., a gene, an open reading frame (ORF), or a coding sequence for a protein or RNA, e.g., a non-coding RNA (including siRNA, miRNA, shRNA, antisense RNA, or precursors thereof). However, in certain situations or contexts, the term GOI also loosely refers to a protein (encoded by the GOI), or a disease or indication that can be repaired by the GOI, or a disease or indication that can be (but is not necessarily) caused by loss of function of the GOI.

[0137] For example, and by way of example only, the gene GALGT2 encodes the protein GalNAc transferase (β-1,4-N-acetylgalactosamine galactosyltransferase), an enzyme that transfers complex sugar molecules to several specific proteins, including dystroglycan. Under normal circumstances, GalNAc transferase is found only at the neuromuscular junction (NMJ), and several components of the dystroglycan-associated protein complex are distinct from other locations in muscle. Importantly, at the NMJ, utrophin is present in place of dystrophin. In the mdx mouse model of muscular dystrophy, viral gene transfer of GALGT2 results in expression of GalNAc transferase throughout the muscle membrane and upregulation of utrophin throughout muscle fibers, instead of solely at the normal expression domain of the NMJ. In mdx mice, this expression can correct muscle functional deficits to the same extent as microdystrophin gene expression. Furthermore, overexpression of GALGT2 corrects muscle pathology in mouse models of other muscular dystrophies, including LGMD2A and congenital muscular dystrophy (MDC1A). Thus, GALGT2 is a GOI for treating muscular dystrophies such as DMD, BMD, LGMD2A, and MDC1A, although GALGT2 is not necessarily defective per se in patients in need of treatment.

[0138] In another example, sarcolipin (SLN) binds to sarcoplasmic / endoplasmic reticulum (SR) Ca. 2+ It inhibits the ATPase (SERCA) and is abnormally elevated in the muscles of DMD patients and animal models, such as the mdx mouse model of DMD. Reducing SLN levels by AAV9-mediated RNA interference has been shown to improve muscle pathology and diaphragm, skeletal muscle, and cardiac function in severe dystrophin / utrophin double mutant (mdx:utr) DMD patients. - / - ) ameliorates dystrophic pathology in mouse models. Thus, the coding sequence for SLN RNAi is a GOI that repairs DMD.

[0139] Thus, a GOI can be a gene (or protein) that, when expressed, replaces a mutated, damaged, or inactive gene or protein. A GOI can be a gene (or protein) that, when expressed, assists an already functioning process that may benefit from further modification for therapy in a disease, disorder, or dysfunction. A GOI can be a gene (or protein) that, when expressed, assists a dysfunctional process that may benefit from further modification for therapy in a disease, disorder, or dysfunction. The GOI nucleic acid sequence can be a DNA, RNA, or synthetic nucleic acid molecule. A GOI can be a protein, enzyme, structural protein, functional protein, or protein adaptable based on cellular function(s). A GOI can provide a therapeutic benefit or treatment modality for a disease, disorder, or dysfunction.

[0140] In certain embodiments, a recombinant DNA vector of the invention comprises multiple copies of a GOI. In some embodiments, a plasmid of the invention comprises multiple copies of a GOI. In certain embodiments, the multiple GOI copies are the same. In some other embodiments, the multiple GOIs are different.

[0141] In certain other embodiments, the GOI may be a CRISPR / Cas effector enzyme, such as a class 2, II, IV, V, or type VI effector enzyme (including CRISPR-Cas9, Cas12, Cas13, etc.). In certain embodiments, the GOI may be a TALEN or other gene-based gene editing protein that functions after intracellular delivery for their intended activity, such as gene editing or gene knockout, in a target cell, tissue, or organism / individual.

[0142] In certain embodiments, the CRISPR / Cas effector enzyme lacks endonuclease activity (dCas, e.g., dCas9).

[0143] In some embodiments, the Cas or dCas is further fused to a base editor, e.g., a cytosine base editor (CBE, e.g., APOBEC, BE1, BE2, BE3, targeted-AID base editor, SaBE3, BE3 PAM variants, BE3 editing window variants, AID, CDA1, APOBEC3G, HF-BE3, BE4, BE4max, and AncBE4max), an adenine base editor (ABE, e.g., ABE7.10, ABE6.3, ABE7.8, ABE7.9, ABEmax, ABE8e (TadA-8e V106W), ABE8 and its variants), or a dual base editor (SPACE, A&C-BEmax).

[0144] Any GOI, as used herein, can benefit from codon optimization for improved expression and activity through known computer-based algorithms or other codon optimization methods, e.g., manual optimization.

[0145] In certain embodiments, the GOI is in a pro-AAV cassette that comprises the GOI operably linked to a promoter.

[0146] In some embodiments, the promoter operably linked to the GOI is a ubiquitous promoter that drives or promotes expression of the GOI in most tissues, such as the elongation factor 1 α-subunit (EF1α) promoter, the cytomegalovirus (CMV) immediate-early enhancer promoter, the chicken β-actin (CBA) and its derivative CAG promoter, the β-glucuronidase (GUSB) promoter, or the ubiquitin C (UBC) promoter.

[0147] In some other embodiments, the promoter operably linked to the GOI is a tissue-specific promoter that can be used to restrict expression to a given cell type, such as, but not limited to, a muscle-specific promoter, a B-cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter (which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes).

[0148] Non-limiting examples of muscle-specific promoters include the mammalian muscle creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the mammalian troponin I (TNNI2) promoter, and the mammalian skeletal alpha-actin (ASKA) promoter.

[0149] In some embodiments, the pro-AAV cassette may further comprise an enhancer that promotes 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.

[0150] In certain embodiments, the pro-AAV cassette comprises transcriptional regulatory elements including promoter and / or enhancer elements from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and / or LSP. These transcriptional 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 et al., Human Gene Therapy 7:637-645, 1996; and LSP: Wang et al., Proc Natl Acad Sci US A. 96(7):3906-3910, 1999, all of which are incorporated herein by reference. Each of these transcriptional regulatory elements can include a promoter, an enhancer, and / or optionally other nucleotides.

[0151] In certain embodiments, a plasmid of the invention comprises a promoter region comprising one or more promoters, wherein the promoter region does not contain non-essential translation initiation codons (e.g., ATG or GTG codons) to prevent unintended / undesired translation initiation. In certain embodiments, the promoter region comprises the p5, p19, and / or p40 promoters, wherein the ATG or GTG codon at one or more positions within these promoters is absent or mutated.

[0152] The GOI can be any gene or coding sequence within the packaging capacity of an AAV, for example, about 4-5 kb, or about 4.7 kb (including ITR sequences), or about 4.4 kb (excluding ITR sequences).

[0153] 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, both AAV ITR sequences flanking the GOI are from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0154] In some embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from the same AAV. In some other embodiments, the AAV ITRs, AAV Rep, and AAV Cap are from different AAVs.

[0155] In certain embodiments, the AAV ITRs are AAV2 ITRs, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0156] In certain embodiments, the tropism of the AAV includes skeletal muscle (e.g., AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9, or a derivative thereof, e.g., SLB-101 (supra)).

[0157] In certain embodiments, an rAAV that can be produced by using a DT vector of the invention (e.g., a DT plasmid) and a suitable host cell (which can supply any useful helper functions for rAAV production in trans) can encode a gene of interest (GOI) that is useful, for example, for gene therapy to treat a disease or condition.

[0158] Representative (but non-limiting) genes of interest (GOI) include genes causing / 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 2I), or genes encoding Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB). IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (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.

[0159] In certain embodiments, the GOI is the microdystrophin gene.

[0160] In some embodiments, the micro-dystrophin gene is any of those described in the following patents: US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO2023 / 018854, or US10,166,272 (each of which is incorporated herein by reference in its entirety). In some embodiments, the GOI is a micro-dystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (incorporated herein by reference). In some embodiments, the micro-dystrophin gene can be packaged into rAAV virions, for example, with a size of about 4.7 kb or less.

[0161] 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 (e.g., as described in U.S. Pat. No. 7,892,824).

[0162] In some embodiments, the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats (i.e., SR1, SR16, SR17, SR23, and SR24, respectively) of a full-length dystrophin protein, such as those described in PCT / US2016 / 013733 (incorporated herein by reference). In some embodiments, the microdystrophin gene does not encode any other spectrin repeats of a full-length dystrophin protein other than SR1, SR16, SR17, SR23, and SR24.

[0163] Diseases or conditions that have the potential to benefit from rAAVs generated by the dual transfection vectors of the present invention include Huntington's disease, X-linked myotubular myopathy (XLM™), acid maltase deficiency (e.g., Pompe disease), spinal muscular atrophy (SMA), myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, mitochondrial myopathies, muscular dystrophies (Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy, and others). Generalized muscular dystrophy (LGMD), facioscapulopuscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), peripheral muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), mucopolysaccharidosis (MPS), metachromatic leukodystrophy (MLD), Batten disease, Rett syndrome, Krabbe disease, Canavan disease, X-linked retinoschisis, color vision deficiency (CNGB3 and CNGA3), X-linked retinitis pigmentosa, age-related macular degeneration, neovascular macular degeneration, Pompe disease, Fabry disease, MPS These include I, II, IIIA, IIIB, Gaucher disease, Danon disease, A1At deficiency, Friedreich's ataxia, Wilson disease, Batten disease (CLN1, CLN3, CLN6, CLN8), Wolman disease, Tay-Sachs, Niemann-Lick type C, CDKL5 deficiency disorders, B-thalassemia, and sickle cell disease.

[0164] In certain embodiments, diseases or conditions that have the potential to benefit from rAAVs generated by the dual transfection vectors of the invention may include Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Bethlem CMD, Fukuyama CMD, muscle-eye-brain disease (MEB), ankylosing spine syndrome, Ullrich CMD, Walker-Warburg syndrome (WWS), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), facioscapulo-peduncular muscular dystrophy (FSHD), limb-girdle muscular dystrophy (LGMD), myotonic dystrophy (DM), oculopharyngeal muscular dystrophy (OPMD), motor neuron diseases including ALS (amyotrophic lateral sclerosis), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA).

[0165] In certain embodiments, diseases or conditions that could potentially benefit from rAAV generated by the dual transfection vectors of the invention may include ion channelopathies, which are typically characterized by muscle weakness, lack of muscle tone, or transient paroxysmal muscle paralysis, including Andersen-Tawil syndrome, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, myotonia congenita, Becker myotonia, Thomsen myotonia, paramyotonia congenita, and potassium-weighted myotonia.

[0166] In certain embodiments, diseases or conditions that could potentially benefit from rAAV generated by the dual transfection vectors of the present invention may include mitochondrial diseases that occur when structures that generate energy for cells are malfunctioning, including Friedreich's ataxia (FA), mitochondrial myopathy, Kearns-Sayre syndrome (KSS), Leigh syndrome (subacute necrotizing encephalomyopathies), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathies, lactic acidosis, and stroke-like episodes (MELAS), mitochondrial neurogastrointestinal encephalomyopathies (MNGIE), myoclonic epilepsy with ragged-red fibers (MERRF), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, and progressive external ophthalmoplegia (PEO).

[0167] In certain embodiments, diseases or conditions that could potentially benefit from rAAVs produced by the dual transfection vectors of the invention may include myopathies, which are muscle disorders in which muscle fibers do not function properly, resulting in muscle weakness. Myopathies include cap myopathy, central core myopathy, congenital myopathy with fiber type imbalance, core myopathy, central core disease, multiminicore myopathy, myosin storage myopathy, myotubular myopathy, nemaline myopathy, distal myopathy, GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Layne distal myopathy, Marcus Berry-Griggs late-onset distal myopathy, Miyoshi myopathy, Wood myopathy / tibial muscular dystrophy, vocal cord and pharyngeal distal myopathy, Welander distal myopathy, endocrine myopathy, and thyroid function myopathy. These include hyperactive myopathy, hypothyroid myopathy, inflammatory myopathy, dermatomyositis, inclusion body myositis, polymyositis, metabolic myopathy, acid maltase deficiency (AMD, Pompe disease), carnitine deficiency, carnitine palmityltransferase deficiency, debranching 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 myopathy (MFM), and scapuloperoneal myopathy.

[0168] In certain embodiments, diseases or conditions that could potentially benefit from rAAV generated by the dual transfection vectors of the present invention may include neuromuscular junction disorders resulting from the disruption, malfunction, or absence of one or more critical proteins involved in transmitting signals between muscles and nerves, including congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndromes (LEMS), and myasthenia gravis (MG).

[0169] In certain embodiments, diseases or conditions that could potentially benefit from rAAV produced by the dual transfection vectors of the invention may include peripheral nerve diseases that affect the motor and sensory nerves that connect the brain and spinal cord to the rest of the body, causing impairment of sensation, movement, or other functions. Such diseases include Charcot-Marie-Tooth disease (CMT), giant axonal neuropathy (GAN), cachexia, and muscle atrophy in aging.

[0170] In certain embodiments, the GOI coding sequence comprises a polyA signal sequence or a polyadenylation site.

[0171] In certain embodiments, the polyadenylation site is the bovine growth hormone (bGH) polyadenylation site.

[0172] In certain embodiments, the polyadenylation site or poly(A) signal sequence is from another suitable source, for example, a synthetic sequence(s) from another eukaryotic gene or virus.

[0173] In certain embodiments, the GOI coding sequence is partially or fully codon-optimized for expression in a mammalian host cell, for example, the 3'-most 300-350 nucleotides of the coding sequence may be codon-optimized for expression in a mammalian host cell.

[0174] 4. Compositions or kits of two-vector (e.g., two-plasmid) systems One embodiment of the present invention provides a composition or kit comprising a two-vector (e.g., two-plasmid) system comprising, consisting essentially of, or consisting of any one of the dual recombinant DNA vectors (dual transfection vectors or "DT vectors") of the present invention and a helper plasmid sufficient to support AAV packaging in the presence of the DT vector of the present invention.

[0175] The two-vector (two-plasmid) system in the composition or kit of the present invention is useful for producing rAAV. The two-vector (two-plasmid) system in the composition or kit of the present invention is suitable for use in producing rAAV. The two-vector (two-plasmid) system in the composition or kit of the present invention is for producing rAAV. The two-vector (two-plasmid) system in the composition or kit of the present invention is for producing rAAV suitable for use in gene therapy. The two-vector (two-plasmid) system in the composition or kit of the present invention is for producing rAAV for use in gene therapy.

[0176] The phrase "two-vector (two-plasmid) system" refers to a system that includes two vectors (e.g., plasmids) and can be used together to generate rAAV without the need for additional plasmids (i.e., they are sufficient in combination for AAV production). In certain embodiments, a two-vector (plasmid) system can be used to generate rAAV without the need for a helper virus, e.g., adenovirus. In certain embodiments, a two-vector (plasmid) system can be used to generate rAAV without the need for genetic material originating from a host cell, optionally with the exception of genes encoding El A / B. However, the vector system in a composition / kit of the invention can include additional non-plasmid components. Optionally, a composition or kit of the invention that includes a two-vector (plasmid) system of the invention does not include a helper virus.

[0177] In certain embodiments, a two-vector (plasmid) system of the invention contains all of the necessary genetic information for the production of an rAAV. For example, a two-vector (plasmid) system of the invention can contain at least one rep gene, at least one cap gene, and at least one helper gene. In certain embodiments, a two-plasmid system of the invention contains all of the necessary genetic information useful for the production of an rAAV suitable for use in gene therapy. For example, a two-plasmid system of the invention can contain an expression cassette comprising at least one rep gene, at least one cap gene, at least one helper gene, and a transgene operably linked to at least one regulatory control element.

[0178] One of the vectors / plasmids in the compositions or kits of the invention is a vector / plasmid containing AAV Rep, Cap and GOI (flanked by AAV ITR sequences), and the other vector / plasmid in the compositions or kits of the invention is a helper plasmid / vector.

[0179] In some embodiments, the helper plasmid / vector may contain at least one helper virus gene. AAV can only grow in the presence of a helper virus. Examples of helper viruses include adenovirus and herpesvirus.

[0180] In certain embodiments, the helper plasmids of the invention contain sufficient helper genes to enable AAV replication and packaging. In certain embodiments, the helper plasmids or vectors that can be used with the rDNA vectors of the invention provide all of the helper genes / functions for AAV packaging (except for the ITR sequences flanking the GOI).

[0181] For example, for host cells that express the adenovirus E1A / B genes, such as HEK293T cells, the remaining adenovirus helper genes encode E4, E2A, and VA RNAs I and II. These helper genes can be included in any order or orientation in the helper plasmid or vector, so long as all of these genes can be transcribed and expressed to facilitate AAV packaging.

[0182] Whether a helper plasmid contains sufficient helper genes to facilitate AAV production can be assessed using any art-recognized AAV production assay to demonstrate sufficient titer of the AAV so produced.

[0183] In one embodiment, helper gene products will be considered to facilitate (sufficient for) AAV production if they support rAAV production at a level that is at or at least 25%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or at least 95% of the level supported by adenoviral helper genes encoding, for example, E4, E2A, and VA RNA I and II, or at any level within a range delimited by any pair of the foregoing values, e.g., 50%-95%, 60%-80%, or 80%-95%. In some embodiments, the yield of the produced rAAV is, for example, 25% or at least 25%, 40% or at least 40%, 50% or at least 50%, 60% or at least 60%, 70% or at least 70%, 80% or at least 80%, 90% or at least 90%, 95% or at least 95%, or a level within any range delimited by any pair of the aforementioned values, e.g., 50%-95%, 60%-80%, or 80%-95%, or a level within any range delimited by any pair of the aforementioned values, e.g., 50%-95%, 60%-80%, or 80%-95%, of the yield of rAAV produced using a reference two-plasmid system (one of which is a helper plasmid containing adenoviral helper genes encoding E4, E2A, and VA RNA I and II).

[0184] The helper plasmid encodes genes that allow for efficient AAV production, so the addition of a helper virus is not required (i.e., the composition or kit of the invention comprising the two vectors / plasmids of the invention is sufficient to support AAV production and packaging).

[0185] In some embodiments, the at least one helper virus gene comprises an adenovirus gene. Adenovirus is a virus known to support the propagation of AAV (Xiao et al., J. Virol., 72:2224-2232, 1998). In some embodiments, the at least one helper virus gene comprises an adenovirus 5 gene or an adenovirus 2 gene. In some embodiments, the adenovirus helper genes encode E1A, E1B, E4, E2A, and VA RNA I and II.

[0186] E1A is encoded by nucleotides 560 to 1545 of the adenovirus 5 genome. E1A can optionally lack a non-essential intron at nucleotides 1113 to 1228.

[0187] E1B is actually two proteins, E1B 19K and E1B 55K, which act together to block apoptosis in adenovirus-infected cells. E1B is encoded by nucleotides 1714 to 2244 (E1B 19K) and by nucleotides 2019 to 3509 (E1B55K) of the adenovirus 5 genome.

[0188] E4 is encoded by multiple different open reading frames (ORFs) in the adenovirus 5 genome. E4 ORF 6 / 7 is encoded by nucleotides 32914 to 34077, which may optionally lack the intron between nucleotides 33193 and 33903. E4 34K is encoded by nucleotides 33193 to 34077 in the adenovirus 5 genome. E4 ORF 4 is encoded by nucleotides 33998 to 34342 in the adenovirus 5 genome. E4 ORF 3 is encoded by nucleotides 34353 to 34703 in the adenovirus 5 genome. E4 ORF B is encoded by nucleotides 34700 to 35092 in the adenovirus 5 genome. E4 ORF 1 is encoded by nucleotides 35140 to 35526 in the adenovirus 5 genome. Because only the amino acids encoded by ORFs 6 and 7 are essential for activity, a functional E4 protein can include only the amino acids encoded by ORFs 6 and 7. In certain embodiments, a functional E4 protein includes the polypeptide sequence encoded by all or a significant portion of ORFs 6 and 7. Optionally, a functional E4 protein does not include the polypeptide sequence encoded by all or a portion of ORFs 1-4 and 34K.

[0189] In certain embodiments, the amino acids encoded by ORFs 1-3 and 34K of E4 are not required but improve the activity of the E4 protein, and therefore a functional E4 protein includes amino acids encoded by ORFs 1-7.

[0190] The E2 (E2A) gene is encoded by nucleotides 22443 to 24032 of the adenovirus 5 genome.

[0191] VA RNA I and II are encoded by nucleotides 10589 to 11044 of the adenovirus 5 genome.

[0192] E1B and E4 are thought to improve AAV mRNA accumulation, while E2A and VA RNA I and II are thought to improve AAV mRNA splicing and translation. E1B, E4, and E2A are proteins encoded by genes present in the adenoviral genome, while 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 not translated into amino acid sequences. Thus, the VA nucleic acid does not encode a protein but "encodes" or "corresponds to" an RNA; i.e., while the term "encodes" is used, it will be understood that the VA nucleic acid is a nucleic acid sequence that is not translated.

[0193] Of the five adenoviral genes, in some embodiments, it is possible to omit E1A or E1B from the helper plasmid because some host cell lines (e.g., HEK293 cells) constitutively express one or more of E1A or E1B. In certain embodiments, the helper plasmid does not contain genes encoding functional adenoviral E1A / B proteins.

[0194] In one embodiment, the at least one helper virus gene includes (a) a VA (virus-associated) nucleic acid encoding functional VA RNA I and II; (b) an E2A gene encoding a functional E2A protein; and / or (c) an E4 gene encoding a functional E4 protein.

[0195] In certain embodiments, the at least one helper virus gene comprises a VA nucleic acid (eg, VA RNA I and / or II), an E2A gene, and an E4 gene.

[0196] "Functional" VA RNA I and II, E2A protein, or E4 protein can facilitate the production of AAV.

[0197] One skilled in the art can determine whether a given VA RNA I and II, E2A protein, or E4 protein is functional, for example, by determining whether the VA RNA I and II, E2A protein, or E4 protein supports AAV production using an AAV production assay such as those described above. In some embodiments, a VA RNA I and II, E2A protein, or E4 protein is considered functional if it supports rAAV production at, for example, 25% or at least 25%, 40% or at least 40%, 50% or at least 50%, 70% or at least 70%, 80% or at least 80%, 90% or at least 90%, 95% or at least 95%, or a level within any range delimited by any pair of the foregoing values, e.g., 50%-95%, 60%-80%, or 80%-95%, of the level supported by wild-type (e.g., found in native adenovirus 5) VA RNA I and II, E2A protein, or E4 protein. In certain embodiments, an E4 protein will be considered "functional" if it supports rAAV production at levels at or at least 70%, 80%, or at least 80%, 90%, or at least 90%, 95%, or at least 95% of the levels supported by wild-type E4 protein.

[0198] In certain embodiments, the E4 gene is not located between the VA nucleic acid and the E2A gene, i.e., the sequence of the plasmid is such that the E4 gene sequence does not appear between the VA nucleic acid sequence and the E2A gene sequence in the plasmid, hi certain other embodiments, the E2A gene is located between the VA nucleic acid and the E4 gene.

[0199] Any of the genes on the helper plasmid can be in any orientation regardless of the other genes.

[0200] In certain embodiments, the VA nucleic acid has an activity level that is 75% or at least 75%, 80% or at least 80%, 90% or at least 90%, 95% or at least 95%, or 95-100% of the activity of a wild-type VA nucleic acid from Adenovirus 5. The activity level of the VA nucleic acid can be determined, for example, by measuring rAAV yield using the AAV production assay described above.

[0201] Expression of the E4 gene is driven by a promoter (designated the "E4 promoter"). A fully active promoter includes nucleotides corresponding to 35793 to 35848 of the Adenovirus 5 genome. In certain embodiments, the E4 gene is operably linked to an E4 promoter having at least 50%, at least 70%, or at least 90% of the activity of the wild-type promoter from Adenovirus 5. The activity of the E4 promoter can be determined by testing its ability to drive protein expression. In one embodiment, the E4 promoter has 25% or at least 25%, 40% or at least 40%, 50% or at least 50%, 70% or at least 70%, 90% or at least 90% of the activity of the wild-type E4 promoter from Adenovirus 5 when it supports rAAV production at a level of 25%, 40%, or at least 40%, 50%, or at least 50%, 60%, or at least 60%, 70%, or at least 70%, 90% or at least 90% of the level supported by the wild-type E4 gene promoter, i.e., when the yield of rAAV produced is 50%, or at least 50%, 70%, or at least 70%, 90% or at least 90% of the yield of rAAV produced using a Reference 2 plasmid system containing wild-type helper genes with the wild-type promoter. Preferably, an E4 promoter will be considered to facilitate AAV production if it supports rAAV production at a level at or at least 70%, 80%, 90%, 95%, or at least 95% of the yield of rAAV produced using a Reference 2 plasmid system containing wild-type helper genes with the wild-type promoter.

[0202] 5.Host cells One embodiment of the present invention includes a host cell comprising a dual recombinant DNA vector (double transfection vector) of the present invention.

[0203] Generally, host cells of the invention are capable of or suitable for the production of rAAV and are typically derived from eukaryotic cell lines, such as vertebrate cell lines, including mammalian cell lines (e.g., human cell lines).

[0204] In certain embodiments, the host cell is a cell selected from the group consisting of HEK293T cells, HEK293 cells, HEK293EBNA cells, CAP cells, CAP-T cells, AGE1.CR cells, PerC6 cells, C139 cells, EB66 cells, BHK cells, COS cells, Vero cells, Hela cells, and A549 cells.

[0205] In certain embodiments, the host cells are selected from the group consisting of HEK293T cells, HEK293 cells, HEK293EBNA cells, CAP cells, CAP-T cells, AGE1.CR cells, PerC6 cells, C139 cells, and EB66 cells.

[0206] In certain embodiments, the host cell is selected from the group consisting of HEK293T cells, HEK293 cells, and HEK293EBNA cells.

[0207] In certain embodiments, the host cell is a HEK293T cell.

[0208] In certain embodiments, the host cell is a cell that expresses functional adenoviral E1A / B proteins. For example, the host cell can contain a chromosome that includes a gene encoding a functional adenoviral E1A / B protein.

[0209] A host cell is considered suitable or capable of producing recombinant AAV if it supports AAV production at or at least 30%, 40%, 50%, 70%, 80%, 90%, 95% or at least 95% of the level supported by HEK293T cells, in which case the Test 2 vector / plasmid system of the invention can be introduced / transfected into the host cell being tested for suitability for recombinant AAV production, and the Reference 2 vector / plasmid system can be introduced / transfected into the HEK293T cells.

[0210] In certain embodiments, the dual transfection vector and / or the helper plasmid (or equivalent functional DNA coding sequence) are stably integrated into the host cell genome.

[0211] In certain embodiments, the host cells are derived from a vertebrate, such as a human, monkey, cow, pig, horse and other equine, dog, cat, sheep, goat, mouse, rat, rabbit, mink, opossum, camel and other camelid, chicken and other bird, armadillo, frog, or reptile, or are derived from an insect cell.

[0212] In certain embodiments, the host cells are cell lines suitable for AAV packaging, eg, Expi cells, HEK293 cells (or derivatives thereof, eg, HEK293T cells).

[0213] In certain embodiments, the host cell is a HEK293 cell (eg, an Expi293F cell), a HeLa cell, an A549 cell, a BHK cell, or an insect cell (eg, an Sf9 cell).

[0214] In certain embodiments, the host cells are HEK293 (human embryonic kidney), which can be grown using standard tissue culture medium, such as DMEM supplemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin.

[0215] In some embodiments, HEK293 cells are grown on solid supports, including tissue culture plates, dishes, flasks, and bottles. To grow adherent HEK293 cells, the percentage of FBS can be reduced during rAAV production to limit contamination with animal-derived components.

[0216] In some embodiments, HEK293 cells are adapted to grow in suspension.

[0217] In certain embodiments, the host cells are Vero cells, such as Vero 75.4 or V75 cells described herein. Such cells can be grown on solid supports, including tissue culture plates, dishes, flasks, bottles, and microcarriers, which allow adherent Vero cells to grow in suspension-like conditions.

[0218] In certain embodiments, the host cells are BHK (baby hamster kidney) cells, e.g., BHK21 or sBHK27. In certain embodiments, the BHK cells are adapted to grow in serum-free suspension.

[0219] In some embodiments, the host cells are HEK293 cells. In some embodiments, HEK293 cells are adapted for growth in serum-free medium (e.g., F17 or Expi293 medium) and in suspension, making them suitable for large-scale growth in bioreactors. See, e.g., Grieger et al. (Mol. Ther. 24:287-297, 2016, incorporated herein by reference).

[0220] In certain embodiments, the HEK293 cells are HEK293T cells that express the SV40 T antigen (temperature sensitive allele tsA1609) and the neomycin / geneticin resistance gene.

[0221] In certain embodiments, for production of rAAV particles, the host cell contains helper virus proteins useful (e.g., required) for AAV packaging.

[0222] In some embodiments, the coding sequence for the helper virus protein is introduced into the host cell on a plasmid via transfection, hi some other embodiments, the coding sequence for the helper virus protein is integrated into the host cell genome.

[0223] In certain embodiments, the host cells of the invention can be adapted for use in producing recombinant AAV vectors encoding a gene of interest (GOI) that can be used in gene therapy. See the section entitled "Recombinant AAV Production" below. In such embodiments, one or more rAAV-producing cell lines can be infected with a DT vector of the invention, e.g., a vector or plasmid encoding the AAV Rep and Cap proteins.

[0224] In certain embodiments, such producer / host cell lines for rAAV production are HeLa or A549 derived cell lines transfected with the DT vectors / plasmids of the present invention, optionally containing drug selection markers.

[0225] In certain embodiments, such a producer cell line for rAAV production is Vero cells.

[0226] In certain embodiments, such a producer cell line for rAAV production is BHK cells.

[0227] In certain embodiments, such a producer cell line for rAAV production is HEK293 cells.

[0228] In some embodiments, such producer / host cell lines for rAAV production comprise a DT vector / vector of the present invention, which comprises a GOI flanked by AAV ITR sequences. The GOI can be any one of the GOIs described herein useful for gene therapy, such as the dystrophin minigene or microdystrophin gene described in US 7,906,111; US ​​7,001,761; US ​​7,510,867; US 6,869,777; US 8,501,920; US 7,892,824; WO2020 / 086844; or US 10,166,272, WO2023 / 018854, or PCT / US2016 / 013733 (all of which are incorporated herein by reference).

[0229] For example, PCT / US2016 / 013733 (WO2016 / 115543A2) provides a microdystrophin gene operably linked to a regulatory cassette, the microdystrophin gene encoding a protein comprising an amino-terminal actin-binding domain, a β-dystroglycan-binding domain, and a spectrin-like repeat domain (comprising at least four spectrin-like repeats), two of which comprise a neuronal nitric oxide synthase-binding domain. In certain embodiments, the at least four spectrin-like repeats include spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), and spectrin-like repeat 24 (SR24). In some embodiments, the protein encoded by the microdystrophin 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 some embodiments, the microdystrophin gene comprises, in order from N to C terminus: a hinge 1 domain (H1); a spectrin-like repeat 1 (SR1); a spectrin-like repeat 16 (SR16); a spectrin-like repeat 17 (SR17); a spectrin-like repeat 24 (SR24); and a hinge 4 domain (H4). In some embodiments, H1 is directly connected to SR1. In some embodiments, SR1 is directly connected to SR16. In some embodiments, SR16 is directly connected to SR17. In some embodiments, SR17 is directly connected to SR24. In some embodiments, SR24 is directly connected to H4. In some embodiments, the protein encoded by the microdystrophin gene further comprises, in N- to C-terminal order, spectrin-like repeat 2 (SR2) and spectrin-like repeat 3 (SR3) between SR1 and SR16. In some embodiments, SR1 is directly connected to SR2, and SR2 is further connected to SR3.In certain embodiments, H1 is directly connected to SR1, SR1 is directly connected to SR16, SR16 is directly connected to SR17, SR17 is directly connected to SR23, SR23 is directly connected to SR24, and SR24 is directly connected to H4.

[0230] In some embodiments, the regulatory cassette is selected from the group consisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter. In some embodiments, the protein encoded by the microdystrophin gene has between 5 and 8 spectrin-like repeats. In some 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 / 115543A2 (incorporated herein by reference).

[0231] In certain embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO2023 / 018854 (hereby incorporated by reference).

[0232] 6. Recombinant AAV Generation The recombinant DNA vectors (dual transfection vectors) and production cell lines of the present invention can be used for large-scale production of recombinant AAV vectors (rAAV) useful for gene therapy. In certain embodiments, the system and associated methods of use can be used to propagate / amplify / produce AAV viral particles containing a GOI flanked by ITRs.

[0233] Recombinant replication-deficient AAV vectors that can be produced using the present recombinant DNA vectors (double transfection vectors) and production cell lines typically contain a gene of interest (GOI) and expression control elements (e.g., a promoter for the GOI) in place of the wild-type AAV viral rep and cap open reading frames (ORFs). The AAV rep and cap ORFs, optionally their native promoters p5, p19, and p40, are supplied by the double transfection vectors of the present invention, while other helper functions useful for AAV packaging are supplied by separate constructs or production cell lines. The rep ORF encodes the four nonstructural Rep proteins involved in the AAV viral life cycle, and the cap ORF encodes the three structural proteins (i.e., VP1, VP2, and VP3) that form the icosahedral AAV capsid. Typically, the only AAV viral sequences retained in the rAAV vector genome are the inverted terminal repeats (ITRs), the minimal cis-acting elements for AAV DNA replication and packaging.

[0234] In certain embodiments, methods for propagating / amplifying / producing replication-deficient AAV particles harboring a GOI comprise introducing the recombinant DNA of the invention into host cells before, simultaneously with, or after introducing AAV helper genes for viral packaging.

[0235] In some embodiments, the recombinant DNA vector is introduced into the host cell by transient transfection.

[0236] A method for producing replication-deficient AAV particles involves simultaneously co-transfecting mammalian cells, such as, but not limited to, HEK293 cells, with a construct comprising the recombinant DNA of the present invention and AAV helper genes.

[0237] In some embodiments, the AAV helper genes are introduced into the host cell via 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 dual transfection vector of the invention into the host cell.

[0238] In certain embodiments, the host cells are derived from vertebrates, such as humans, monkeys, cattle, pigs, horses and other equines, dogs, cats, sheep, goats, mice, rats, rabbits, mink, opossums, camels and other camelids, chickens and other birds, armadillos, frogs, or reptiles, or are derived from insect cells. Human cells include BHK cells, Vero cells, HEK293 cells, etc.

[0239] In certain embodiments, the host cell is a HEK293 cell (eg, an Expi293F cell or a VP2 cell), a HeLa cell, an A549 cell, a BHK cell, or an insect cell (eg, an Sf9 cell).

[0240] In certain embodiments, the host cells are HEK293 (human embryonic kidney), which can be grown using standard tissue culture medium, such as DMEM supplemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin.

[0241] In some embodiments, HEK293 cells are grown on solid supports, including tissue culture plates, dishes, flasks, and bottles. To grow adherent HEK293 cells, the percentage of FBS can be reduced during rAAV production to limit contamination with animal-derived components.

[0242] In some embodiments, HEK293 cells are adapted to grow in suspension.

[0243] In certain embodiments, HEK293 cells are adapted for growth in serum-free medium (e.g., F17 or Expi293 medium) and suspension, and are therefore suitable for large-scale growth in bioreactors. See, e.g., Grieger et al. (Mol. Ther. 24:287-297, 2016, incorporated herein by reference).

[0244] In certain embodiments, the HEK293 cells are HEK293T cells that express the SV40 T antigen (temperature sensitive allele tsA1609) and the neomycin / geneticin resistance gene.

[0245] In certain embodiments, the host cells are Vero cells, such as Vero 75.4 or V75 cells described herein. Such cells can be grown on solid supports, including tissue culture plates, dishes, flasks, bottles, and microcarriers, which allow adherent Vero cells to grow in suspension-like conditions.

[0246] In certain embodiments, the host cells are BHK (baby hamster kidney) cells, e.g., BHK21 or sBHK27. In certain embodiments, the BHK cells are adapted to grow in serum-free suspension.

[0247] In some embodiments, the method further comprises recovering the replication-deficient AAV particles comprising the GOI. In some embodiments, the method comprises purifying the recombinant replication-deficient AAV particles.

[0248] Genes of interest (GOI) may include genes useful for gene therapy in the treatment of a given disease or condition. Exemplary (non-limiting) GOIs include genes causing / 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 2I), or genes responsible for / defective in Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB). IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (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.

[0249] Suitable microdystrophin genes include those described in the following patents: US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO2023 / 018854; US10,166,272; WO2020 / 086844 (all of which are incorporated herein by reference). In some embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (which is incorporated herein by reference).

[0250] Diseases or conditions that could potentially benefit from rAAVs generated by the DT vectors of the present invention-based systems include Huntington's disease, X-linked myotubular myopathy (XLM™), acid maltase deficiency (e.g., Pompe disease), spinal muscular atrophy (SMA), myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, mitochondrial myopathies, muscular dystrophies (Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy (BMD), limb-girdle myopathy, and others. Generalized muscular dystrophy (LGMD), facioscapulopuscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), peripheral muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), mucopolysaccharidosis (MPS), metachromatic leukodystrophy (MLD), Batten disease, Rett syndrome, Krabbe disease, Canavan disease, X-linked retinoschisis, color vision deficiency (CNGB3 and CNGA3), X-linked retinitis pigmentosa, age-related macular degeneration, neovascular macular degeneration, Pompe disease, Fabry disease, MPS These include I, II, IIIA, IIIB, Gaucher disease, Danon disease, A1At deficiency, Friedreich's ataxia, Wilson disease, Batten disease (CLN1, CLN3, CLN6, CLN8), Wolman disease, Tay-Sachs disease, Niemann-Lick type C, CDKL5 deficiency disorders, B-thalassemia, and sickle cell disease.

[0251] Wild-type AAV, a naturally replication-deficient human parvovirus, integrates into host cell chromosomes at its genome site-specifically for replication in the absence of helper assistance, where it persists indefinitely unless rescued through cell infection with a helper virus. Introduction of a helper virus into a host cell triggers AAV replication and the production of progeny virions. In the case of rAAV virions useful for gene therapy, introduction of helper virus functions into a suitable host cell triggers packaging of a GOI in the rAAV virion if the necessary rep and cap coding sequences are also provided in the same system.

[0252] Thus, the production of recombinant AAV depends on (1) the presence of AAV rep and cap coding sequences, and (2) helper virus function.

[0253] In certain embodiments, the AAV tropism includes serotypes such as AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9, AAV10, AAV11, preferably AAV9, AAVrh74, AAVrh10, etc. In certain embodiments, the AAV capsid may be genetically modified, or the capsid may be engineered as described (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 of which are incorporated herein by reference), can be synthetic designer capsids that enhance tissue-specific or physiological compartmental delivery of GOIs to specific tissues, such as muscle, skeletal muscle, cardiac muscle, smooth muscle, etc., and / or detarget certain tissues. AAV tropism through pseudotyping, or mixing capsids and genomes from different viral serotypes, can also be used. These serotypes are indicated using a slash, such as AAV2 / 5, which indicates a virus containing a serotype 2 genome packaged in a capsid from serotype 5. The use of these pseudotyped viruses can improve transduction efficiency and also 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 AAVs include AAV2 / 6. In certain embodiments, in silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical use.This effort resulted in the generation of nine putative functional ancestral AAVs and the identification of Anc80, the predicted ancestor of AAV serotypes 1, 2, 8, and 9, which have been widely studied as highly potent in vivo gene therapy vectors 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).

[0254] In some embodiments, the tropism of the AAV includes skeletal muscle (e.g., AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9). In some embodiments, the tropism of the AAV includes cardiac muscle (e.g., AAV1, AAV5, AAV6, AAV8, AAV9, AAVrh74, AAV2-8, AAV2-9). In some embodiments, the AAV detargets a predetermined tissue, e.g., the liver. In some embodiments, the tropism of the AAV targets and detargets a predetermined tissue(s).

[0255] In certain embodiments, the gene of interest (GOI) includes genes causing / 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 2I), or genes causing / defective in Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB). IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (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.

[0256] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0257] In certain embodiments, the GOI is the microdystrophin gene.

[0258] In some embodiments, the microdystrophin gene is described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO2023 / 018854 or US10,166,272 (each of which is incorporated herein by reference in its entirety).In some embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (incorporated herein by reference in its entirety).

[0259] In certain embodiments, the microdystrophin gene comprises coding sequences for the R16 and R17 spectrin-like repeats (e.g., as described in US 7,892,824) for a full-length dystrophin protein.

[0260] In some embodiments, the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats of a full-length dystrophin protein (e.g., the microdystrophin gene described in PCT / US2016 / 013733).

[0261] In certain embodiments, the micro-dystrophin gene does not include coding sequences for spectrin repeats of the full-length dystrophin protein other than SR1, SR16, SR17, SR23, and SR24 repeats (e.g., in that order).

[0262] In certain embodiments, the rAAV vectors of the invention are produced under in vitro culture conditions, e.g., in a bioreactor (e.g., a 0.5 L, 1 L, 2 L, 3 L, 5 L, 10 L, 20 L, 50 L, 100 L, 250 L, 500 L, or 1,000 L functional volume bioreactor), e.g., a CelliGen Plus packed bed bioreactor (New Brunswick Scientific) for fed-batch vector production, for 3 days post-infection.

[0263] In some embodiments, recombinant, replication-deficient AAV viral particles are produced in in vitro culture on anchorage-dependent cell lines, such as Vero and Vero-derived cell lines or HEK293 and HEK293-derived cells, that rely on a solid support. In some embodiments, the solid support is a tissue culture surface, e.g., a tissue culture dish, plate, bottle, flask, cell factory, etc. In some embodiments, the solid support is a microcarrier, e.g., Cytodex1 (GE Healthcare Life Sciences, Piscataway, NJ); a macrocarrier, e.g., FibraCel (New Brunswick Scientific, Edison, NJ), or a multi-layer culture vessel, e.g., CellCube (Corning Life Sciences, Lowell, MA), which allows for medium perfusion.

[0264] In certain embodiments, recombinant replication-deficient AAV viral particles are produced in vitro in eukaryotic cells adapted to grow in suspension, for example, suspension cultures of BHK or HEK293 cell lines adapted for growth in suspension.

[0265] In certain embodiments, the culture supernatant contains 1×10 10 Plaque-forming units (PFU) of rAAV, 1 x 10 11 Plaque-forming units (PFU) of rAAV, 1 x 10 12 Plaque-forming units (PFU) of rAAV, 1 x 10 13 Plaque-forming units (PFU) of rAAV, 1 x 10 14 Plaque-forming units (PFU) of rAAV in excess of, or within any range delimited by any pair of the aforementioned values, e.g., 1 x 10 10 PFU ~ 1 × 10 14 PFU, 1 × 10 11 PFU ~ 1 × 10 13 PFU, or 1 × 10 12 PFU ~ 1 × 10 14 Bringing PFU etc.

[0266] In certain embodiments, the vector stock is produced by one or more downstream processing steps, such as filtration and / or concentration (e.g., depth filtration, dead-end filtration, tangential flow filtration (TFF), and diafiltration), multi-column chromatographic purification, final concentration / buffer exchange, etc., to obtain a vector stock of sufficient purity for administration to animals, including humans. In certain embodiments, the purification process and purified vector stock meet GMP standards.

[0267] In certain embodiments, the titer of the rAAV vector stock is about 1-2 x 10 7 PFU / ml, approximately 1~2×10 8 PFU / ml, approximately 1~2×10 9 PFU / ml, approximately 1~2×10 10 PFU / ml, approximately 1~2×10 11 PFU / ml, approximately 1~2×10 12 PFU / ml, or any range of titers bounded by any pair of the aforementioned values.

[0268] In certain embodiments, the total yield of rAAV vector stock is about 1-25 x 10 14 Total VG purified rAAV, approximately 1-10 x 10 14 Total VG purified rAAV, approximately 1-5 x 10 14 Total VG purified rAAV, approximately 2-4 x 10 14 Total VG purified rAAV, or total yield within any range bounded by any pair of the aforementioned values.

[0269] In certain embodiments, the rAAV vectors so produced are further purified from the crude cell lysate by ion exchange chromatography and / or iodixanol density gradient centrifugation to ensure high final product purity, hi certain embodiments, the rAAV vectors so produced are qualified as clinical-grade vector batches.

[0270] In certain embodiments, the AAV production methods of the invention further comprise determining the titer, purity, and / or potency of the rAAV vector so produced, which may include characterizing purified rAAV stocks using, for example, silver staining of SDS-PAGE separation of proteins to determine purity.

[0271] 7. Treatment of muscular dystrophy using AAV The DT vector of the present invention-based system can be used for large-scale production of rAAV, which in turn can be used in gene therapy to treat various forms of muscular dystrophy, such as Duchenne muscular dystrophy (DMD), myotonic dystrophy, Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), facioscapulopuscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), peripheral muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), etc. In certain embodiments, the muscular dystrophy is DMD or BMD.

[0272] Accordingly, another aspect of the present invention provides a method of treating muscular dystrophy (e.g., 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 a gene defective in muscular dystrophy, e.g., the microdystrophin gene, wherein the rAAV is produced by a method of the present invention using a DT vector and a complementation system of the present invention.

[0273] In some embodiments, the microdystrophin gene is described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2020 / 086844; WO2016115543; WO2023 / 018854, or US10,166,272 (all of which are incorporated herein by reference). In some embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 in WO2023 / 018854 (which is incorporated herein by reference).

[0274] In some embodiments, the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats of a full-length dystrophin protein (e.g., as described in PCT / US2016 / 013733).

[0275] In certain embodiments, the method further comprises producing rAAV by a method of the invention using a DT vector system of the invention prior to administering the rAAV so produced to a subject.

[0276] 8. Treatment of Cardiomyopathy Using AAV The DT vector of the present invention-based system can be used for large-scale production of rAAV, which can then be used in gene therapy to treat various forms of cardiomyopathies. Cardiomyopathy refers to a diverse range of conditions of the myocardium and is the second most common cause of heart disease in subjects, with medical management of secondary symptoms being 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 may thicken, stiffen, thin, or become filled with body-produced substances that do not belong to the myocardium. As a result, the ability of the heart muscle to pump blood is reduced, which can lead to arrhythmias, blood stagnation in the lungs or the rest of the body, and heart failure. Cardiomyopathy can be acquired or inherited. While the cause is not always known, there is progress in understanding the genetic basis of inherited forms of the disease. Gene transfer strategies have been shown to ameliorate heart disease.

[0277] Cardiomyopathy is a class of myocardial diseases that adversely affect the heart's ability to circulate blood through the cardiovascular system. Various types of cardiomyopathy exist, including dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (AVC), left ventricular compaction cardiomyopathy (LVNC), and restrictive cardiomyopathy (RCM). There are numerous genetic forms of cardiomyopathy, including but not limited to DCM associated with Duchenne and Becker muscular dystrophy. In certain forms of Becker muscular dystrophy, and in most cases of Duchenne muscular dystrophy, cardiomyopathy can ultimately limit the patient's survival. Therefore, some embodiments of the present invention provide AAV or rAAV vectors for delivering transgenes to a subject's heart, whether directly, systemically, or by both methods.

[0278] Accordingly, another aspect of the present 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 a gene deficient in the cardiomyopathy, e.g., a gene such as the BAG3 gene, the MYBPC gene, the TMEM43 gene, the TNNT2 gene, the RBM20 gene, the RYR2 gene, or the CASQ2 gene, wherein the rAAV is produced by a method of the present invention using a DT vector and a complementation system of the present invention.

[0279] Other genes associated with various cardiomyopathies include: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Example]

[0280] Example 1 AAV production using a dual transfection system Transfection and sampling Approximately 24 hours before transfection, HEK293 cells (Expi293™ Working Cell Bank) were plated at 1.5 x 10 in 50 mL of fresh Freestyle™ F17 medium (supplemented with 4 mM Glutamax, 0.1% F68) in a shake flask. 6Cells were seeded at a concentration of 1.5E6 cells / mL. On the day of transfection, the target cell density was 2.5E6-3.5E6 cells / mL. Plasmid cocktails were prepared by adding the plasmids to 15 mL of unsupplemented Freestyle™ F17 medium in a tube, mixing, adding PEI (polyethyleneimine) transfection reagent, incubating the mixture for 5 minutes, and remixing the cocktail by inverting the tube. Each plasmid cocktail was added to the cells in each shake flask. The cells and plasmid cocktail were gently mixed together and then incubated at 110 RPM, 5% CO2, and 37°C. Approximately 2-20 hours after transfection, the plasmids were neutralized with 50 μL of Gibco™ anti-aggregation agent.

[0281] Samples were collected from flasks 2–4 days after transfection, and cells were lysed by Triton-X treatment. After centrifugation, the supernatant of lysed cells was collected and stored at −80°C for ddPCR analysis.

[0282] AAV9 generation using a dual transfection system The dual plasmid containing the RepCap gene for AAV9 and the microdystrophin variant as the gene of interest (GOI) was transfected into HEK293 cells together with the helper plasmid at a dual:helper (w / w) ratio of 1:1 or 1.5:1 as described above. The experimental details and results are shown in Figure 2.

[0283] As shown in Figure 2, double transfection using the dual vector of the present invention containing both RepCap and GOI sequences in one plasmid was sufficient to generate AAV particles.

[0284] Example 2 Comparison between double and triple transfection methods - titers Example 2A AAV9 and AAV-SLB101 production using double transfection (DT) and triple transfection (TT) methods were performed using the transfection method described above in Example 1, with a double:helper (w / w) ratio of 1:1 and a RepCap:GOI:helper (w / w) ratio of 1:1:1, respectively. The amount of each plasmid added to HEK293 cells for transfection was 0.25 pg / cell. The produced AAV9 and AAV-SLB101 were determined by ddPCR at 24, 48, and 72 hours after transfection.

[0285] Although AAV9 and SLB101 were used in this example to demonstrate the efficacy of the dual plasmid system of the present invention useful for AAV production and the surprisingly high titers compared to triple transfection methods, it should be understood that the capsids that can be used in such dual transfection vectors / plasmids are not limiting.

[0286] Virus production was compared between the two transfection methods and the results are shown in Figure 3A.

[0287] Strikingly, both AAV9 and AAV-SLB101 viral titers were at least 2-3 times (i.e., 200-300%) higher in the double transfection method compared to the triple transfection method at all measured time points (see Figure 3A).

[0288] This experiment suggests that the double transfection method using the DT-vector of the present invention (containing both the GOI and Rep / Cap sequences) results in surprisingly high virus yields compared to the conventional triple transfection method (in which the GOI, RepCap, and helper are encoded by three separate plasmids).

[0289] Example 2B Using the AMBR® 15 cell culture, a high-throughput automated advanced microbioreactor system for 48 parallel cultures at a 10-15 mL microbioreactor scale, a vector containing AAV-SLB101 with the micro-dystrophin GOI (AV-SLB101-uDys) was produced by both the double transfection system and triple transfection system of the present invention according to the parameters in Table 2 below. [Table 2]

[0290] Figure 3B shows the titers for each sample four days after transfection using FectoVIR. Viral titers using triple transfection (TT) ranged from less than 3.2E10 vg / mL to a maximum of less than 1E11 vg / mL. However, viral titers using the double transfection (DT) method of the present invention ranged from a minimum of 1.85E11 vg / mL to a maximum of 3.83E11 vg / mL (an increase of approximately 100% to approximately 400% relative to the titers from the triple transfection method).

[0291] Example 2C The AAV vector in Example 2B (AAV-SLB101-uDys) was produced at large scale using a 2 L bioreactor according to the parameters in Table 3 below. [Table 3]

[0292] The results are shown in Figure 3C, where all samples achieved at least 4.55E11 vg / mL and reached as high as 1E12 vg / mL (Figure 3C). When compared to Figure 3B, yields translate well from 10-15 mL microbioreactors (Example 2B) to 2 L bioreactors, confirming the scalability of the dual transfection system of the present invention.

[0293] Example 3 Comparison between double and triple transfection methods - Mispackaging rates Kanamycin titers were measured using two commercially available transfection reagents (Gibco AAV-MAX transfection kit (ThermoFisher Scientific) and FECTOVIR® (AAV transfection reagent for large-scale rAAV production) (Polyplus, NY)) in AMBR® 15 cell culture (see above), and the kanamycin titers relative to the GOI titers were calculated and compared between the DT system of the present invention and the triple transfection system.

[0294] Figure 4 shows that kanamycin titers at recovery were much lower using the DT plasmid system of the present invention compared to the TT system, based on the kanamycin-to-GOI ratio by ddPCR using both AAV-MAX and FectoVIR as transfection reagents.

[0295] Example 4 Comparison of GOI titer and p5 promoter packaging into GOI Both GOI titers and P5:GOI (as a percentage) were measured by ddPCR in the DT plasmid system of the present invention using the P5 promoter and modified P5 promoter.

[0296] For the DT plasmid system with a modified P5 promoter, the wild-type AAV2 P5 promoter in the RepCap cassette was modified by inserting an exogenous 5-nucleotide spacer sequence (P5SPC promoter) between the REP and YY1 binding sites of the wild-type P5 promoter. The DT plasmids of the present invention containing the P5SPC promoter driving transcription of the RepCap cassette are designated "P5SPC-SLB101" (DT-P5SPC-SLB101-1 and DT-P5SPC-SLB101-2) in Figure 5. Mispackaging of DNA upstream of the P5 promoter (exemplified by the KanR coding sequence) in the plasmid vectors for DT-P5SPC-SLB101-1 and DT-P5SPC-SLB101-2 was significantly reduced compared to other samples, while maintaining recombinant AAV titers.

[0297] Figure 5 shows that kanamycin relative to GOI was lower (approximately 2.6%) for the DT plasmid with the P5SPC promoter (DT-P5SPC-SLB101) relative to the DT plasmid with the wild-type P5 promoter (DT-SLB101, approximately 15%) using both AAV-MAX and FectoVIR transfection reagents (see DT-SLB101-1 and DT-SLB101-2 vs. DT-P5SPC-SLB101-3 and DT-P5SPC-SLB101-4). See also the data summarized in Table 4 below. [Table 4]

[0298] On the other hand, using both AAV-MAX and FectoVIR transfection reagents, the titers for AAV viruses generated using the DT plasmid with the P5SPC promoter and the DT plasmid with the wild-type P5 promoter were significantly higher than the titers for AAV generated using triple transfection (Figure 5).

Claims

1. 1. A recombinant DNA vector comprising: (a) a gene of interest (GOI) flanked by 5′ adeno-associated virus (AAV) ITR sequences and 3′ ITR sequences; (b) a coding sequence for AAV Rep that is compatible with the 5′ AAV ITR and the 3′ AAV ITR, optionally wherein the coding sequence for AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3′ to the GOI; and (c) a coding sequence for AAV Cap; wherein expression of the AAV Rep in a host cell containing AAV helper genes is sufficient to package an AAV vector genome (vg) containing the GOI flanked by the 5'- and 3'-ITR sequences into an AAV capsid containing the AAV Cap; However, the recombinant DNA vector is not a herpes simplex virus (HSV) vector. The recombinant DNA vector.

2. The vector of claim 1 which is a plasmid.

3. A vector comprising: (a) a gene of interest (GOI) flanked by adeno-associated virus (AAV) ITRs; (b) a coding sequence for AAV Rep that is compatible with the ITRs, optionally under the transcriptional control of an AAV P5 promoter located downstream or 3′ to the GOI; and (c) a coding sequence for AAV Cap; wherein the vector is not a herpes simplex virus (HSV) vector. The vector.

4. A plasmid comprising: (a) A gene of interest (GOI) flanked by (AAV) ITRs; (b) a coding sequence for AAV Rep that is compatible with the (AAV) ITRs, optionally under the transcriptional control of an AAV P5 promoter located downstream or 3′ to the GOI; and (c) Coding sequence for AAV Cap Including, The plasmid.

5. A plasmid comprising: (a) a gene of interest (GOI) flanked by 5′ adeno-associated virus (AAV) ITR sequences and 3′ ITR sequences; (b) a coding sequence for AAV Rep that is compatible with the 5′ AAV ITR and the 3′ AAV ITR, optionally under the transcriptional control of an AAV P5 promoter located downstream or 3′ to the GOI; and (c) a coding sequence for AAV Cap; wherein expression of the AAV Rep in a host cell containing AAV helper genes is sufficient to package an AAV vector genome (vg) containing the GOI flanked by the 5'- and 3'-ITR sequences into an AAV capsid containing the AAV Cap. The plasmid.

6. A two-plasmid system comprising a vector plasmid and a helper plasmid, wherein the vector plasmid comprises a GOI, a rep gene encoding a functional Rep protein, and a cap gene encoding a functional Cap protein.

7. The vector of any one of claims 1 to 6, wherein the GOI comprises a pro-AAV cassette comprising the GOI operably linked to a promoter.

8. The pro-AAV cassette comprises: (1) an enhancer that promotes the transcription of the GOI from the promoter; (2) 5′UTR; (3) Kozak sequence; (4) a heterologous intron that facilitates transcription and / or translation of the GOI; (5) 3′UTR; (6) a WPRE sequence; and / or (7) Poly(A) signal sequence The vector of claim 7 further comprising:

9. 9. The vector of any one of claims 1 to 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 RepCap promoter (e.g., the AAV P5 promoter).

10. The RepCap cassette and the pro-AAV cassette are i) immediately adjacent to each other (e.g., with substantially no intervening polynucleotide sequences); ii) are not directly adjacent to each other; iii) have the same transcriptional direction; or iv) have opposite transcription directions; The vector described in claim 9.

11. a bacterial replication Ori gene, a selectable marker (e.g., an antibiotic resistance gene, e.g., Kan) under the transcriptional control of a selectable marker promoter (e.g., a bacterial promoter); Ｒ or Amp Ｒ 11. The vector of claim 10, further comprising:

12. The vector of any one of claims 1 to 11, wherein the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

13. The GOI may include genes causing / 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 2I), or genes encoding Naglu (α-N-acetylglucosaminidase, in the case of Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase, or SGSH (in the case of mucopolysaccharidosis type IIIA or MPS IIIB) IIIA), Factor IX, Factor VIII, myotubularin 1 (MTM1), survival of motor neurons (SMN, spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, Pompe disease), alpha-galactosidase A or GLA (Fabry disease), glucocerebrosidase, dystrophin or microdystrophin, The vector according to any one of claims 1 to 12.

14. The GOI is a microdystrophin gene (e.g., those described in US 7,906,111; US ​​7,001,761; US ​​7,510,867; US 6,869,777; US 8,501,920; US 7,892,824; WO2016115543; WO2023 / 018854, or US 10,166,272), The vector according to any one of claims 1 to 13.

15. 15. The vector of claim 14, wherein the microdystrophin gene comprises coding sequences for R16 and R17 spectrin-like repeats for a full-length dystrophin protein (e.g., as described in US 7,892,824).

16. 16. The vector of claim 15, wherein the microdystrophin gene comprises coding sequences for the R1, R16, R17, R23, and R24 spectrin-like repeats of a full-length dystrophin protein (e.g., the microdystrophin gene described in WO2016115543 or SEQ ID NO: 1 of WO2023 / 018854).

17. 17. The vector of any one of claims 1 to 16, wherein the 5' and 3' 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.

18. The vector of claim 17, wherein the tropism of the AAV includes skeletal muscle (e.g., AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9).

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

20. 20. The vector of claim 19, wherein the AAV ITR is an AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or a derivative thereof.

21. 21. The vector of any one of claims 1 to 20, wherein the coding sequences for AAV Rep and Cap proteins are under the transcriptional control of a promoter, such as the AAV P5 promoter, an upstream HSV promoter, a modified P5 promoter lacking RBE (Rep binding element), the HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (such as a CMV promoter, an EF1a promoter, a CAG promoter, a CB promoter, etc.).

22. 22. The vector 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-Yang1 (YY1) binding site, the modified P5 promoter comprising an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the spacer is 5 to 100 nucleotides in length (e.g., about 5 nt).

23. 23. The vector of any one of claims 1 to 22, wherein the AAV helper genes comprise adenovirus, herpesvirus, or papillomavirus viral genes for AAV packaging (e.g., E1A, E1B, E2A, E4, and VA RNAs), optionally operably linked to a promoter as a single transcription unit.

24. (1) A composition or kit comprising the vector according to any one of claims 1 to 23, and (2) a helper plasmid.

25. 25. The composition or kit of claim 24, wherein the helper plasmid comprises helper virus genes that support AAV packaging.

26. The helper virus gene is (i) an adenovirus gene, optionally an adenovirus 5 or adenovirus 2 gene; and / or 26. The composition or kit of claim 25, comprising (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.

27. A host cell comprising a vector or two-plasmid system according to any one of claims 1 to 23, or a composition according to any one of claims 24 to 26.

28. 28. The host cell of claim 27, wherein the host cell is a HEK293 cell (e.g., an Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

29. 24. A method for propagating / amplifying / producing recombinant replication-deficient AAV viral particles harboring a GOI according to any one of claims 1 to 23, the method comprising introducing into a host cell a vector according to any one of claims 1 to 23 before, simultaneously with or after introducing into the host cell the AAV helper genes according to any one of claims 1 to 23, thereby propagating / amplifying / producing the recombinant replication-deficient AAV viral particles.

30. 30. The method of claim 29, further comprising recovering the recombinant, replication-defective AAV viral particles from the host cell.

31. 31. The method of claim 29 or 30, wherein the host cell is a HEK293 cell (e.g., Expi293F cell), a VP2 cell, a Vero cell, a HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (e.g., an Sf9 cell).

32. The method of any one of claims 29 to 31, wherein the recombinant DNA vector is introduced into the host cell by transient transfection.

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