Dual transfection vector

Abstract

The present invention described herein provides an improved system and method for rAAV generation. The system of the invention comprises a recombinant DNA vector comprising a gene of interest (GOI) to be delivered to a target cell and Rep and Cap coding sequences for rAAV packaging.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit and priority of the filing date of U.S. Provisional Application No. 63 / 413,887, filed on October 6, 2022, the entire content of which is incorporated herein by reference. Background of the Invention

[0003] In the early stage of rAAV production, cultured cells are first infected with adenovirus and then transfected with two additional DNA plasmids: one plasmid contains a pair of essential rAAV genes (e.g., Rep / Cap genes), and the other plasmid carries the sequence of the gene of interest (GOI). This method produces a reasonable yield of rAAV particles, but there is a risk of adenovirus contaminating the preparation, which may pose risks to patients treated with rAAV.

[0004] In 1998, different research groups determined that the key genes of adenovirus could be transferred to a third "helper" plasmid, and all three plasmids were co-transfected into a preferred host cell to produce rAAV particles, thus avoiding potential contamination of rAAV. Since then, this method, known as the "triple transfection" (TT) technique, has been widely used as the main method for producing recombinant adeno-associated virus (rAAV), including for the commercial manufacture of AAV virus stocks for gene therapy, and may also be referred to herein as traditional triple transfection or conventional triple transfection.

[0005] However, a long-standing problem with AAV-based gene therapy is the need for a large number of suitable AAV virus particles (e.g., in the range of about 1×10 14 to 1×10 15 vector genomes) to achieve effective treatment.

[0006] Today, rAAV is regarded as a key platform for delivering multiple genes for gene therapy, and the number of rAAV-based therapies under development continues to increase. Therefore, there is a need to further improve large-scale rAAV production. Summary of the Invention

[0007] One aspect of the present invention provides a recombinant DNA vector comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (such as AAV2 5' ITR) sequences and 3' ITR sequences (such as AAV2 3' ITR); (b) a coding sequence of AAV Rep (such as AAV2 Rep or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein, in a host cell comprising AAV helper genes, the expression of the AAV Rep is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid comprising the AAV Cap, provided that the recombinant DNA vector is not a herpes simplex virus (HSV) vector.

[0008] Another aspect of the present invention provides a vector comprising (a) a gene of interest (GOI) flanked by adeno-associated virus (AAV) ITR; (b) a coding sequence of AAV Rep (such as AAV2 Rep or Rep2) compatible with the ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein the vector is not a herpes simplex virus (HSV) vector.

[0009] In certain embodiments, the recombinant DNA vector or vector is a plasmid.

[0010] In another aspect, the present invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by (AAV) ITR (such as AAV2 5' ITR sequences and AAV2 3' ITR sequences); (b) a coding sequence of AAV Rep (such as AAV2 Rep or Rep2) compatible with the (AAV) ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAVP5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap.

[0011] In another aspect, the present invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (such as AAV2 5' ITR) sequences and 3' ITR sequences (such as AAV2 3' ITR); (b) a coding sequence of an AAV Rep (such as AAV2 Rep or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein, in a host cell comprising AAV helper genes, the expression of the AAV Rep is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid comprising the AAV Cap.

[0012] In certain embodiments, the plasmid of the present invention does not contain a polynucleotide sequence encoding a helper gene.

[0013] In yet another aspect, the present invention provides a dual 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.

[0014] In some embodiments, the expression of AAV Rep in a host cell comprising AAV helper genes is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by 5'- and 3-ITR sequences into an AAV capsid comprising the AAV Cap.

[0015] In certain embodiments, the GOI is located within a pro-AAV cassette comprising the GOI operably linked to a promoter.

[0016] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that promotes transcription of the GOI from the promoter; (2) a 5' UTR; (3) a Kozak sequence; (4) a heterologous intron that promotes transcription and / or translation of the GOI; (5) a 3' UTR; (6) a WPRE sequence; and / or (7) a polyA signal sequence.

[0017] In certain embodiments, the coding sequences of the AAV Rep and the AAV Cap are located within a RepCap cassette comprising an operably linked RepCap promoter (such as the AAV P5 promoter).

[0018] In certain embodiments, the RepCap cassette and the pro-AAV cassette: i) are adjacent to each other (e.g., with substantially no intervening polynucleotide sequence); ii) are not adjacent to each other; iii) have the same transcriptional orientation; iv) have opposite transcriptional orientations.

[0019] In certain embodiments, the recombinant DNA vector further comprises a bacterial replication Ori gene, a selectable marker (such as an antibiotic resistance gene, e.g., Kan R or Amp R ), which is under the transcriptional control of a selectable marker promoter (such as a bacterial promoter).

[0020] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin mini-gene encoding a functional micro-dystrophin).

[0021] In certain embodiments, the GOI includes genes that cause / are defective in the following diseases: 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 or coding sequences for the following: NAGLU (α-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid α-glucosidase (GAA, for Pompe disease), α-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or micro-dystrophin.

[0022] In certain embodiments, the GOI is the microdystrophin gene (e.g., the gene described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854 or US10,166,272, each of which is incorporated herein by reference). In certain embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0023] In certain embodiments, the microdystrophin gene comprises the coding sequences of the R16 and R17 spectrin-like repeats of full-length dystrophin (such as the sequences described in US7,892,824).

[0024] In certain embodiments, the microdystrophin gene comprises the coding sequences of the R1, R16, R17, R23 and R24 spectrin-like repeats of full-length dystrophin (such as the microdystrophin gene described in PCT / US2016 / 013733).

[0025] In certain embodiments, both the 5' and 3' 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.

[0026] In certain embodiments, both the 5' and 3' AAV ITR sequences flanking the GOI are from AAV2.

[0027] In certain embodiments, the tropism of the AAV includes skeletal muscle (such as AAV1, AAV6, AAV7, AAV8 or AAV9, preferably AAV9).

[0028] In certain embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from the same or different AAVs.

[0029] In certain embodiments, the AAV ITR is the AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or its derivatives (such as its spectral or transcriptional derivatives).

[0030] In certain embodiments, the coding sequences of 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 the REP binding site and the YY1 binding site where the transcription start site is located, or a ubiquitous promoter (such as the CMV promoter, EF1a promoter, CAG promoter, CB promoter, etc.).

[0031] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter. In some embodiments, the modified P5 promoter contains the Ying-Yang 1 (YY1) binding site where the REP binding site and the transcription start site are located. In some embodiments, the modified P5 promoter contains an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site. In some embodiments, the length of the spacer is from 5 nucleotides to 100 nucleotides (e.g., about 5 nt).

[0032] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter containing the Ying-Yang 1 (YY1) binding site where the REP binding site and the transcription start site are located, wherein the modified P5 promoter contains an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the length of the spacer is from 5 nucleotides to 100 nucleotides (e.g., about 5 nt).

[0033] In certain embodiments, the AAV helper genes include adenovirus, herpesvirus, or papillomavirus genes (such as E1A, E1B, E2A, E4, and VA RNA) that can be used for AAV packaging, which are optionally operably linked to a promoter as a transcriptional unit.

[0034] Another aspect of the present invention provides a composition or kit that contains the recombinant DNA vector of the present invention and a helper plasmid.

[0035] In certain embodiments, the helper plasmid contains helper virus genes sufficient to support AAV packaging.

[0036] In certain embodiments, the helper virus genes include: (i) adenovirus genes, optionally adenovirus 5 or adenovirus 2 genes; and / or (ii) VA nucleic acids encoding functional VA RNA I and II, the E2A gene encoding a functional E2A protein, and the E4 gene encoding a functional E4 protein.

[0037] Another aspect of the present invention provides a host cell that contains the recombinant DNA vector of the present invention or the composition of the present invention.

[0038] In certain embodiments, the host cell is a HEK293 cell (such as 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 (such as Sf9).

[0039] Another aspect of the present invention provides a method for propagating / amplifying / generating recombinant replication-defective AAV viral particles encapsulating the GOI of the present invention, the method comprising: introducing the recombinant DNA vector of the present invention into the host cell before, simultaneously with, or after introducing the AAV helper gene of the present invention into the host cell, thereby propagating / amplifying / generating recombinant replication-defective AAV viral particles.

[0040] In certain embodiments, the method further comprises harvesting the recombinant replication-defective AAV viral particles of the present invention from the host cell.

[0041] In certain embodiments, the host cell is a HEK293 cell (such as 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 (such as Sf9).

[0042] In certain embodiments, the recombinant DNA vector is introduced into the host cell by transient transfection.

[0043] It should be understood that any one embodiment of the present invention, including those described only in one of the examples, claims, or subsections, can be combined with any other one or more embodiments, unless such combination is inappropriate or expressly disclaimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A A schematic diagram (not drawn to scale) of an enlarged portion of an exemplary vector of the present invention is shown, showing the Pro-AAV cassette and the RepCap cassette (which may or may not be directly adjacent and may or may not be in the same transcriptional orientation, as shown here). pZ-KanR (a kanamycin resistance gene under the transcriptional control of a bacterial promoter) is also an optional feature. GOI: gene of interest.

[0045] Figure 1BPlasmid maps of two exemplary plasmids containing the dual-transfected GOI / RepCap vector of the present invention for generating AAV9 (left) and AAV-SLB101 (right) viral particles are shown, where the GOI is a variant of microdystrophin, as a non-limiting example of the GOI. μDys: microdystrophin. In both constructs, the Pro-AAV cassette is upstream / 5' of the RepCap cassette, and its transcription is driven by the p5 promoter in the RepCap cassette. Thus, in the circular plasmid vector, the KanR coding sequence is upstream / 5' of the p5 promoter compared to the equivalent KanR coding sequence in the RepCap plasmid (where there is no Pro-AAV cassette) used in triple transfection.

[0046] Figure 2 Results of generating rAAV by dual transfection of the GOI / RepCap plasmid of the present invention and a suitable helper plasmid using Expi cells are shown, where the exemplary GOI:helper plasmid (w / w) ratios are 1:1 and 1.5:1, and the DNA amounts are 0.5 pg / cell and 0.75 pg / cell, respectively.

[0047] Figure 3A Shows that, compared to the traditional triple transfection (TT) plasmid, using the dual transfection (DT) plasmid of the present invention (see Figure 1B ), there are surprisingly high titers (e.g., 200 - 300%) of rAAV viral particles of AAV9 and AAV-SLB101 viral particles produced in Expi cells. At 24 hours, 48 hours, and 72 hours after transfection, the viral titers were determined by ddPCR by lysing the infected Expi cells.

[0048] Figure 3B Shows that, compared to the triple transfection method (the first two bars from the left), higher yields of AAV are produced by various methods using the DT plasmid of the present invention (the 3rd to 5th bars from the left).

[0049] Figure 3C Shows the scalable high-yield production of AAV in a 2L bioreactor using the DT plasmid of the present invention.

[0050] Figure 4 Shows that, based on the ddPCR and KanR / GOI ratio, using two commercially available transfection reagents, the mispackaging rate of KanR is reduced using the DT plasmid of the present invention compared to the TT plasmid. AAVMAX: Gibco AAV-MAX transfection kit (ThermoFisher Scientific). FectoVIR: -AAV (Polyplus, NY).

[0051] Figure 5 It is shown that the modified p5 promoter further reduces the mispackaging rate (measured as % of p5-related mispackaging / GOI) and increases the GOI titer. Detailed Description

[0052] 1. General Overview

[0053] Traditional AAV production systems utilize three plasmid vectors (the so-called triple transfection vector system): one plasmid vector carries the gene of interest (GOI), flanked by AAV ITR sequences, for packaging into AAV viral particles; another plasmid vector carries an expression cassette encoding AAV Rep and Cap proteins, which can be used for AAV packaging; and yet another plasmid vector provides useful helper genes from other viruses such as adenovirus, herpesvirus, or papillomavirus to enable the AAV production life cycle.

[0054] The recombinant dual transfection vector of the present invention described herein improves upon the traditional system by inserting the GOI cassette and the rep-cap expression cassette into a single plasmid to create the dual transfection vector (e.g., plasmid) of the present invention, which can be used for dual transfection together with a helper plasmid (e.g., the same helper plasmid used in the traditional triple transfection method).

[0055] Surprisingly, at appropriate (e.g., identical / equimolar) ratios of Rep, Cap, and helper genes, the dual transfection system of the present invention produces rAAV viral particles at levels of about 200 - 300% compared to the traditional triple transfection system that has been used for commercial production of rAAV viral particles for decades.

[0056] Furthermore, in some embodiments, using the dual vector / plasmid system of the present invention for AAV production can reduce the frequency of mispackaging. Such exemplary embodiments of the rDNA vector of the present invention include the configurations as Figure 1A shown.

[0057] Accordingly, 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 of AAV Rep (such as AAV2 Rep or Rep2) compatible with the ITRs, optionally, the coding sequence of the AAV Rep being under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein the vector is not a herpes simplex virus (HSV) vector.

[0058] Another aspect of the present invention provides a recombinant DNA (rDNA) vector comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (such as AAV2 5' ITR) sequences and 3' ITR sequences (such as AAV2 3' ITR); (b) a coding sequence for an AAV Rep (such as AAV2 Rep or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally, the coding sequence for the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence for AAV Cap; wherein in a host cell containing AAV helper genes, the expression of the AAV Rep is sufficient to package an AAV vector genome (vg) containing the GOI flanked by 5'- and 3'-ITR sequences into an AAV capsid containing the AAV Cap, provided that the recombinant DNA vector is not a herpes simplex virus (HSV) vector.

[0059] In certain embodiments, the vector or recombinant DNA vector is a plasmid.

[0060] In certain embodiments, the vector is suitable for mammalian cells. Vectors suitable for mammalian cells are widely described and 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 that can be used for replication of the vector in prokaryotic and / or eukaryotic cells, selection of the vector, and expression of sequences in a variety of host cells. For example, the vectors of the present disclosure may include a prokaryotic replicon (i.e., a sequence having the ability to direct extrachromosomal autonomous replication and maintenance of the vector in a prokaryotic host cell, such as a bacterial host cell). Such replicons are well known in the art. In some embodiments, the vector may include a shuttle element that renders the vector suitable for replication and integration in both prokaryotes and eukaryotes. In addition, the vector may also include a gene whose expression confers a detectable marker, such as a drug resistance gene, which allows for selection and maintenance of the host cell. The vector may also have a reporter marker, such as a gene encoding a fluorescent or other detectable protein.

[0061] The vectors of the present invention may also include transcriptional enhancers, translation signals, and transcriptional and translational termination signals. Examples of transcriptional termination signals include, but are not limited to, polyadenylation signal sequences such as bovine growth hormone (BGH) poly(A), SV40 late poly(A), rabbit β-globin (RBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof.

[0062] In certain embodiments, the dual-transfection recombinant DNA vector (dual-transfection (DT) vector) of the present invention comprises a gene of interest (GOI) flanked by 5' and 3' adeno-associated virus (AAV, such as AAV2) ITRs, a coding sequence of AAV Rep (such as AAV2 Rep), and a coding sequence of AAV Cap (such as AAV9 or its derivative) compatible with the AAV ITR. In a host cell containing AAV helper genes (which can be introduced into the host cell by any art-recognized method, such as infecting the host cell with an adenovirus or transfecting a helper plasmid containing the genes required for AAV packaging), the expression of AAV Rep and Cap proteins from the dual-transfection vector is sufficient to package an AAV vector genome (vg) containing the GOI flanked by 5' and 3' AAV ITRs.

[0063] In certain embodiments, the recombinant DNA vector is a plasmid, which can be suitable for transfection into an AAV packaging cell line, such as Expi, HEK293 (such as HEK293T cells) or other commonly used AAV packaging cell lines.

[0064] In a related aspect, the present invention provides a plasmid comprising (a) a gene of interest (GOI) flanked by (AAV) ITRs; (b) a coding sequence of AAV Rep (such as AAV2 Rep or Rep2) compatible with the (AAV) ITRs, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap.

[0065] In another related aspect, the present invention provides a plasmid comprising: (a) a gene of interest (GOI) flanked by 5' adeno-associated virus (AAV) ITR (such as AAV2 5' ITR) sequence and 3' ITR sequence (such as AAV2 3' ITR); (b) a coding sequence of AAV Rep (such as AAV2 Rep or Rep2) compatible with the 5' AAV ITR and the 3' AAV ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein, in a host cell containing AAV helper genes, the expression of the AAV Rep is sufficient to package an AAV vector genome (vg) containing the GOI flanked by 5'- and 3-ITR sequences into an AAV capsid containing the AAV Cap.

[0066] In some embodiments, the plasmid of the present invention does not contain a polynucleotide sequence encoding a helper gene.

[0067] 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.

[0068] As used herein, the term "plasmid" includes nucleic acid molecules capable of replicating independently of the cell chromosome. The term "plasmid" is intended to include circular nucleic acid molecules and linear nucleic acid molecules. In addition, 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, the plasmid is a circular nucleic acid (DNA) molecule. In certain embodiments, the plasmid is a nucleic acid molecule of bacterial origin.

[0069] In some embodiments, the GOI is located within a pro-AAV cassette that contains the GOI operably linked to a promoter.

[0070] In certain embodiments, the rDNA vector contains two or more copies of the pro-AAV cassette. In certain embodiments, all copies of the pro-AAV cassette contain the same GOI. In certain embodiments, at least two of the pro-AAV cassettes contain different GOIs. The latter embodiment may be useful, for example, if an AAV vector is used to deliver different parts of the same functional component, such as the coding sequences of a CRISPR / Cas effector enzyme and one or more guide RNAs.

[0071] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that promotes transcription of the GOI from the promoter; (2) a 5'UTR; (3) a Kozak sequence; (4) a heterologous intron that promotes transcription and / or translation of the GOI; (5) a 3'UTR; (6) a WPRE sequence; and / or (7) a polyA signal sequence.

[0072] In some embodiments, the coding sequences for AAV Rep and AAV Cap are located within a RepCap cassette that contains an operably linked RepCap promoter. In certain embodiments, the operably linked RepCap promoter includes the AAVP5 promoter.

[0073] In some embodiments, the RepCap cassette and the pro-AAV cassette are adjacent to each other (e.g., with substantially no intervening polynucleotide sequence).

[0074] In some other embodiments, the RepCap cassette and the pro-AAV cassette are not adjacent to each other.

[0075] In some embodiments, the RepCap cassette and the pro-AAV cassette have the same transcriptional orientation.

[0076] In some other embodiments, the RepCap cassette and the pro-AAV cassette have opposite transcriptional orientations.

[0077] In some embodiments, the pro-AAV cassette is upstream of the RepCap cassette.

[0078] In some other embodiments, the pro-AAV cassette is downstream of the RepCap cassette.

[0079] In some embodiments, the GOI is before the coding sequence of AAV Rep. In some embodiments, the GOI is before the coding sequence of AAV Cap. In some embodiments, the GOI is before the coding sequences of both AAV Rep and AAV Cap.

[0080] In certain embodiments, the coding sequence of AAV Rep is under the transcriptional control of an AAV P5 promoter that is downstream or 3' of the GOI.

[0081] 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.

[0082] In some embodiments, the recombinant DNA vector (dual transfection vector) also contains a bacterial replication Ori gene, a selectable marker (such as an antibiotic resistance gene, e.g., KanR or AmpR), which is under the transcriptional control of a selectable marker promoter (such as a bacterial promoter).

[0083] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a dystrophin minigene encoding functional microdystrophin).

[0084] In certain embodiments, the GOI includes genes that cause or are defective in the following diseases: 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 or coding sequences for the following: NAGLU (α-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid α-glucosidase (GAA, for Pompe disease), α-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or utrophin.

[0085] In certain embodiments, the GOI is the utrophin gene (e.g., the gene described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854 or US10,166,272, each of which is incorporated herein by reference). In certain embodiments, the GOI is the utrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0086] In certain embodiments, the utrophin gene contains the coding sequence of the R16 and R17 spectrin-like repeats of full-length dystrophin (such as the sequence described in US7,892,824).

[0087] In certain embodiments, the utrophin gene contains the coding sequence of the R1, R16, R17, R23 and R24 spectrin-like repeats of full-length dystrophin (such as the utrophin gene described in PCT / US2016 / 013733).

[0088] In certain embodiments, the 5' and 3' AAV ITR sequences flanking the GOI are each from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0089] In certain embodiments, the tropism of the AAV includes skeletal muscle (such as AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9).

[0090] In certain embodiments, the AAV ITR, AAV Rep, and AAV Cap are from the same or different AAVs.

[0091] In certain embodiments, the AAV ITR is the AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or a derivative thereof (such as a spectral or transcriptional derivative).

[0092] In certain embodiments, the coding sequences of 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 YY1 binding site positioned at the transcription start site, or a ubiquitous promoter (such as the CMV promoter, EF1a promoter, CAG promoter, CB promoter, etc.).

[0093] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter. In some embodiments, the modified P5 promoter contains a REP binding site and a Ying-Yang 1 (YY1) binding site positioned at the transcription start site. In some embodiments, the modified P5 promoter contains an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site. In some embodiments, the length of the spacer is from 5 nucleotides to 100 nucleotides (e.g., about 5 nt).

[0094] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter containing a REP binding site and a Ying-Yang 1 (YY1) binding site positioned at the transcription start site, wherein the modified P5 promoter contains an exogenous spacer sequence inserted between the REP binding site and the YY1 binding site; optionally, the length of the spacer is from 5 nucleotides to 100 nucleotides (e.g., about 5 nt).

[0095] In certain embodiments, AAV helper genes include adenovirus, herpesvirus, or papillomavirus genes (such as E1A, E1B, E2A, E4, and VA RNA) that can be used for AAV packaging, optionally operably linked to a promoter as a single transcription unit.

[0096] Another aspect of the invention provides a composition or kit that contains the recombinant DNA vector and helper plasmid of the invention.

[0097] In certain embodiments, the helper plasmid contains helper virus genes sufficient to support AAV packaging.

[0098] In certain embodiments, the helper virus genes include: (i) adenovirus genes, optionally adenovirus 5 or adenovirus 2 genes; and / or (ii) VA nucleic acids encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein.

[0099] Another aspect of the invention provides a host cell that contains the recombinant DNA vector or the composition of the invention.

[0100] In certain embodiments, the host cell is a HEK293 cell (such as 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 (such as Sf9).

[0101] In another aspect, the invention provides a method for propagating, amplifying, or producing recombinant replication-defective AAV virus particles encapsulating a GOI, which includes introducing the recombinant DNA vector (dual transfection vector) of the invention (for example, in a plasmid) into a host cell before, simultaneously with, or after introducing an AAV helper gene (for example, in a plasmid) into the host cell.

[0102] In some embodiments, the dual vector and the AAV helper gene in two separate plasmids are co-transfected into a host cell to produce recombinant replication-defective AAV virus particles encapsulating a GOI.

[0103] In certain embodiments, the method further includes harvesting the recombinant replication-defective AAV virus particles from the host cell.

[0104] In certain embodiments, the host cell is a HEK293 cell (such as 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 (such as Sf9).

[0105] In certain embodiments, a recombinant DNA vector is introduced into a host cell by transient transfection.

[0106] Based on the general principles of the invention set forth herein, the following sections provide further detailed descriptions of various aspects of the invention. It should be understood that any one embodiment of the invention can be combined with any one or more other embodiments of the invention, including those described in different parts of this application, as well as those described only in the examples, drawings, or claims.

[0107] Furthermore, all disclosures relating to "recombinant DNA vectors" also apply to the "vectors" and "plasmids" described herein.

[0108] 2. Rep and Cap genes

[0109] In certain embodiments, the coding sequences of AAV Rep and Cap proteins and the gene of interest (GOI) flanked by AAV ITR sequences are integrated into a single dual transfection (DT) vector of the invention, such as a DT plasmid.

[0110] The Rep proteins are essential for replication and packaging, while the capsid proteins assemble to produce the protein coat or AAV capsid of AAV, which forms the outer capsid shell protecting the viral genome and actively participates in cell binding and internalization. Alternative splicing and alternative start codons and promoters result in the production of four different Rep proteins (Rep78, Rep68, Rep52, and Rep40) from a single open reading frame and three capsid proteins (VP; VP1 / VP2 / VP3) from a single open reading frame. Although not wishing to be bound by theory, AAV capsid proteins typically contain a molar ratio of VP1:VP2:VP3 of 1:1:10. As used herein, "AAV serotype" is primarily defined by the AAV capsid. In some cases, the ITR is also specifically described by the AAV serotype.

[0111] In certain embodiments, the AAV ITR, AAV Rep, and AAV Cap are from the same or different AAV serotypes.

[0112] In some embodiments, the AAV ITR, AAV Rep, and AAV Cap are from AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9 K449R, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5,AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, 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 / hu.6, AAV3.1 / hu.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, AAV16.12 / 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, AAV52 / 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, AAVF3, AAVF5, AAVH2, AAVrh.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.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.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.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.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, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, 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.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAVShuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61AAV, 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.29AAV128.1 / hu.43, True-to-type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotype, AAVCBr-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, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAVCHt-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, AAVClv1-7, 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-D3AAV 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, AAV F1 / HSC1, AAV F11 / HSC11, AAV F12 / HSC12, AAV F13 / HSC13, AAV F14 / HSC14, AAV F15 / HSC15, AAV F16 / HSC16, AAV F17 / HSC17, AAV F2 / HSC2, AAV F3 / HSC3, AAV F4 / HSC4, AAV F5 / HSC5, AAV F6 / HSC6, AAV F7 / HSC7, AAV F8 / HSC8, and / or AAV F9 / HSC9 and their variants.,

[0113] In certain embodiments, the AAV Cap is a derivative of wild-type AAV9. In certain embodiments, the derivative comprises the insertion of a short peptide (e.g., 3, 4, 5, 6, 7, 8, or 9 residues) between residues 588 and 589 of the wild-type AAV9 capsid VP1. In certain embodiments, the insertion comprises inserting RGDLGLS into residues 588 and 589 of the wild-type AAV9 capsid VP1, consisting essentially of, or consisting of, the same.

[0114] In certain embodiments, the rDNA vector / plasmid of the present invention comprises a cap gene promoter. The cap gene promoter can be operably linked to the cap gene. In certain embodiments, the cap gene promoter is a native cap gene promoter.

[0115] 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, the rDNA vector / plasmid of the present invention comprises a cap gene promoter, such as the AAV p40 promoter, p5 promoter, and / or p19 promoter.

[0116] The native p40 promoter is contained within the native rep gene. In some embodiments, the p40 promoter has the sequence of AAV2 or a sequence having at least 95%, at least 98%, or 99% identity to the sequence of AAV2. In some embodiments, at least one cap gene promoter is contained within a promoter region comprising the p40 promoter, p5 promoter, and p19 promoter.

[0117] The native p5 promoter is located upstream of the native rep gene. In some embodiments, the p5 promoter has the sequence of AAV2 or a sequence having at least 95%, at least 98%, or 99% identity to the sequence of AAV2.

[0118] In certain embodiments, the wild-type or native p5 promoter is modified to produce 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 Ying-Yang 1 (YY1) binding site positioned at the transcription start site. In certain embodiments, the modified P5 promoter comprises a REP binding site and a Ying-Yang 1 (YY1) binding site positioned at the transcription start site and a foreign 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).

[0119] Without being bound by a particular theory, inserting a spacer between the P5 REP binding sequence and the YY1 box at the transcription start site of the P5 promoter may result in or contribute to reducing or eliminating mispackaging of vector / plasmid DNA upstream of the P5 promoter while maintaining recombinant AAV titers.

[0120] In certain embodiments, the spacer includes a foreign spacer sequence inserted between the REP binding site and the YY1 binding site. In certain embodiments, no foreign spacer is included, and 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 higher sequence identity with the wild-type P5 promoter, or a length within any range defined by any pair of the foregoing values, such as, for example, between 80% and 99%, between 90% and 99%, or between 95% and 99%. In certain embodiments, the modified P5 promoter with a foreign spacer inserted has a nucleotide sequence corresponding to at least about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 nucleotides, or a length within any range defined by any pair of the foregoing values, such as between 40 and 50, between 50 and 160, or between 80 and 130, for example, adjacent to (or upstream of) the transcription start site of the large REP proteins (i.e., REP78 and REP68) in the AAV genome, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or higher (including the range) sequence identity therewith, wherein the promoter drives the transcription of the large REP proteins, i.e., REP78 and REP68.

[0121] In some instances, the modified P5 promoter has a sequence of a region having a transcription start site of REP78 and REP68 from among those derived from the vicinity of adeno-associated virus (AAV) serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), or any one of 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.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant.

[0122] In certain embodiments, the modified P5 promoter comprises or consists of a sequence of about 160 nucleotides corresponding to or adjacent to the transcription start site of REP78 and REP68 in an AAV genome such as AAV1, 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, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant or AAVrh8R R533A mutant genome.

[0123] As used herein, "REP binding sequence", "REP binding site", or "REP binding element" are used interchangeably herein and refer to an element of the P5 promoter consisting of two to four imperfect GAGC repeats, which repeats are located downstream, i.e., 3', of the TATA box and upstream of the transcription start site of the P5 promoter. Without being bound by a particular theory, Rep-mediated repression is thought to result from the binding of Rep to the REP binding element. In certain embodiments, the P5 REP binding sequence is or comprises GCCCGAGTGAGCACGC.

[0124] As used herein, "Ying-Yang 1 binding site", "YY1 binding site", or "YY1 box" refers to the YY1 recognition sequence which, when bound by YY1, supports initiation from the transcription start site. The +1 YY1 binding of the P5 promoter is located at the transcription start site and has the consensus sequence CCAT.

[0125] As used herein, the term "exogenous spacer sequence" refers to a sequence that is not native to the P5 promoter.

[0126] The exogenous spacer sequence can be inserted at any position between the REP binding site and the YY1 binding site, as described herein. In some embodiments, the exogenous spacer sequence is inserted at or replaces one or more of the seven 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 sequence AGGGTCT of the AAV8 P5 promoter.

[0127] In some embodiments, the total length of the exogenous spacer is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more nucleotides, or a length within any range defined by any pair of the foregoing values, e.g., such as between 5 and 120, between 10 and 100, or between 5 and 100. Desirably, the length of the exogenous spacer is 5 to 100 nucleotides. As will be understood by those skilled in the art, the sequence of the spacer is not important. The sequence of the spacer can be a random artificial sequence, a sequence derived from AAV, or a sequence derived from other sources.

[0128] The p19 promoter is contained within the native rep gene. In some embodiments, the p19 promoter has the sequence of AAV2 or a sequence having at least 95%, at least 98%, or 99% identity to the sequence of AAV2.

[0129] In some embodiments, the AAV ITR, AAV Rep, and AAV Cap are from the same AAV. In some other embodiments, the AAV ITR, 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 aforementioned wt AAV9 derivative, and including SLB-101 (see SEQ ID NO:14 and FIG. 6 of WO2021 / 072197, which is incorporated herein by reference).

[0130] In certain embodiments, the AAV ITR is the AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0131] In certain embodiments, the coding sequence of the AAV Rep protein encodes wild-type Rep40, Rep52, Rep68, and / or Rep78 of AAV (such as AAV2). In certain embodiments, these Rep proteins are transcribed from one or more rep promoters p5, p19, and / or p40.

[0132] The rep gene encodes at least one functional Rep protein (Rep78, Rep68, Rep52, and Rep40). This gene region is under the control of the p5 and p19 promoters. When the p5 promoter is used, the sequences encoding Rep78 and Rep68 are transcribed. Rep78 and Rep68 are two alternative splicing variants (Rep78 contains an intron excised in Rep68). Similarly, when the p19 promoter is used, the sequences encoding Rep52 and Rep40 are transcribed. Rep52 and Rep40 are alternative splicing variants (Rep52 contains an intron excised in Rep40). Four Rep proteins are known to be involved in the replication and packaging of the viral genome and are thus useful in rAAV production.

[0133] In certain embodiments, it is not necessary for all four Rep proteins to be present. In certain embodiments, there is at least one encoded large Rep protein (Rep78 or Rep68) and one encoded small Rep protein (Rep52 or Rep40). Rep78 can be toxic to cells, and Rep78 is not required for AAV replication. 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.

[0134] In certain embodiments, the rDNA vector / plasmid of the present invention comprises at least one rep gene encoding the following proteins: (a) a functional Rep52 protein; (b) a functional Rep40 protein; and / or (c) a functional Rep68 protein.

[0135] As used herein, a "functional" Rep protein is a protein that permits the production of AAV particles. Specifically, Rep78 or Rep68 (large Rep proteins) are thought to be involved in the replication of the AAV genome, while Rep52 and Rep40 (small Rep proteins) are thought to be involved in packaging the AAV genome into the capsid. One of ordinary skill in the art can readily determine whether a given Rep protein is functional, such as by using the AAV production assay described above to determine, for example, whether the Rep protein supports AAV production.

[0136] In some embodiments, at least one rep gene encodes a "functional" Rep protein if the level of rAAV production supported by the Rep protein is, for example, the level supported by wild-type Rep protein or at least 25% thereof, the level supported by wild-type Rep protein or at least 40% thereof, the level supported by wild-type Rep protein or at least 50% thereof, the level supported by wild-type Rep protein or at least 70% thereof, the level supported by wild-type Rep protein or at least 80% thereof, the level supported by wild-type Rep protein or at least 90% thereof, the level supported by wild-type Rep protein or at least 95% thereof, or a level within any range defined by any pair of the foregoing values, such as between 25% and 95%, between 40% and 90%, or between 25% and 70%.

[0137] In certain embodiments, the Rep protein is compatible with the ITRs surrounding the GOI. Some Rep proteins may be able to package genomic material (such as an expression cassette) only when flanked by ITRs of the same serotype as the Rep protein. Other Rep proteins have cross-compatibility and can package genomic material flanked by ITRs of different serotypes.

[0138] In certain embodiments, the rDNA vector / plasmid of the present invention contains two or more rep genes separated on the vector / plasmid. For example, one separate rep gene may encode Rep 68 (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 gene may encode Rep 40 (e.g., using the p19 promoter or a different promoter located near the normal position of the p19 promoter). One rep gene encoding Rep 40 may also encode Rep 52 since Rep 52 and Rep 40 are alternatively spliced variants.

[0139] In certain embodiments, if two genes encode a Rep 40 protein, one of the two genes encoding a functional Rep 40 protein may contain an intron. In one embodiment, both genes encoding a functional Rep 40 protein contain introns. In another embodiment, only one of the genes encoding a functional Rep 40 protein contains an intron.

[0140] For example, to avoid at least one rep gene encoding Rep 78, the rep gene can be split into two genes by partial duplication. One gene may contain nucleotides corresponding to the full-length native rep gene with the sequences corresponding to introns removed. Such a gene will encode Rep 68 and Rep 40, but not Rep 78 or Rep 52, because a portion of each of the Rep 78 and Rep 52 proteins is encoded by sequences that act as introns in the case of rep 40. The second gene can contain nucleotides corresponding to the region of the native rep gene downstream of the p19 promoter, which will encode Rep 52 (with the intron spliced in) and Rep 40 (with the intron spliced out).

[0141] 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 the expression of the cap gene but is not required for rep gene expression. A functional p40 promoter is a promoter capable of driving cap gene expression.

[0142] In certain embodiments, the coding sequences of 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 (such as the CMV promoter, EF1a promoter, CAG promoter, CB promoter, etc.).

[0143] In any of the above embodiments, in the rDNA vector of the present invention, the rep gene or the cap gene or both can be replaced by a cloning site (e.g., a multiple cloning site or MCS) such that a suitable rep and / or cap gene can be cloned / inserted into the corresponding cloning site. This will facilitate the exchange of any desired rep or cap gene into the rDNA vector / plasmid of the present invention.

[0144] 3. Genes of interest (GOI) in rAAV and treatable diseases

[0145] The systems and methods of the present invention can be used to produce recombinant AAV vectors carrying a gene of interest (GOI) flanked by AAV ITR sequences.

[0146] In certain embodiments, the rDNA vector (plasmid) of the present invention contains ITR sequences derived from AAV1, AAV2, AAV4, and / or AAV6. In certain embodiments, the ITR sequence is the AAV2 ITR sequence.

[0147] As used herein, "gene of interest" or GOI or goi generally refers to a nucleic acid or polynucleotide sequence, such as a gene, an open reading frame (ORF), or the coding sequence of a protein or RNA, such as non-coding RNA, which includes siRNA, miRNA, shRNA, antisense RNA, or its precursor. However, in certain cases or situations, the term GOI also broadly refers to the protein (encoded by the GOI), or a disease or indication that can be treated by the GOI, or a disease or indication that can (but not necessarily) be caused by a loss of function of the GOI.

[0148] For example, and for illustrative purposes only, the gene GALGT2 encodes the protein GalNAc transferase (β-1,4-N-acetylgalactosamine galactosyltransferase), which is an enzyme that transfers complex sugar molecules to some specific proteins (including dystroglycan). Under normal circumstances, GalNAc transferase is only present at the neuromuscular junction (NMJ), where some components of the dystroglycan-associated protein complex are different from other parts of the muscle. Importantly, in the NMJ, utrophin is present instead of dystrophin. In the mdx mouse model of muscular dystrophy, viral gene transfer of GALGT2 results in the expression of GalNAc transferase throughout the sarcolemma, not just in the normal expression domain of the NMJ, and the upregulation of utrophin in the entire muscle fiber. In mdx mice, this expression can correct muscle function defects to the same extent as microdystrophin gene expression. In addition, overexpression of GALGT2 can correct muscle pathology in other muscular dystrophy mouse models (including LGMD2A and congenital muscular dystrophy (MDC1A)). Therefore, GALGT2 is a GOI for treating muscular dystrophy (such as DMD, BMD, LGMD2A, and MDC1A), even if the patient in need of treatment does not necessarily have a defect in GALGT2 itself.

[0149] In another example, sarcolipin (SLN) inhibits the sarcoplasmic / endoplasmic reticulum (SR) Ca 2+ 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 can improve dystrophic pathology in the severe dystrophin / utrophin double mutant (mdx:utr - / - ) DMD mouse model, including reducing muscle pathology and improving diaphragm, skeletal muscle, and heart function. Therefore, the coding sequence of SLN RNAi is a GOI for treating DMD.

[0150] Thus, the GOI can be a gene (or protein) that, when expressed, can replace a mutated, damaged, or inactivated gene or protein. The GOI can be a gene (or protein) that, when expressed, can assist a process that is already functioning and that can benefit from further modification for treating a disease, disorder, or dysfunction. The GOI can be a gene (or protein) that, when expressed, can assist a dysfunctional process that can benefit from further modification for treating a disease, disorder, or dysfunction. The GOI nucleic acid sequence can be DNA, RNA, or a synthetic nucleic acid molecule. The GOI can be a protein, enzyme, structural protein, functional protein, or cell function-based adaptive protein. The GOI can provide a therapeutic benefit or modality for a disease, disorder, or dysfunction.

[0151] In certain embodiments, the recombinant DNA vector of the present invention comprises more than one copy of the GOI. In some embodiments, the plasmid of the present invention comprises more than one copy of the GOI. In certain embodiments, the multiple copies of the GOI are identical. In some other embodiments, the multiple GOIs are different.

[0152] In some other embodiments, the GOI can be a CRISPR / Cas effector enzyme, such as a type II, II, IV, V, or VI effector enzyme, including CRISPR-Cas9, Cas 12, Cas 13, etc. In certain embodiments, the GOI can be a TALEN or other gene-based gene editing protein that functions to achieve its intended activity upon intracellular delivery, such as gene editing or gene knockout in a target cell, tissue, or organism / individual.

[0153] In certain embodiments, the CRISPR / Cas effector enzyme lacks endonuclease activity (dCas, such as dCas9).

[0154] In certain embodiments, the Cas or dCas is further fused with a base editor, such as a cytosine base editor (CBE, e.g., APOBEC, BE1, BE2, BE3, Target-AID base editor, SaBE3, BE3 PAM variant, BE3 editing window variant, AID, CDA1, APOBEC3G, HF-BE3, BE4, BE4max, and AncBE4max), an adenine base editor (ABE, e.g., ABE7.10, ABE 6.3, ABE7.8, ABE7.9, ABEmax, ABE8e (TadA-8e V106W), ABE8 and its variants), or a dual base editor (SPACE, A&C-BEmax).

[0155] Any and all GOIs as used herein may benefit from codon optimization by known computer-based algorithms or other codon optimization methods such as manual optimization to enhance expression and activity.

[0156] In certain embodiments, the GOI is within a pro-AAV cassette that contains the GOI operably linked to a promoter.

[0157] In some embodiments, the promoter operably linked to the GOI is a ubiquitous promoter that drives or facilitates expression of the GOI in most tissues. In some embodiments, the promoter is the elongation factor 1α 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.

[0158] In some other embodiments, the promoter operably linked to the GOI is a tissue-specific promoter, which can be used to restrict expression to certain cell types, such as but not limited to muscle-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters, which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0159] 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 α-actin (ASKA) promoter.

[0160] In some embodiments, the pro-AAV cassette may further contain an enhancer, 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 that promotes transcription of the GOI from the promoter.

[0161] In certain embodiments, the pro-AAV cassette comprises transcriptional regulatory elements, which comprise promoter elements 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 U S A. 96(7):3906-3910, 1999 (all incorporated herein by reference). Each of these transcriptional regulatory elements may comprise a promoter, an enhancer, and / or optionally other nucleotides.

[0162] In certain embodiments, the plasmid of the present invention comprises a promoter region containing one or more promoters, and 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, and the ATG or GTG codons at one or more positions within these promoters are deleted or mutated.

[0163] The GOI can be any gene or coding sequence within the AAV packaging capacity, such as about 4-5 kb, or about 4.7 kb (including the ITR sequences), or about 4.4 kb (excluding the ITR sequences).

[0164] The AAV ITR sequences flanking the GOI can be from any AAV ITR and can also be from the same or different AAV serotypes. In certain embodiments, the AAV ITR sequences flanking the GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0165] In some embodiments, the AAV ITR, AAV Rep, and AAV Cap are from the same AAV. In some other embodiments, the AAV ITR, AAV Rep, and AAV Cap are from different AAVs.

[0166] In certain embodiments, the AAV ITR is the AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0167] In certain embodiments, the tropism of the AAV includes skeletal muscle (such as AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9 or a derivative thereof, such as SLB-101 (above)).

[0168] In certain aspects, rAAV produced by using the DT vector (e.g., DT plasmid) of the present invention and a suitable host cell (which can provide any useful helper function for rAAV production in trans) can encode a gene of interest (GOI), such as a gene that can be used in gene therapy to treat a disease or disorder.

[0169] Representative (non-limiting) genes of interest (GOIs) can include: genes that cause / are defective in the following diseases: 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 or coding sequences for the following: NAGLU (α-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid α-glucosidase (GAA, for Pompe disease), α-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin, or utrophin.

[0170] In certain embodiments, the GOI is the utrophin gene.

[0171] In certain embodiments, the minidystrophin gene is any such minidystrophin gene described in the following patents: US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854 or US10,166,272, each of which is incorporated herein by reference). In certain embodiments, the GOI is a minidystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference). In certain embodiments, the minidystrophin gene is capable of being packaged into an rAAV virion, e.g., having a size not exceeding about 4.7 kb.

[0172] In certain embodiments, the minidystrophin gene contains spectrin-like repeats R16 and R17 in its coding sequence that are capable of restoring nitric oxide synthase (nNOS) activity in the sarcolemma (such as those described in US7,892,824).

[0173] In certain embodiments, the minidystrophin gene contains the coding sequences of the R1, R16, R17, R23, and R24 spectrin-like repeats of full-length dystrophin (i.e., SR1, SR16, SR17, SR23, and SR24, respectively), such as the sequences described in PCT / US2016 / 013733 (incorporated herein by reference). In certain embodiments, in addition to SR1, SR16, SR17, SR23, and SR24, the minidystrophin gene does not encode any other spectrin-like repeats of full-length dystrophin.

[0174] Diseases or disorders that may benefit from rAAV produced by the dual transfection vectors of the present invention include: Huntington's disease, X-linked myotubular myopathy (XLMTM), acid maltase deficiency (e.g., Pompe disease), spinal muscular atrophy (SMA), myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, mitochondrial myopathy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), mucopolysaccharidosis (MPS), metachromatic leukodystrophy (MLD), Batten disease, Rett syndrome, Krabbe disease, Canavan disease, X-linked retinoschisis, achromatopsia (CNGB3 and CNGA3), X-linked retinitis pigmentosa, age-related macular degeneration, neovascular macular degeneration, Pompe disease, Fabry disease, MPS I, II, IIIA, IIIB, Gaucher's disease, Dannon disease, A1At deficiency, Friedreich's ataxia, Wilson disease, Batten disease (CLN1, CLN3, CLN6, CLN8), Wolman disease, Tay-Sachs disease, Niemann-Pick disease type C, CDKL5 deficiency, beta-thalassemia, sickle cell disease.

[0175] In certain embodiments, diseases or disorders that may benefit from rAAV produced by the dual transfection vectors of the present invention may include: Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), Bethlem CMD, Fukuyama CMD, muscle-eye-brain disease (MEB), rigid spine syndrome, Ullrich CMD, Walker-Warburg syndromes (WWS), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), facioscapulohumeral 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), spinal muscular atrophy (SMA).

[0176] In certain embodiments, diseases or disorders that may benefit from rAAV produced from the dual transfection vectors of the present invention can include ion channel diseases, which are generally characterized by muscle weakness, hypotonia, or episodic muscle paralysis. They include Andersen-Tawil syndrome, hyperkalemic periodic paralysis, hypokalemic periodic paralysis, myotonia congenita, Becker myotonia, Thomsen myotonia, paramyotonia congenita, potassium-aggravated myotonia.

[0177] In certain embodiments, diseases or disorders that may benefit from rAAV produced from the dual transfection vectors of the present invention can include mitochondrial diseases, which occur when there is a dysfunction in the structures that produce energy for the cell. Such diseases include: Friedreich ataxia (FA), mitochondrial myopathy, Kearns-Sayre syndrome (KSS), Leigh syndrome (subacute necrotizing encephalomyopathy), mitochondrial DNA depletion syndrome, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), myoclonic epilepsy with ragged red fibers (MERRF), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, progressive external ophthalmoplegia (PEO).

[0178] In certain embodiments, diseases or disorders that may benefit from rAAV produced from the dual transfection vectors of the invention may include myopathies, which are muscle diseases in which muscle fibers do not function properly, resulting in muscle weakness. Myopathies include: Cap myopathies, central core myopathies, congenital myopathies with fiber type disproportion, axial myopathies, central axial diseases, multi-minicore myopathies, myosin storage myopathies, myotubular myopathies, nemaline myopathies, distal myopathies, GNE myopathy / Nonaka myopathy / hereditary inclusion body myopathy (HIBM), Laing distal myopathy, Markesberg-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy / tibial muscular dystrophy, vocal cord and pharyngeal distal myopathy, Welander distal myopathy, endocrine myopathies, hyperthyroid myopathy, hypothyroid myopathy, inflammatory myopathies, dermatomyositis, inclusion body myositis, polymyositis, metabolic myopathies, acid maltase deficiency (AMD, Pompe disease), carnitine deficiency, carnitine palmitoyltransferase 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), scapuloperoneal myopathy.

[0179] In certain embodiments, diseases or disorders that may benefit from rAAV produced from the dual transfection vectors of the invention may include neuromuscular junction diseases, which are caused by the disruption, dysfunction, or absence of one or more key proteins involved in signal transmission between muscle and nerve. Such diseases include: congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), myasthenia gravis (MG).

[0180] In certain embodiments, diseases or disorders that may benefit from rAAV produced from the dual transfection vectors of the invention may include peripheral nerve diseases, in which the motor and sensory nerves that connect the brain and spinal cord to the rest of the body are affected, resulting in impaired sensation, movement, or other functions. Such diseases include: Charcot-Marie-Tooth disease (CMT), giant axonal neuropathy (GAN), cachectic amyotrophy and aging.

[0181] In certain embodiments, the GOI coding sequence contains a polyA signal sequence or polyadenylation site.

[0182] In certain embodiments, the polyadenylation site is the bovine growth hormone (bGH) polyadenylation site.

[0183] In certain embodiments, the polyadenylation site or poly(A) signal sequence is from other suitable sources, such as synthetic sequences or sequences from other eukaryotic genes or viruses.

[0184] In certain embodiments, the GOI coding sequence is partially or fully codon-optimized for expression in mammalian host cells. For example, the 300-350 nucleotides closest to the 3' end of the coding sequence can be codon-optimized for expression in mammalian host cells.

[0185] 4. Composition or kit of a dual-vector (such as dual-plasmid) system

[0186] One aspect of the present invention provides a composition or kit comprising a dual-vector (e.g., dual-plasmid) system, which system comprises any one of the dual recombinant DNA vectors (dual transfection vectors or "DT vectors") of the present invention and an auxiliary plasmid sufficient to support AAV packaging in the presence of the DT vector of the present invention, consisting essentially of or consisting of the same.

[0187] The dual-vector (dual-plasmid) system in the composition or kit of the present invention can be used to produce rAAV. The dual-vector (dual-plasmid) system in the composition or kit of the present invention is suitable for producing rAAV. The dual-vector (dual-plasmid) system in the composition or kit of the present invention is used to produce rAAV. The dual-vector (dual-plasmid) system in the composition or kit of the present invention is used to produce rAAV suitable for gene therapy. The dual-vector (dual-plasmid) system in the composition or kit of the present invention is used to produce rAAV for gene therapy.

[0188] The phrase "dual-vector (dual-plasmid) system" refers to a system that comprises two vectors (e.g., plasmids) and can be used together without additional plasmids to produce rAAV (i.e., they are combined and sufficient for AAV production). In certain embodiments, the dual-vector (plasmid) system can be used to produce rAAV without an auxiliary virus (such as adenovirus). In certain embodiments, the dual-vector (plasmid) system can be used to produce rAAV without genetic material derived from the host cell, optionally excluding the gene encoding El A / B. However, the vector system in the composition / kit of the present invention can comprise additional non-plasmid components. Optionally, the composition or kit of the present invention comprising the dual-vector (plasmid) system of the present invention does not contain an auxiliary virus.

[0189] In certain embodiments, the dual-vector (plasmid) system of the present invention contains all the necessary genetic information for generating rAAV. For example, the dual-vector (plasmid) system of the present invention may contain at least one rep gene, at least one cap gene, and at least one helper gene. In certain embodiments, the dual-plasmid system of the present invention contains all the necessary genetic information for generating rAAV suitable for gene therapy. For example, the dual-plasmid system of the present invention may contain at least one rep gene, at least one cap gene, at least one helper gene, and an expression cassette containing a transgene operably linked to at least one regulatory element.

[0190] One of the vectors / plasmids in the composition or kit of the present invention is a vector / plasmid containing AAV Rep, Cap, and GOI (flanked by AAV ITR sequences). Another vector / plasmid in the composition or kit of the present invention is a helper plasmid / vector.

[0191] In certain embodiments, the helper plasmid / vector may contain at least one helper virus gene. AAV can only replicate in the presence of a helper virus. Examples of helper viruses include adenovirus and herpesvirus.

[0192] In certain embodiments, the helper plasmid of the present invention contains sufficient helper genes to allow AAV replication and packaging. In certain embodiments, the helper plasmid or vector that can be used with the rDNA vector of the present invention provides all the helper genes / functions for AAV packaging (except for the ITR sequences flanking the GOI).

[0193] For example, for host cells expressing the adenovirus E1A / B genes, such as HEK293T cells, the remaining adenovirus helper genes encode E4, E2A, and VA RNA I and II. These helper genes can be included in the helper plasmid or vector in any order or orientation as long as all of these genes can be transcribed and expressed to facilitate AAV packaging.

[0194] Any AAV production assay recognized in the art can be used to evaluate whether the helper plasmid contains sufficient helper genes to promote AAV production, thereby showing a sufficient titer of the AAV produced in this way.

[0195] In one embodiment, if the level of rAAV production supported by the accessory gene product is, for example, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 25% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 40% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 50% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 60% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 70% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 80% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 90% thereof, the level supported by adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II, or at least 95% thereof, or a level within any range defined by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, then the accessory gene product will be considered (sufficient to) facilitate AAV production. In certain embodiments, if the yield of rAAV produced is at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%, or at least 90%, or at least 95% of the yield of rAAV produced using a reference dual-plasmid system, or a level within any range defined by any pair of the foregoing numerical values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%, or a level within any range defined by any pair of the foregoing numerical values, such as, for example, between 50% and 95%, between 60% and 80%, or between 80% and 95%, where one plasmid is an accessory plasmid containing adenoviral accessory genes encoding E4, E2A, and VA RNAs I and II.

[0196] Because the accessory plasmid encodes genes that permit efficient AAV production, there is no need to add accessory virus (i.e., the compositions or kits of the invention containing the dual-vector / plasmids of the invention are sufficient to support AAV production and packaging).

[0197] In certain embodiments, at least one accessory viral gene includes an adenoviral gene. Adenovirus is a virus known to be able to assist AAV dissemination (Xiao et al., J. Virol, 72:2224 - 2232, 1998). In certain embodiments, at least one accessory viral gene includes an adenovirus 5 gene or an adenovirus 2 gene. In certain embodiments, the accessory genes of adenovirus encode E1A, E1B, E4, E2A, and VA RNAs I and II.

[0198] E1A is encoded by nucleotides 560 - 1545 of the adenovirus 5 genome. E1A may optionally lack the non-essential intron at nucleotides 1113 - 1228.

[0199] E1B is actually two proteins, E1B 19K and E1B 55K, which act together to block apoptosis in cells infected with adenovirus. E1B is encoded by nucleotides 1714 - 2244 (E1B 19K) and nucleotides 2019 - 3509 (E1B 55K) of the adenovirus 5 genome.

[0200] E4 is encoded by multiple different open reading frames (ORFs) of the adenovirus 5 genome. E4 ORF 6 / 7 is encoded by nucleotides 32914 - 34077, which may optionally lack the intron between nucleotides 33193 and 33903. E4 34K is encoded by nucleotides 33193 - 34077 of the adenovirus 5 genome. E4 ORF 4 is encoded by nucleotides 33998 - 34342 of the adenovirus 5 genome. E4 ORF 3 is encoded by nucleotides 34353 - 34703 of the adenovirus 5 genome. E4 ORF B is encoded by nucleotides 34700 to 35092 of the adenovirus 5 genome. E4 ORF 1 is encoded by nucleotides 35140 - 35526 of the adenovirus 5 genome. A functional E4 protein may only contain the amino acids encoded by ORF6 and 7, as only the amino acids encoded by ORF 6 and 7 are required for activity. In certain embodiments, the functional E4 protein comprises a polypeptide sequence encoded by all or most of ORF 6 and 7. Optionally, the functional E4 protein does not comprise a polypeptide sequence encoded by all or part of ORF 1 - 4 and 34K.

[0201] In certain embodiments, the functional E4 protein comprises the amino acids encoded by ORF 1 - 7, as the amino acids encoded by ORF1 - 3 and 34K of E4, while not essential, do increase the activity of the E4 protein.

[0202] The E2 (E2A) gene is encoded by nucleotides 22443 - 24032 of the adenovirus 5 genome.

[0203] VA RNA I and II are encoded by nucleotides 10589 - 11044 of the adenovirus 5 genome.

[0204] E1B and E4 are thought to enhance AAV mRNA accumulation, while E2A and VA RNAs I and II are thought to enhance AAV mRNA splicing and translation. E1B, E4, and E2A are proteins encoded by genes present in the adenovirus genome, while the VA nucleic acid encodes two RNA transcripts, called VA RNA I and VA RNA II. The transcripts themselves have functions in the cell and are never translated into an amino acid sequence. Thus, it should be understood that the VA nucleic acid does not encode a protein but "encodes" or "corresponds to" RNA, i.e., although the term "encodes" is used, the VA nucleic acid is a non-translated nucleic acid sequence.

[0205] Among the five adenovirus genes, in some embodiments, E1A or E1B may not be included in the helper plasmid because some host cell lines (such as HEK293 cells) constitutively express one or more of E1A or E1B. In certain embodiments, the helper plasmid does not contain a gene encoding a functional adenovirus E1A / B protein.

[0206] In one embodiment, at least one helper virus gene comprises: (a) a VA (virus-associated) nucleic acid encoding functional VA RNAs I and II; (b) an E2A gene encoding a functional E2A protein; and / or (c) an E4 gene encoding a functional E4 protein.

[0207] In certain embodiments, at least one helper virus gene comprises VA nucleic acids (such as VA RNA I and / or II), an E2A gene, and an E4 gene.

[0208] "Functional" VA RNAs I and II, E2A protein, or E4 protein are capable of promoting AAV production.

[0209] One of ordinary skill in the art can determine whether a given VA RNA I and II, E2A protein, or E4 protein is functional by, for example, using the AAV production assay described above to determine whether the VA RNA I and II, E2A protein, or E4 protein supports AAV production. In some embodiments, a VA RNA I and II, E2A protein, or E4 protein is considered functional if the level of rAAV production supported by the VA RNA I and II, E2A protein, or E4 protein is the level supported by wild-type (e.g., as found in native adenovirus 5) VA RNA I and II, E2A protein, or E4 protein or at least 25% thereof, the level supported by wild-type VA RNA I and II, E2A protein, or E4 protein or at least 40% thereof, the level supported by VA RNA I and II, E2A protein, or E4 protein or at least 50% thereof, the level supported by VA RNA I and II, E2A protein, or E4 protein or at least 70% thereof, the level supported by VA RNA I and II, E2A protein, or E4 protein or at least 80% thereof, the level supported by VA RNA I and II, E2A protein, or E4 protein or at least 90% thereof, the level supported by VA RNA I and II, E2A protein, or E4 protein or at least 95% thereof, or a level within any range defined by any pair of the foregoing values, such as between 50% and 95%, between 60% and 80%, or between 80% and 95%. In certain embodiments, an E4 protein is considered "functional" if the level of rAAV production supported by the E4 protein is the level supported by wild-type E4 protein or at least 70% thereof, the level supported by wild-type E4 protein or at least 80% thereof, the level supported by wild-type E4 protein or at least 90% thereof, the level supported by wild-type E4 protein or at least 95% thereof.

[0210] 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 occur between the VA nucleic acid sequence and the E2A gene sequence in the plasmid. In certain other embodiments, the E2A gene is located between the VA nucleic acid and the E4 gene.

[0211] Any gene on the helper plasmid can be in any orientation, regardless of the other genes.

[0212] In certain embodiments, the VA nucleic acid has an activity level that is the activity of the wild-type VA nucleic acid from adenovirus 5 or at least 75% thereof, the activity of the wild-type VA nucleic acid from adenovirus 5 or at least 80% thereof, the activity of the wild-type VA nucleic acid from adenovirus 5 or at least 90% thereof, the activity of the wild-type VA nucleic acid from adenovirus 5 or at least 95% thereof, or between 95% and 100% of the activity of the wild-type VA nucleic acid from adenovirus 5. The activity level of the VA nucleic acid can be determined, for example, by measuring rAAV production, such as using the AAV production assay described above.

[0213] The expression of the E4 gene is driven by a promoter (designated as the "E4 promoter"). The fully active promoter contains nucleotides corresponding to 35793-35848 of the adenovirus 5 genome. In certain embodiments, the E4 gene is operably linked to an E4 promoter that has at least 50%, at least 70%, or at least 90% of the activity of 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, if the E4 promoter supports the production level of rAAV to be the level supported by the wild-type E4 gene promoter or at least 25% thereof, the level supported by the wild-type E4 gene promoter or at least 40% thereof, the level supported by the wild-type E4 gene promoter or at least 50% thereof, the level supported by the wild-type E4 gene promoter or at least 60% thereof, the level supported by the wild-type E4 gene promoter or at least 70% thereof, the level supported by the wild-type E4 gene promoter or at least 90% thereof, i.e., if the yield of the produced rAAV is the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 50% thereof, the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 70% thereof, the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 90% thereof, then the E4 promoter has the activity of the wild-type E4 promoter from adenovirus 5 or at least 25% of said activity, has the activity of the wild-type E4 promoter from adenovirus 5 or at least 40% of said activity, has the activity of the wild-type E4 promoter from adenovirus 5 or at least 50% of said activity, has the activity of the wild-type E4 promoter from adenovirus 5 or at least 70% of said activity, has the activity of the wild-type E4 promoter from adenovirus 5 or at least 90% of said activity. Preferably, if the E4 promoter supports the production level of rAAV to be the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 70% thereof, the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 80% thereof, the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 90% thereof, the yield of rAAV produced using a reference dual plasmid system comprising wild-type helper genes and a wild-type promoter or at least 95% thereof, then the E4 promoter is considered to facilitate AAV production.

[0214] 5. Host cell

[0215] One aspect of the invention includes a host cell comprising the double recombinant DNA vector (double transfection vector) of the invention.

[0216] Generally, the host cells of the present invention are capable of or suitable for producing rAAV. Host cells are typically derived from eukaryotic cell lines, such as vertebrate cell lines, including mammalian cell lines (e.g., human cell lines).

[0217] 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.

[0218] In certain embodiments, the host cell is selected from the group consisting of: HEK293T cells, HEK293 cells, HEK293EBNA cells, CAP cells, CAP-T cells, AGEl.CR cells, PerC6 cells, C139 cells, and EB66 cells.

[0219] In certain embodiments, the host cell is selected from the group consisting of: HEK293T cells, HEK293 cells, and HEK293EBNA cells.

[0220] In certain embodiments, the host cell is HEK293T cells.

[0221] In certain embodiments, the host cell is a cell that expresses a functional adenovirus E1A / B protein. For example, the host cell may contain a chromosome containing a gene encoding a functional adenovirus E1A / B protein.

[0222] If the level of AAV production supported by the host cell is the level supported by HEK293T cells or at least 30% thereof, the level supported by HEK293T cells or at least 40% thereof, the level supported by HEK293T cells or at least 50% thereof, the level supported by HEK293T cells or at least 70% thereof, the level supported by HEK293T cells or at least 80% thereof, the level supported by HEK293T cells or at least 90% thereof, the level supported by HEK293T cells or at least 95% thereof, then the host cell is considered suitable or capable of producing recombinant AAV. In this case, the test dual-vector / plasmid system of the present invention can be introduced / transfected into the host cell to be tested for its suitability for producing recombinant AAV, and the reference dual-vector / plasmid system can be introduced / transfected into HEK293T cells.

[0223] 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.

[0224] In certain embodiments, the host cell is derived from a vertebrate, such as a human, monkey, bovine, porcine, equine, canine, feline, ovine, caprine, murine, rat, rabbit, mink, opossum, camel, chicken, or other avian, armadillo, frog, or reptile, or from an insect cell.

[0225] In certain embodiments, the host cell is a cell line suitable for AAV packaging, such as Expi cells, HEK293 cells (or derivatives thereof such as HEK293T cells).

[0226] In certain embodiments, the host cell is a HEK293 cell (such as Expi293F cells), HeLa cell, A549 cell, BHK cell, or an insect cell (such as Sf9).

[0227] In certain embodiments, the host cell is HEK293 (human embryonic kidney), which can be grown using standard tissue culture media (such as DMEM supplemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin).

[0228] In some embodiments, HEK293 cells are grown on a solid support, which includes tissue culture plates, dishes, flasks, and bottles. For adherent HEK293 cells in growth, the percentage of FBS can be reduced during rAAV production to limit contamination from animal-derived components.

[0229] In some embodiments, HEK293 cells are suitable for suspension growth.

[0230] In certain embodiments, the host cell is a Vero cell, such as Vero75.4 or V75 cells described herein. Such cells can be grown on a solid support, which includes tissue culture plates, dishes, flasks, bottles, and microcarriers that allow adherent Vero cells to grow in suspension-like conditions.

[0231] In certain embodiments, the host cell is a BHK (baby hamster kidney) cell, such as BHK21 or sBHK27. In certain embodiments, BHK cells are suitable for growth in serum-free suspension.

[0232] In certain embodiments, the host cell is a HEK293 cell. In certain embodiments, HEK293 cells are suitable for growth in serum-free media (such as F17 or Expi293 medium) and suspension, and thus are suitable for large-scale growth in a bioreactor. See, e.g., Grieger et al. (Mol. Ther. 24:287-297, 2016, which is incorporated herein by reference).

[0233] 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.

[0234] In certain embodiments, to produce rAAV particles, the host cell contains helper virus proteins useful (e.g., required) for AAV packaging.

[0235] In some embodiments, the coding sequences of the helper virus proteins are introduced into the host cell in a plasmid by transfection. In some other embodiments, the coding sequences of the helper virus proteins are integrated into the host cell genome.

[0236] In certain embodiments, the host cells of the present invention can be adapted to produce recombinant AAV vectors encoding a gene of interest (GOI), which can be used for gene therapy. See the section entitled "Recombinant AAV Production" below. In such embodiments, one or more rAAV-producing cell lines can be infected with the DT vectors of the present invention (such as vectors or plasmids encoding AAV Rep and Cap proteins).

[0237] 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, which optionally contain a drug selection marker.

[0238] In certain embodiments, such producer cell lines for rAAV production are Vero cells.

[0239] In certain embodiments, such producer cell lines for rAAV production are BHK cells.

[0240] In certain embodiments, such producer cell lines for rAAV production are HEK293 cells.

[0241] In certain embodiments, such producer / host cell lines for rAAV production contain the DT vectors / vectors of the present invention, which contain a GOI flanked by AAV ITR sequences. The GOI can be any of the GOIs described herein that can be used for gene therapy, such as the dystrophin minigene or microminidystrophin gene described in US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2020 / 086844; or US10,166,272, WO 2023 / 018854 or PCT / US2016 / 013733 (each incorporated herein by reference).

[0242] For example, PCT / US2016 / 013733 (WO2016 / 115543A2) provides a dystrophin gene operably linked to a regulatory cassette, wherein the dystrophin gene encodes a protein that comprises: an amino-terminal actin-binding domain; a β-dystroglycan-binding domain; and a spectrin-like repeat domain comprising at least four spectrin-like repeats, wherein two of the at least four spectrin-like repeats comprise a neuronal nitric oxide synthase-binding domain. In certain embodiments, the at least four spectrin-like repeats include spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16), spectrin-like repeat 17 (SR17), and spectrin-like repeat 24 (SR24). In certain embodiments, the protein encoded by the dystrophin gene further comprises at least a portion of a hinge domain, such as at least one of hinge 1 domain, hinge 2 domain, hinge 3 domain, hinge 4 domain, and hinge-like domain. In certain embodiments, the dystrophin gene comprises, in N-terminal to C-terminal order: hinge 1 domain (H1); spectrin-like repeat 1 (SR1); spectrin-like repeat 16 (SR16); spectrin-like repeat 17 (SR17); spectrin-like repeat 24 (SR24); and hinge 4 domain (H4). In certain embodiments, H1 is directly coupled to SR1. In certain embodiments, SR1 is directly coupled to SR16. In certain embodiments, SR16 is directly coupled to SR17. In certain embodiments, SR17 is directly coupled to SR24. In certain embodiments, SR24 is directly coupled to H4. In certain embodiments, the protein encoded by the dystrophin gene further comprises, in N-terminal to C-terminal order, spectrin-like repeat 2 (SR2) and spectrin-like repeat 3 (SR3) between SR1 and SR16. In certain embodiments, SR1 is directly coupled to SR2, and SR2 is further coupled to SR3. In certain embodiments, H1 is directly coupled to SR1, SR1 is directly coupled to SR16, SR16 is directly coupled to SR17, SR17 is directly coupled to SR23, SR23 is directly coupled to SR24, and SR24 is directly coupled to H4.

[0243] In certain embodiments, the regulatory cassette is selected from the group consisting of the CK8 promoter and the cardiac troponin T (cTnT) promoter. In certain embodiments, the protein encoded by the minidystrophin gene has five spectrin-like repeats to eight spectrin-like repeats. In certain embodiments, the protein encoded by the minidystrophin gene has at least 80% or 90% sequence identity with the amino acid sequence of SEQ ID NO:4 or 5 in WO2016 / 115543A2 (incorporated herein by reference).

[0244] In certain embodiments, the GOI is the minidystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0245] 6. Recombinant AAV production

[0246] The recombinant DNA vectors (dual transfection vectors) of the present invention, as well as the production cell lines, can be used for large-scale production of recombinant AAV vectors (rAAV) that can be used in gene therapy. In certain embodiments, the system and related methods of use can be used to propagate / amplify / produce AAV viral particles containing a GOI flanked by ITRs.

[0247] The recombinant replication-defective AAV vectors that can be produced using the recombinant DNA vectors (dual transfection vectors) and production cell lines of the present invention generally contain a gene of interest (GOI) and an expression regulator (such as a promoter of 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 provided by the dual transfection vectors of the present invention, and other auxiliary functions for AAV packaging are provided by another construct or production cell line. The rep ORF encodes four non-structural Rep proteins involved in the AAV viral life cycle, and the cap ORF encodes three structural proteins (i.e., VP1, VP2, and VP3) that form the icosahedral AAV capsid. Generally, 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.

[0248] In certain aspects, the method for propagating / amplifying / producing replication-defective AAV particles encapsulating a GOI includes introducing the recombinant DNA of the present invention into a host cell before, simultaneously with, or after introducing AAV helper genes for viral packaging.

[0249] In some embodiments, the recombinant DNA vector is introduced into the host cell by transient transfection.

[0250] Methods for generating replication-deficient AAV particles include the following steps: co-transfecting mammalian cells, such as but not limited to HEK293 cells, with the recombinant DNA of the present invention and a construct containing AAV helper genes.

[0251] In some embodiments, the AAV helper genes are introduced into the host cells by transfection (e.g., transient transfection). In some other embodiments, the AAV helper genes are integrated into the host cell genome before introducing the dual transfection vector of the present invention into the host cells.

[0252] In certain embodiments, the host cells are derived from vertebrates, such as humans, monkeys, cows, pigs, horses and other equids, canids, felids, sheep, goats, murids, rats, rabbits, minks, opossums, camels and other camelids, chickens and other avians, armadillos, frogs or reptiles, or are derived from insect cells. Human cells include BHK cells, Vero cells, HEK293 cells, etc.

[0253] In certain embodiments, the host cells are HEK293 cells (such as Expi293F cells or VP2 cells), HeLa cells, A549 cells, BHK cells or insect cells (such as Sf9).

[0254] In certain embodiments, the host cells are HEK293 (human embryonic kidney), which can be grown using standard tissue culture media, such as DMEM supplemented with L-Gln, 5-10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0255] In some embodiments, the HEK293 cells are grown on solid supports, which include tissue culture plates, culture dishes, flasks and bottles. For adherent HEK293 cells during growth, the percentage of FBS can be reduced during the rAAV production process to limit contamination caused by animal-derived components.

[0256] In some embodiments, the HEK293 cells are suitable for suspension growth.

[0257] In certain embodiments, the HEK293 cells are suitable for growth in serum-free media (such as F17 or Expi293 medium) and in suspension, and thus are suitable for large-scale growth in bioreactors. See, for example, Grieger et al. (Mol. Ther. 24:287-297, 2016, which is incorporated herein by reference).

[0258] In certain embodiments, the HEK293 cells are HEK293T cells expressing the SV40 T antigen (temperature-sensitive allele tsA1609) and the neomycin / geneticin resistance gene.

[0259] In certain embodiments, the host cell is a Vero cell, such as Vero75.4 or V75 cells as described herein. Such cells can be grown on solid supports, including tissue culture plates, dishes, flasks, bottles, and microcarriers that allow adherent Vero cells to grow under suspension-like conditions.

[0260] In certain embodiments, the host cell is a BHK (baby hamster kidney) cell, such as BHK21 or sBHK27. In certain embodiments, BHK cells are suitable for growth in serum-free suspension.

[0261] In some embodiments, the method further comprises harvesting replication-defective AAV particles comprising the GOI. In some embodiments, the method comprises purifying recombinant replication-defective AAV particles.

[0262] The gene of interest (GOI) may include genes that can be used in gene therapy to treat certain diseases or disorders. Representative (non-limiting) GOIs may include genes that cause / are defective in the following diseases: 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 or coding sequences for the following: NAGLU (α-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid α-glucosidase (GAA, for Pompe disease), α-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin, or utrophin.

[0263] Suitable dystrophin 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 (each incorporated herein by reference). In certain embodiments, the GOI is a dystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0264] Diseases or disorders that may benefit from rAAV generated from the DT vectors of the systems according to the invention include: Huntington's disease, X-linked myotubular myopathy (XLMTM), acid maltase deficiency (e.g., Pompe disease), spinal muscular atrophy (SMA), myasthenia gravis (MG), amyotrophic lateral sclerosis (ALS), Friedreich's ataxia, mitochondrial myopathy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), facioscapulohumeral muscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), mucopolysaccharidosis (MPS), metachromatic leukodystrophy (MLD), Batten disease, Rett syndrome, Krabbe disease, Canavan disease, X-linked retinoschisis, achromatopsia (CNGB3 and CNGA3), X-linked retinitis pigmentosa, age-related macular degeneration, neovascular macular degeneration, Pompe disease, Fabry disease, MPS I, II, IIIA, IIIB, Gaucher disease, Dannon disease, A1At deficiency, Friedreich's ataxia, Wilson disease, Batten disease (CLN1, CLN3, CLN6, CLN8), Wolman disease, Tay-Sachs disease, Niemann-Pick disease type C, CDKL5 deficiency, β-thalassemia, sickle cell disease, etc.

[0265] As a naturally occurring replication-defective human parvovirus, wild-type AAV will integrate its genome site-specifically into the host cell chromosome in the absence of a helper virus to assist replication, where it will persist indefinitely in the host cell chromosome unless rescued by infection of the cell with a helper virus. Introduction of the helper virus into the host cell triggers AAV replication and the production of progeny virions. In the case of rAAV virions used for gene therapy, introduction of the helper virus functions into a suitable host cell triggers packaging of the GOI in the rAAV virions, while also providing the necessary rep and cap coding sequences in the same system.

[0266] In other words, the production of recombinant AAV depends on (1) the presence of the AAV rep and cap coding sequences, and (2) the functions of a helper virus.

[0267] In certain embodiments, the tropism of AAV includes the following serotypes, such as AAV1, AAV2, AAV6, AAV7, AAV8, or AAV9, AAV10, AAV11, preferably AAV9, AAVrh74, AAVrh10. In certain embodiments, the AAV capsid can be genetically modified, or the capsid can be a synthetic, designed capsid that enhances tissue specificity or physiological compartmentalization to deliver the GOI to specific tissues, such as muscle, skeletal muscle, cardiac muscle, smooth muscle, etc., as described (see, e.g., Zinn and Grimm, High-Throughput Dissection of AAV–Host Interactions: The Fast and the Curious, JMB 430(17):2626-2640, 2018; Kotterman and Schaffer, Engineering adeno-associated viruses for clinical gene therapy. Nature Reviews Genetics (2014) 4445-4451, both of which are incorporated herein by reference) and / or does not target certain tissues. The tropism of AAV can also be achieved by pseudotyping, or mixing capsids and genomes from different viral serotypes. These serotypes are indicated by a slash, so AAV2 / 5 represents a virus containing a genome of serotype 2 packaged in a capsid of serotype 5. The use of these pseudotyped viruses can improve transduction efficiency and 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, computer-derived sequences are de novo synthesized and characterized for biological properties relevant to clinical applications.This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80 (the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9) as a highly efficient in vivo gene therapy vector for targeting the 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).

[0268] In certain embodiments, the tropism of AAV includes skeletal muscle (such as AAV1, AAV6, AAV7, AAV8, or AAV9, preferably AAV9). In certain embodiments, the tropism of AAV includes cardiac muscle (such as AAV1, AAV5, AAV6, AAV8, AAV9, AAVrh74, AAV2 - 8, AAV2 - 9). In certain embodiments, AAV does not target certain tissues, such as the liver. In certain embodiments, the tropism of AAV targets certain tissues and does not target certain tissues.

[0269] In certain embodiments, the gene of interest (GOI) includes genes that cause or are defective in the following diseases: 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 or coding sequences for the following: NAGLU (α - N - acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain - 3 (CAPN - 3), acid α - glucosidase (GAA, for Pompe disease), α - galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin, or minidystrophin.

[0270] In certain embodiments, the GOI is a functional equivalent of dystrophin (e.g., a minidystrophin gene encoding functional minidystrophin).

[0271] In certain embodiments, the GOI is the minidystrophin gene.

[0272] In certain embodiments, the minidystrophin gene is one described in the following: US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023 / 018854 or US10,166,272 (each of which is incorporated herein by reference). In certain embodiments, the GOI is the minidystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0273] In certain embodiments, the minidystrophin gene comprises the coding sequences of the R16 and R17 spectrin-like repeats of full-length dystrophin (such as the sequences described in US7,892,824).

[0274] In certain embodiments, the minidystrophin gene comprises the coding sequences of the R1, R16, R17, R23 and R24 spectrin-like repeats of full-length dystrophin (such as the minidystrophin gene described in PCT / US2016 / 013733).

[0275] In certain embodiments, the minidystrophin gene does not comprise the coding sequences and spectrin-like repeats of full-length dystrophin other than the SR1, SR16, SR17, SR23 and SR24 repeats (e.g., in this order).

[0276] In certain embodiments, the rAAV vectors of the invention are produced under in vitro culture conditions, such as in a bioreactor (e.g., a 0.5L, 1L, 2L, 3L, 5L, 10L, 20L, 50L, 100L, 250L, 500L or 1,000L working volume bioreactor), such as a CelliGen Plus packed bed bioreactor (New Brunswick Scientific) for fed-batch vector production within 3 days after infection.

[0277] In certain embodiments, recombinant replication-deficient AAV viral particles are produced as an in vitro culture on an adhesion-dependent cell line that is dependent on a solid support, such as a Vero and Vero-derived cell line or a HEK293 cell and HEK293-derived cell. In certain embodiments, the solid support is a tissue culture surface, such as a tissue culture dish, plate, flask, bottle, cell factory, etc. In certain embodiments, the solid support is a microcarrier, such as Cytodex 1 (GE Healthcare Life Sciences, Piscataway, NJ); a macrocarrier, such as FibraCel (New Brunswick Scientific, Edison, NJ), or a multi-layer culture vessel, such as a CellCube (Corning Life Sciences, Lowell, MA) that allows medium perfusion.

[0278] In certain embodiments, recombinant replication-deficient AAV viral particles are produced as an in vitro culture in eukaryotic cells adapted to suspension growth, such as a suspension culture of a BHK cell line or a HEK293 cell line adapted to suspension growth.

[0279] In certain embodiments, the culture supernatant yields more than 1×10 10 plaque-forming units (PFU) of rAAV, 1×10 11 plaque-forming units (PFU) of rAAV, 1×10 12 plaque-forming units (PFU) of rAAV, 1×10 13 plaque-forming units (PFU) of rAAV, 1×10 14 plaque-forming units (PFU) of rAAV, or yields within any range defined by any pair of the foregoing values, e.g., such as between 1×10 10 PFU and 1×10 14 PFU, between 1×10 11 PFU and 1×10 13 PFU, or between 1×10 12 PFU and 1×10 14 PFU.

[0280] In certain embodiments, the vector bulk is produced by one or more post-treatment steps, such as filtration and / or concentration (e.g., depth filtration, dead-end filtration, tangential flow filtration (TFF), and diafiltration), multi-column chromatography purification, final concentration / buffer exchange, etc., to obtain a vector bulk with sufficient purity to be administered to animals (including humans). In certain embodiments, the purification process and the purified vector bulk meet GMP standards.

[0281] In certain embodiments, the titer of the rAAV vector stock solution is about 1 - 2×10 7 PFU / ml, about 1 - 2×10 8 PFU / ml, about 1 - 2×10 9 PFU / ml, about 1 - 2×10 10 PFU / ml, about 1 - 2×10 11 PFU / ml, about 1 - 2×10 12 PFU / ml, or a titer within any range defined by any pair of the foregoing values.

[0282] In certain embodiments, the total yield of the rAAV vector stock solution is about 1 - 25×10 14 total VG of purified rAAV, about 1 - 10×10 14 total VG of purified rAAV, about 1 - 5×10 14 total VG of purified rAAV, about 2 - 4×10 14 total VG of purified rAAV, or a total yield within any range defined by any pair of the foregoing values.

[0283] In certain embodiments, the rAAV vector thus produced is further purified from the crude cell lysate by ion exchange chromatography and / or by iodixanol density gradient centrifugation to ensure high purity of the final product. In certain embodiments, the rAAV vector thus produced qualifies as a clinical-grade vector batch.

[0284] In certain embodiments, the method for producing AAV of the present invention further includes determining the titer, purity, and / or potency of the rAAV vector thus produced. This may include characterizing the purified rAAV stock solution by determining purity using, for example, silver staining of proteins separated by SDS-PAGE.

[0285] 7. Treatment of muscular dystrophy using AAV

[0286] The DT vector of the system based on the present invention can be used to produce rAAV on a large scale, which can then 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), facioscapulohumeral muscular dystrophy (FSH), congenital muscular dystrophy (CDM), oculopharyngeal muscular dystrophy (OPMD), distal muscular dystrophy, Emery-Dreifuss muscular dystrophy (EDMD), etc. In certain embodiments, the muscular dystrophy is DMD or BMD.

[0287] Accordingly, another aspect of the present invention provides a method of treating a subject in need of treatment for muscular dystrophy (such as DMD and BMD), the method comprising administering to the subject a therapeutically effective amount of a recombinant AAV (rAAV) encoding a functional form of a gene defective in muscular dystrophy, such as the microdystrophin gene, wherein the rAAV is produced by the method of the present invention using the DT vector and complementary system of the present invention.

[0288] In certain embodiments, the microdystrophin gene is one described in the following: US7,906,111; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2020 / 086844; WO2016115543; WO 2023 / 018854 or US10,166,272 (each document is incorporated herein by reference). In certain embodiments, the GOI is the microdystrophin gene having the nucleotide sequence of SEQ ID NO:1 of WO 2023 / 018854 (incorporated herein by reference).

[0289] In certain embodiments, the microdystrophin gene comprises the coding sequences of the R1, R16, R17, R23, and R24 spectrin-like repeats of full-length dystrophin (such as the sequences described in PCT / US2016 / 013733).

[0290] In certain embodiments, the method further comprises producing rAAV by the method of the present invention using the DT vector system of the present invention, and then administering the so-produced rAAV to the subject.

[0291] 8. Treatment of cardiomyopathy using AAV

[0292] The DT vector of the system based on the present invention can be used for large-scale production of rAAV, which can in turn be used in gene therapy for treating various forms of cardiomyopathy. Cardiomyopathy represents a collection of multiple disorders of the myocardium and is the second most common cause of heart disease in subjects, and medical management of its secondary signs is the only treatment option. These diseases have multiple causes, symptoms, and treatments, and can affect people of all ages and races. When cardiomyopathy occurs, the normal muscle of the heart thickens, hardens, thins, or fills with substances produced by the body that do not belong to the myocardium. As a result, the heart's ability to pump blood decreases, leading to arrhythmias, blood reflux to the lungs or other parts of the body, and heart failure. Cardiomyopathy can be acquired or genetic. Its cause is not always known, but the understanding of the genetic basis of the genetic form of the disease is increasing. Gene transfer strategies have been shown to improve heart diseases.

[0293] Cardiomyopathy is a class of heart muscle diseases that adversely affect the heart's ability to circulate blood through the cardiovascular system. There are various types of cardiomyopathy, including dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic cardiomyopathy (AVC), left ventricular non-compaction cardiomyopathy (LVNC), and restrictive cardiomyopathy (RCM). There are multiple genetic forms of cardiomyopathy, including but not limited to DCM associated with Duchenne muscular dystrophy and Becker muscular dystrophy. In some cases of Becker-type muscular dystrophy and in most cases of Duchenne muscular dystrophy, cardiomyopathy ultimately limits the patient's survival. Accordingly, some aspects of the present invention provide AAV or rAAV vectors for directly, systemically, or by both methods delivering a transgene into the heart of a subject.

[0294] 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 that encodes a functional form of a gene defective in cardiomyopathy, such as the BAG3 gene, the MYBPC gene, the TMEM43 gene, the TNNT2 gene, the RBM20 gene, the RYR2 gene, the CASQ2 gene, wherein the rAAV is produced by the method of the present invention using the DT vector and complementary system of the present invention.

[0295] Other genes associated with various cardiomyopathies include the following:

[0296] Table 1

[0297]

[0298]

[0299]

[0300]

[0301]

[0302] Examples

[0303] Example 1 AAV production using a dual transfection system

[0304] Transfection and Sampling

[0305] Approximately 24 hours prior to transfection, HEK293 cells (Expi293 TM working cell bank) were seeded into 50 mL of fresh Freestyle TMIn F17 medium (supplemented with 4 mM Glutamax, 0.1% F68), at a concentration of 1.5×10 6 (1.5E6) cells / mL. On the day of transfection, the target cell density was 2.5E6 - 3.5E6 cells / mL. By adding the plasmid to 15 mL of unsupplemented Freestyle TM F17 medium in a test tube, mixing, adding PEI (polyethyleneimine) transfection reagent, incubating the mixture for 5 minutes, and then mixing the mixture again by inverting the test tube, a plasmid mixture was prepared. Each plasmid mixture was added to the cells in each shake flask. The cells and the plasmid mixture were gently mixed together and then incubated at 110 RPM, 5% CO2, and 37 °C. Approximately 2 - 20 hours after transfection, 50 μL of Gibco TM anti - cell clumping agent was used to neutralize the plasmid.

[0306] Samples were collected from the flasks 2 - 4 days after transfection and the cells were lysed by treatment with Triton - X. After centrifugation, the supernatant of the centrifuged lysed cells was collected and stored at - 80 °C for ddPCR analysis.

[0307] Production of AAV9 using a dual - transfection system

[0308] A dual plasmid containing the RepCap gene of AAV9 and a micro - dystrophin variant as the gene of interest (GOI) was transfected into HEK293 cells at a dual plasmid:helper plasmid (w / w) ratio of 1:1 or 1.5:1 as described above. Experimental details and results are described in Figure 2 in.

[0309] As Figure 2 shown, dual transfection using the dual vector of the present invention containing the RepCap and GOI sequences in one plasmid is sufficient to produce AAV particles.

[0310] Example 2 Comparison of dual - transfection and triple - transfection methods - titer

[0311] Example 2A

[0312] Methods for the production of AAV9 and AAV - SLB101 using dual - transfection (DT) and triple - transfection (TT) were carried out using the transfection method described in Example 1 above, where the dual:helper (w / w) ratio was 1:1 and the RepCap:GOI:helper (w / w) ratio was 1:1:1. The amount of each plasmid added to HEK293 cells for transfection was 0.25 pg / cell. The produced AAV9 and AAV - SLB101 were assayed 24, 48, and 72 hours after transfection as measured by ddPCR.

[0313] It should be understood that although AAV9 and SLB101 are used in this embodiment to demonstrate the effectiveness of the dual plasmid system of the present invention for AAV production and the surprisingly higher titers compared to the triple transfection method, the capsids that can be used for such dual transfection vectors / plasmids are not limited.

[0314] Virus production between the two transfection methods was compared, and the results are shown in Figure 3A .

[0315] Surprisingly, at all measured time points, the virus titers of AAV9 and AAV - SLB101 in the dual transfection method were at least 2 to 3 times (i.e., 200 - 300%) that of the triple transfection method. See Figure 3A .

[0316] This experiment shows that compared to the traditional triple transfection method (where the GOI, RepCap, and helper are encoded by 3 independent plasmids), the dual transfection method using the DT vector of the present invention (containing the GOI and Rep / Cap sequences) produced a surprisingly higher virus yield.

[0317] Example 2B

[0318] Using the dual transfection system and triple transfection system of the present invention, 15 cell cultures were used to produce vectors containing AAV - SLB101 (AV - SLB101 - uDys) with the micro - dystrophin GOI, which is a high - throughput, automated advanced microbial reactor system for 48 parallel cultures at a scale of 10 - 15 mL microbial reactors and according to the parameters in Table 2 below.

[0319] Table 2

[0320]

[0321]

[0322] Figure 3B Shows the titers of each sample on the 4th day after transfection with FectoVIR. The virus titer using triple transfection (TT) only reached a maximum of less than 1E11 vg / mL from less than 3.2E10 vg / mL. However, the virus titers using the dual transfection (DT) method of the present invention ranged from a minimum of 1.85E11 vg / mL to as high as 3.83E11 vg / mL, representing an almost 100% to almost 400% increase in titer compared to the triple transfection method.

[0323] Example 2C

[0324] The AAV vector (AAV-SLB101-uDys) in Example 2B was produced on a larger scale using a 2L bioreactor according to the parameters in Table 3 below.

[0325] Table 3

[0326] Batch generator Scale Culture medium Quenching USP-276-V02 2L Expi293 Yes USP-276-V03 2L Expi293 Yes USP-276-V04 2L Expi293 Yes USP-276-V06 2L Expi293 Yes USP-278-V01 2L F17 No USP-278-V03 2L F17 No USP-278-V06 2L F17 No USP-278-V07 2L Expi293 No USP-278-V08 2L Expi293 No USP-272-V01 2L Expi293 Yes USP-272-V02 2L Expi293 Yes USP-272-V03 2L Expi293 Yes USP-272-V08 2L Expi293 Yes USP-272-V09 2L Expi293 Yes USP-271-V03 2L Expi293 Yes USP-271-V05 2L Expi293 Yes

[0327] Results are shown in Figure 3C where all samples reached at least 4.55E11 vg / mL and up to 1E12 vg / mL ( Figure 3C ). Compared to Figure 3B , the yield was well converted from a 10 - 15 mL microbial reactor (Example 2B) to a 2L bioreactor, supporting the scalability of the dual transfection system of the present invention.

[0328] Example 3 Comparison between dual transfection and triple transfection methods - mispackaging rate

[0329] Using 15 cell cultures (see above), the kanamycin titer was measured, the kanamycin titer relative to the GOI titer was calculated, and a comparison was made between the DT system and the triple transfection system of the present invention, where two commercially available transfection reagents were used, namely the Gibco AAV-MAX transfection kit (ThermoFisher Scientific) and -AAV transfection reagent (Polyplus, NY) for large-scale rAAV production.

[0330] Figure 4 It was shown that based on the ratio of kanamycin to GOI determined by ddPCR using AAV-MAX and FectoVIR as transfection reagents, the kanamycin titer at harvest using the DT plasmid system of the present invention was much lower than that of the TT system.

[0331] Example 4 Comparison of GOI titer and packaging of p5 promoter into GOI

[0332] In the DT plasmid systems of the present invention with P5 promoter and modified P5 promoter, the GOI titer and P5:GOI percentage were measured by ddPCR.

[0333] For the DT plasmid system with the modified P5 promoter, the wild-type AAV2 P5 promoter in the RepCap cassette was modified by inserting an exogenous 5-nucleotide spacer sequence between the REP binding site and the YY1 binding site of the wild-type P5 promoter (P5SPC promoter). In Figure 5Among them, the DT plasmid of the present invention containing the P5SPC promoter that drives the transcription of the RepCap cassette is designated as "P5SPC-SLB101" (DT-P5SPC-SLB101-1 and DT-P5SPC-SLB101-2). Compared with other samples, for DT-P5SPC-SLB101-1 and DT-P5SPC-SLB101-2, the mis-packaging of the DNA upstream of the P5 promoter in the plasmid vector (taking the KanR coding sequence as an example) is significantly reduced while maintaining the recombinant AAV titer.

[0334] Figure 5 It shows that for the DT plasmid with the P5SPC promoter (DT-P5SPC-SLB101), it is relatively low for GOI kanamycin (about 2.6%), compared with the DT plasmid with the wild-type P5 promoter (DT-SLB101, about 15%), where AAV-MAX and FectoVIR transfection reagents are used (see DT-SLB101-1 and DT-SLB101-2 compared with DT-P5SPC-SLB101-3 and DT-P5SPC-SLB101-4). See also the data summarized in Table 4 below.

[0335] Table 4

[0336]

[0337]

[0338] Meanwhile, using the DT plasmid with the P5SPC promoter and the DT plasmid with the wild-type P5 promoter, the titers of the AAV viruses produced using the AAV-MAX and FectoVIR transfection reagents are significantly higher than the titers of the AAVs produced using triple transfection ( Figure 5 ).

Claims

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

2. The vector according to claim 1, which is a plasmid.

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

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

5. A plasmid comprising: (a) a gene of interest (GOI) flanked by a 5' adeno-associated virus (AAV) ITR sequence and a 3' ITR sequence; (b) a coding sequence of AAV Rep compatible with the 5' AAV ITR and the 3' AAV ITR, optionally, the coding sequence of the AAV Rep is under the transcriptional control of an AAV P5 promoter located downstream or 3' of the GOI; and (c) a coding sequence of AAV Cap; wherein in a host cell comprising AAV helper genes, the expression of the AAV Rep 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.

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 according to any one of claims 1-6, wherein the GOI is located within a pro-AAV cassette comprising the GOI operably linked to a promoter.

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

9. The vector according to any one of claims 1-8, wherein the coding sequence of the AAV Rep and the coding sequence of the AAV Cap are located within a RepCap cassette comprising an operably linked RepCap promoter (such as the AAV P5 promoter).

10. The vector according to claim 9, wherein the RepCap cassette and the pro-AAV cassette: i) are adjacent to each other (e.g., there is substantially no intervening polynucleotide sequence); ii) are not adjacent to each other; iii) have the same transcriptional orientation; or iv) have opposite transcriptional orientations.

11. The vector according to claim 10, further comprising a bacterial replication Ori gene, a selection marker (such as an antibiotic resistance gene, e.g., Kan R or Amp R ), which is under the transcriptional control of a selection marker promoter (such as a bacterial promoter).

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

13. The vector according to any one of claims 1-12, wherein the GOI comprises a gene that causes / has a defect in the following diseases: 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 or coding sequence of the following: NAGLU (α-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPSIIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), factor IX, factor VIII, myotubularin 1 (MTM1), survival motor neuron protein (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid α-glucosidase (GAA, for Pompe disease), α-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or micro-dystrophin.

14. The vector according to any one of claims 1-13, wherein the GOI is the microdystrophin gene (e.g., the genes 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).

15. The vector according to claim 14, wherein the microdystrophin gene comprises the coding sequences of the R16 and R17 spectrin-like repeats of full-length dystrophin (such as the sequences described in US7,892,824).

16. The vector according to claim 15, wherein the microdystrophin gene comprises the coding sequences of the R1, R16, R17, R23 and R24 spectrin-like repeats of full-length dystrophin (such as the microdystrophin gene described in WO2016115543 or SEQ ID NO:1 of WO 2023 / 018854).

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

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

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

20. The vector according to claim 19, wherein the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or its derivatives.

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

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

23. The vector according to any one of claims 1-22, wherein the AAV helper genes include adenovirus, herpesvirus, or papillomavirus genes for AAV packaging (such as E1A, E1B, E2A, E4, and VA RNA), which are optionally operably linked to a promoter as a transcription unit.

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

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

26. The composition or kit according to claim 25, wherein the helper virus genes include: (i) adenovirus genes, optionally adenovirus 5 or adenovirus 2 genes; and / or (ii) VA nucleic acids 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 the vector according to any one of claims 1-23 or the dual plasmid system, or the composition according to any one of claims 24-26.

28. The host cell according to claim 27, which is a HEK293 cell (such as 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 (such as Sf9).

29. A method for propagating / amplifying / generating recombinant replication-deficient AAV viral particles encapsulating a GOI of any one of claims 1-23, the method comprising: Before, simultaneously with, or after introducing the AAV helper gene according to any one of claims 1-23 into the host cell, introducing the vector according to any one of claims 1-23 into the host cell, thereby propagating / amplifying / producing the recombinant replication-defective AAV virus particles.

30. The method according to claim 29, further comprising harvesting the recombinant replication-defective AAV virus particles from the host cell.

31. The method according to claim 29 or 30, wherein the host cell is a HEK293 cell (such as 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 (such as Sf9).

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

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