Bidirectional rep-cap plasmid for production of aav

Abstract

The present disclosure provides improved adenoviral plasmids for the production of recombinant adeno-associated viruses.

Description

Attorney Docket No. 2013906-0060BIDIRECTIONAL REP-CAP PLASMID FOR PRODUCTION OF AAVCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 708,096 filed on October 16, 2024, the entire contents of which are hereby incorporated by reference in its entirety.BACKGROUND

[0002] Adeno-associated virus (AAV) technology has quickly become a dominant form of gene therapy for genetic diseases. Recombinant adeno-associated viruses (rAAVs) can be produced on a large scale in a variety of host cell systems.SUMMARY

[0003] AAVs can be produced on a large scale in a variety of host cell systems, including mammalian cells, e.g., HEK293 cells and insect cells, e.g., Sf9 cells. Traditionally, AAV production in cells involves the introduction of several plasmids to the host cells, the plasmids encoding, for example, a human gene or genes of interest flanked by AAV ITR sequences, and various viral genes critical for viral replication and packaging. Due to the number of genes required for viral DNA replication, capsid formation, and packaging, these are traditionally delivered on two or three separate plasmids.

[0004] The present disclosure, among other things, provides certain insights and technologies related to improved AAV packaging plasmids, specifically including Rep-Cap plasmids that, in many embodiments, may improve AAV yield.

[0005] Among other things, the present disclosure provides an adenoviral packing plasmid comprising (i) a first expression cassette comprising a Rep gene, and (ii) a second expression cassette comprising a Cap gene, wherein the first and second expression cassette are in an opposite transcriptional orientation relative to one another.

[0006] In some embodiments, a Rep gene described herein is derived from AAV serotype 2 (AAV2). In some embodiments, a Rep gene described herein comprises (i) a Rep78 encodingPage 1 of 9213040886vlAttorney Docket No. 2013906-0060 sequence, (ii) a Rep68 encoding sequence, (iii) a Rep52 encoding sequence, and (iv) a Rep40 encoding sequence.

[0007] In some embodiments, a first expression cassette described herein is truncated. In some embodiments, a Rep gene described herein is truncated. In some embodiments, a Rep78 encoding sequence is truncated by 300 nucleotides as compared to a canonical Rep78 encoding sequence. In some embodiments, a Rep52 encoding sequence is truncated by 300 nucleotides as compared to a canonical Rep52 encoding sequence. In some embodiments, a first expression cassette encodes only Rep78 and Rep 52.

[0008] In some embodiments, a Cap gene described herein is derived from AAV serotype 9 (AAV9). In some embodiments, a Cap gene is derived from AAV serotype rh.74 (AAVrh74). In some embodiments, a Cap gene is derived from AAV serotype rh.10 (AAVrhlO). In some embodiments, a Cap gene is derived from AAV serotype 2 (AAV2). In some embodiments a Cap gene is derived from AAV serotype 2_7m8 (AAV2_7m8). In some embodiments a Cap gene is derived from AAV serotype 3B (AAV3B). In some embodiments a Cap gene is derived from AAV serotype 6 (AAV6).

[0009] In some embodiments, a first expression cassette described herein further comprises a splice donor sequence and one or two splice acceptor sequence(s). In some embodiments, a splice donor sequence is or is the complement of SED ID NO: 1. In some embodiments, a splice donor sequence is or is the complement of SEQ ID NO: 4.

[0010] In some embodiments, a second expression cassette described herein further comprises a splice donor sequence and one or two splice acceptor sequence(s). In some embodiments, a splice donor sequence described herein is or is the complement of SEQ ID NO: 1. In some embodiments, a splice donor sequence is or is the complement of SEQ ID NO: 2. In some embodiments, a splice donor sequence is or is the complement of SEQ ID NO: 3.

[0011] In some embodiments, a first expression cassette described herein further comprises at least one poly(A) sequence element. In some embodiments, a first expression cassette comprises two poly(A) sequence elements.

[0012] In some embodiments, a second expression cassette described herein further comprises at least one poly(A) sequence element. In some embodiments, a second expression cassette comprises two poly(A) sequence elements.Page 2 of 9213040886vlAttorney Docket No. 2013906-0060

[0013] In some embodiments, a first expression cassette described herein comprises a p5 promoter. In some embodiments, a p5 promoter is upstream of a Rep gene. In some embodiments, a p5 promoter is operably linked to a Rep gene described herein.

[0014] In some embodiments, a first expression cassette described herein comprises a pl9 promoter. In some embodiments, a p!9 promoter is within a Rep gene described herein.

[0015] In some embodiments, a first expression cassette described herein comprises a p40 promoter. In some embodiments, a p40 promoter is downstream of a p!9 promoter and upstream of a splice donor sequence described herein.

[0016] In some embodiments, a second expression cassette described herein comprises a p40 promoter. In some embodiments, a p40 promoter described herein is upstream of a Cap gene described herein. In some embodiments, a p40 is operably linked to a Cap gene. In some embodiments, a p40 promoter is upstream of a splice donor sequence described herein.

[0017] Among other things, the present disclosure also provides a method of producing recombinant AAV (rAAV) particles comprising transfecting a producer cell with (i) a plasmid as described herein, (ii) an adenoviral helper plasmid, (iii) an AAV vector plasmid, or (iv) any combination of (i) - (iv). In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is HEK293 or derivative cell line thereof. In some embodiments, an adenoviral helper plasmid described herein comprises a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 5.

[0018] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 6.

[0019] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atPage 3 of 9213040886vlAttorney Docket No. 2013906-0060 least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 7.

[0020] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 8.

[0021] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 9.

[0022] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 10.

[0023] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 11.

[0024] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 12.

[0025] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atPage 4 of 9213040886vlAttorney Docket No. 2013906-0060 least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 13.

[0026] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 14.

[0027] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 15.

[0028] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 16.

[0029] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 17.

[0030] In another aspect, the present disclosure also provides a nucleic acid comprising a nucleotide sequence is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 18.

[0031] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.Page 5 of 9213040886vlAttorney Docket No. 2013906-0060BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A-1F show a comparison of a canonical Rep-Cap plasmid design (labeled “I.”) with a bidirectional Rep-Cap plasmid design (labeled “II ”). Figure 1A shows two expression cassette solutions for an AAV Rep-Cap plasmid. Figure IB shows a map of bidirectional Rep- Cap expression cassette. Figure 1C shows a plasmid map of AAVrep2cap9-insert3C_pUC57- Kan. Figure ID shows a plasmid map of new_ptrl30-cag-egfp-hbgpasv40pa-3schimin. Figure IE shows AAV9 yield obtained (VG / mL) using each Rep-Cap plasmid design. Figure IF shows Rep and Cap protein expression levels during a 96 hour time course.

[0033] Figures 2A-2B shows exemplary expression cassette solutions. Figure 2A shows VP protein ratio regulation using alternative splicing in a natural AAV Cap protein expression cassette. Figure 2B depicts a truncated Rep expression cassette in a bidirectional Rep-Cap plasmid that results in appearance of a Rep protein variant with a higher molecular weight (e.g., a molecular weight higher than 40 kDa).

[0034] Figures 3A-3K shows comparison of exemplary Rep-Cap plasmid designs with a modified Swal site or a modified splice donor site. Figure 3A shows an expression cassette illustration for bidirectional Rep-Cap plasmid pInvRep2Cap9_B comprising a modified splice donor site. Figure 3B shows a plasmid map of pInv2Rep2Cap9_B . Figure 3C shows an expression cassette illustration for bidirectional Rep-Cap plasmid pInvRep2Cap9_C comprising a modified splice donor site. Figure 3D shows a plasmid map of pInv2Rep2Cap9_C. Figure 3E depicts a bidirectional Rep-Cap plasmid pInvRep2Cap9 sequence map with splice donor site modifications for pInvRep2Cap9_B and pInvRep2Cap9_C indicated. Figure 3F shows an expression cassette illustration for bidirectional Rep-Cap plasmid pInvRep2Cap9_A comprising an inactivated Swal site. Figure 3G shows a plasmid map of pInv2Rep2Cap9_A. Figure 3H shows a table of an exemplary canonical Rep-Cap plasmid and exemplary bidirectional Rep-Cap plasmids. Figure 31 shows a table of experimental combinations of exemplary AAV Rep-Cap plasmids with an AAV helper plasmid and an AAV plasmid comprising a gene of interest (GOI). Figure 3 J shows western blotting analysis results for Cap protein (VP1, VP2, VP3) yield of each Rep-Cap plasmid design. Figure 3K shows AAV9 yield obtained (VG / mL) using each Rep-Cap plasmid design.Page 6 of 9213040886vlAttorney Docket No. 2013906-0060

[0035] Figures 4A-4F show a comparison of exemplary Rep-Cap plasmid designs. Figure 4A shows an expression cassette of bidirectional Rep-Cap plasmid p!nvRep2Cap9_F(G) which harbors a stop codon in the Rep gene cassette. Figure 4B shows a plasmid map of p!nv2Rep2Cap9_F. Figure 4C depicts a bidirectional Rep-Cap plasmid p!nvRep2Cap9_F(G) sequence map. Figure 4D shows AAV9 yield obtained (VG / mL) using each Rep-Cap plasmid design. Figure 4E depicts a western blot showing Cap protein (VP1, VP2, VP3) expression from each Rep-Cap plasmid. Figure 4F depicts a western blot showing Rep78 and Rep52 protein (e.g., truncated and untruncated) expression from each Rep-Cap plasmid.

[0036] Figures 5A-5E show a comparison of exemplary Rep-Cap plasmid designs. Figure 5A shows an expression cassette illustration for bidirectional Rep-Cap plasmid p!nvRep2Cap9_N that expresses only Rep78 and Rep52 proteins. Figure 5B depicts a bidirectional Rep-Cap plasmid p!nvRep2Cap9_N sequence map. Figure 5C depicts western blots showing Cap protein (VP1, VP2, VP3) expression and Rep protein expression (Rep78 and Rep52) from each Rep-Cap plasmid. Figure 5D shows AAV9 yield obtained (VG / mL) using each Rep-Cap plasmid design. Figure 5E shows a plasmid map of p!nvRep2Cap9_N.

[0037] Figures 6A-6B show a comparison of exemplary AAVrh74 Rep-Cap plasmid designs. Figure 6A shows a plasmid map of p!nvRep2Cap74_B. Figure 6B shows AAVrh74 yield obtained (VG / mL) using each Rep-Cap plasmid design.

[0038] Figures 7A-7C show a comparison of exemplary AAV and AAVrhlO Rep-Cap plasmid designs. Figure 7A shows a plasmid map of p!nvRep2Cap 2. Figure 7B shows a plasmid map of p!nvRep2CaplO_C. Figure 7C shows AAV2 or AAVrhlO yield obtained (VG / mL) using each Rep-Cap plasmid design.

[0039] Figures 8A-8B show a comparison of exemplary AAV6 Rep-Cap plasmid designs. Figure 8A shows a plasmid map of aavrep2-cap6insert-3cv2_puc57-kan. Figure 8B shows AAV6 yield obtained (VG / mL) using each Rep-Cap plasmid design.

[0040] Figures 9A-9C show a comparison of exemplary AAV2_7m8 and AAV3B Rep-Cap plasmid designs. Figure 9A shows a plasmid map of pInvRep2Cap2_7m8. Figure 9B shows a plasmid map of p!nvRep2Cap3B. Figure 9C shows AAV2_7m8 or AAV3B yield obtained (VG / mL) using each Rep-Cap plasmid design.Page 7 of 9213040886vlAttorney Docket No. 2013906-0060

[0041] Figure 10 shows a table of percent yield increases of a bidirectional Rep-Cap plasmid compared to a canonical Rep-Cap plasmid for certain transgenes.DEFINITIONS

[0042] Agent: In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g., heat, electric current or field, magnetic force or field, etc). In appropriate circumstances, as will be clear from context to those skilled in the art, the term may be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature. In some instances, again as will be clear from context, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. Tn some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as collections or libraries, for example, that may be screened to identify or characterize active agents within them. In some cases, the term “agent” may refer to a compound or entity that is or comprises a polymer; in some cases, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or of one or more particular polymeric moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymeric moiety.

[0043] Approximately / about As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated referencePage 8 of 9213040886vlAttorney Docket No. 2013906-0060 value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0044] Attenuated: As used herein, the term “attenuated” refers to a component, value, activity, or parameter that is lessened or weakened. In certain embodiments, the term “attenuated” refers to a range of values that fall within 55%, 50%, 45%, 44%, 42%, 41%, 40%, 39%, 38%, 37%, 36, 35%, 30%, 25% or less than the stated reference value. For example, in some embodiments, an attenuated Kozak sequence refers to a modified Kozak sequence wherein a translational efficiency range value falls within 45%, 44%, 42%, 41%, 40%, 39%, 38%, 37%, 36, 35% or less than the stated reference value of an unmodified Kozak sequence.

[0045] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0046] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position / identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering systemPage 9 of 9213040886vlAttorney Docket No. 2013906-0060 based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0047] Downstream. As used herein, the term “downstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 3’ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, such that sequence A is downstream of sequence B, transcription of sequence B proceeds toward sequence A.

[0048] Nucleic acid. As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within thePage 10 of 9213040886vlAttorney Docket No. 2013906-0060 scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaad enosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2 ’-fluororibose, ribose, 2’ -deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0049] Operably linked. As used herein, the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element “ operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditionsPage 11 of 9213040886vlAttorney Docket No. 2013906-0060 compatible with the control element. In some embodiments, “operably linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a distance from the functional element of interest.

[0050] Producer cell: As used herein, the term “producer cell” refers to any cell used to produce recombinant AAV (rAAV). In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is a transformed mammalian cell. In some embodiments, a producer cell is a Vero, HeLa, HEK293, HEK293T cell or derivative thereof. In some embodiments, a producer cell is an insect cell. In some embodiments, a producer cell is a transformed insect cell. In some embodiments, a producer cell is an Sf9 cell or derivative thereof.

[0051] Transformation: As used herein, the term “transformation” refers to any process by which exogenous DNA is introduced into a host cell. Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation methodology is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, mating, lipofection. In some embodiments, a “transformed" cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, a transformed cell transiently expresses introduced nucleic acid for limited periods of time.

[0052] Upstream: As used herein, the term “upstream” refers to the location or position of a nucleic acid sequence relative to a reference nucleic acid sequence, particularly a position that, during RNA transcription, is closer to the 5’ end of the transcribed RNA molecule encoded by the reference sequence. For example, for two sequences, A and B, such that sequence A is upstream of sequence B, transcription of sequence B proceeds away from sequence A.

[0053] Vector. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a hostPage 12 of 9213040886vlAttorney Docket No. 2013906-0060 cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0054] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0055] A major challenge in the development and optimization of rAAV vectors for clinical applications is to maximize the amount of virus produced per production run. Due to their nonproliferative nature, rAAV production depends, in part, on the transfection efficiency of parvoviral genomic components into producer cell lines (e.g., HEK293 cells) and properly regulated production of proteins encoded by those components. Thus, it remains of high importance to develop means to increase rAAV production.

[0056] Among other things, the present disclosure describes compositions and methods for production of AAV (e.g., recombinant AAV (rAAV)).

[0057] In some embodiments, a method of producing a rAAV comprises transfection of a producer cell with an AAV vector plasmid, an AAV Rep-Cap expressing plasmid, and anPage 13 of 9213040886vlAttorney Docket No. 2013906-0060 adenoviral helper plasmid. In some embodiments, an AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, an adenoviral helper plasmid is any adenoviral helper plasmid described herein.

[0058] In some embodiments, the present disclosure describes compositions and methods for production of AAV bidirectional Rep-Cap plasmids.AAV Rep-Cap Plasmids

[0059] Among other things, the present disclosure provides recombinant adenoviral Rep-Cap plasmids useful for production of AAV (e.g., rAAV).

[0060] A person of skill in the art will understand that an AAV genome comprises, among other things, a Rep gene and a Cap gene.

[0061] In some embodiments, a Rep gene comprises a Rep78 encoding sequence, a Rep68 encoding sequence, a Rep52 encoding sequence, and a Rep40 encoding sequence.

[0062] In some embodiments, a Cap gene comprises a VP1 encoding sequence, a VP2 encoding sequence, and a VP3 encoding sequence.

[0063] In many embodiments, a Rep gene and a Cap gene are translated to produce multiple distinct proteins.

[0064] In some embodiments, a Rep gene is translated to produce Rep78, Rep68, Rep52, and Rep40 proteins.

[0065] In some embodiments, a canonical Rep78 protein has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 23. In some embodiments a canonical Rep78 protein has an amino acid sequence that is SEQ ID NO: 23.

[0066] In some embodiments a canonical Rep68 protein has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 22. In some embodiments a canonical Rep68 protein has an amino acid sequence that is SEQ ID NO: 22.

[0067] In some embodiments, a canonical Rep52 protein has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 20. In some embodiments a canonical Rep52 protein has an amino acid sequence that is SEQ ID NO: 20.Page 14 of 9213040886vlAttorney Docket No. 2013906-0060

[0068] In some embodiments a canonical Rep40 protein has an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100% identical to SEQ ID NO: 19. In some embodiments a canonical Rep40 protein has an amino acid sequence that is SEQ ID NO: 19.

[0069] In some embodiments, Rep proteins are required for viral genome replication and packaging. See, for example, Brining H et al., “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol Ther Methods Clin Dev. 2019 Jan 26;12:248-265. doi: 10.1016 / j.omtm.2019.01.008, which is incorporated herein by reference in its entirety.

[0070] Without wishing to be bound by any particular theory, in some embodiments, overexpression of Rep proteins may decrease AAV yield. In some embodiments, overexpression ofRep52 may decrease AAV yield. In some embodiments, overexpression ofRep78 may decrease AAV yield. In some embodiments, overexpression of Rep40 may decrease AAV yield. In some embodiments, the present disclosure provides compositions and methods that reduce expression of Rep and increase AAV yield.

[0071] In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is lower compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is reduced by about 2 fold, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, about 2.7 fold, about 2.8 fold, about 2.9 fold, about 3 fold, about 3.1 fold, about 3.2 fold, about 3.3 fold, about 3.4 fold, about 3.5 fold, about 3.6 fold, about 3.7 fold, about 3.8 fold, about 3.9 fold, about 4 fold, about 4.1 fold, about 4.2 fold, about 4.3 fold, about 4.4 fold, about 4.5 fold, about 4.6 fold, about 4.7 fold, about 4.8 fold, about 4.9 fold, about 5 fold, about 5.1 fold, about 5.2 fold, about 5.3 fold, about 5.4 fold, about 5.5 fold, about 5.6 fold, about 5.7 fold, about 5.8 fold, about 5.9 fold, or about 6 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is reduced by 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold,2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold,3.9 fold, 4 fold, 4.1 fold, 4.2 fold, 4.3 fold, 4.4 fold, 4.5 fold, 4.6 fold, 4.7 fold, 4.8 fold, 4.9 fold,5 fold, 5.1 fold, 5.2 fold, 5.3 fold, 5.4 fold, 5.5 fold, 5.6 fold, 5.7 fold, 5.8 fold, 5.9 fold, or 6 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from aPage 15 of 9213040886vlAttorney Docket No. 2013906-0060 bidirectional Rep-Cap plasmid of the present disclosure is reduced by 2 fold, 3 fold, 4 fold, 5 fold, or 6 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is reduced by 2-4, 3-5 fold, or 4-6 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is not lower compared to a canonical Rep-Cap plasmid.

[0072] In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is reduced by about 3 fold to about 4 fold relative to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid is reduced by about 3 fold, about 3.1 fold, about 3.2 fold, about 3.3 fold, about 3.4 fold, about 3.5 fold, about 3.6 fold, about 3.7 fold, about 3.8 fold, about 3.9 fold, or about 4 fold relative to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep52 from a bidirectional Rep-Cap plasmid of the present disclosure is reduced by 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, or 4 fold relative to a canonical Rep-Cap plasmid.

[0073] In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid of the present disclosure is higher compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid of the present disclosure is increased by about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, about 2.7 fold, about 2.8 fold, about 2.9 fold, about 3 fold, about 3.1 fold, about 3.2 fold, about 3.3 fold, about 3.4 fold, about 3.5 fold, about 3.6 fold, about 3.7 fold, about 3.8 fold, about 3.9 fold, or about 4 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid of the present disclosure is increased by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, 2.7 fold, 2.8 fold, 2.9 fold, 3 fold, 3.1 fold, 3.2 fold, 3.3 fold, 3.4 fold, 3.5 fold, 3.6 fold, 3.7 fold, 3.8 fold, 3.9 fold, or 4 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid of the present disclosure is increased by 1-3 fold, 1.5-3.5 fold, 1.5-2.5 fold, or 2-4 fold compared to a canonical Rep-Cap plasmid. In some embodiments expression of Rep78 from bidirectionalPage 16 of 9213040886vlAttorney Docket No. 2013906-0060Rep-Cap plasmid of the present disclosure is not higher compared to a canonical Rep-Cap plasmid.

[0074] In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid of the present disclosure is increased by about 1.1 fold to about 3 fold compared to a canonical Rep- Cap plasmid. In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid is increased by about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2 fold, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, or about 2.7 fold compared to a canonical Rep-Cap plasmid. In some embodiments, expression of Rep78 from a bidirectional Rep-Cap plasmid is increased by 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 2.6 fold, or 2.7 fold compared to a canonical Rep-Cap plasmid.

[0075] In some embodiments, a stoichiometric Rep78:Rep52 protein ratio from a bidirectional Rep-Cap plasmid of the present disclosure is 1 : 1. In some embodiments, Rep40 is not expressed from a bidirectional Rep-Cap plasmid. In some embodiments, Rep68 is not expressed from a bidirectional Rep-Cap plasmid.

[0076] In some embodiments, a Cap gene is translated to produce capsid proteins VP1, VP2, and VP3. Without wishing to be bound by any particular theory, the ratio of AAV capsid expression affects capsid physical features, AAV titer, and AAV infectivity. The present disclosure recognizes that modification of the sequences regulating translation of Cap proteins (e.g., VP1, VP2, and VP3) can improve production of rAAV The present disclosure recognizes that modification of the orientation of sequences or genes near the Cap or Rep sequence in a wild type AAV genome can regulate translation of Cap proteins (e.g., VP1, VP2, and VP3) can improve production of rAAV. In some embodiments, a ratio of expressed Cap protein of 1: 1: 10 VP1 :VP2:VP3 is a desired or theoretical value.

[0077] Among other things, the present disclosure provides an AAV Rep-Cap plasmid comprising a first expression cassette comprising a Rep gene, and a second expression cassette comprising a Cap gene, wherein the first and second expression cassette are in opposite transcriptional orientation relative to one another, or a “bidirectional Rep-Cap plasmid”. In somePage 17 of 9213040886vlAttorney Docket No. 2013906-0060 embodiments, expression of Cap proteins (e.g., VP1, VP2, VP3) from a bidirectional Rep-Cap plasmid is slightly higher compared to a canonical Rep-Cap plasmid.

[0078] In some embodiments, an AAV Rep-Cap plasmids comprise a first expression cassette comprising an AAV Rep gene derived from a particular serotype. In some embodiments, an AAV Rep gene described herein is derived from AAV serotype 2 (AAV2). In some embodiments, an AAV Rep-Cap plasmid described herein comprises a modified AAV2 Rep gene (e.g., modified to comprise one or more nucleic acid substitutions, deletions, or additions). In some embodiments, a first expression cassette described herein is truncated. In some embodiments, an AAV Rep gene of a first expression cassette is truncated.

[0079] In some embodiments, a Rep78 encoding sequence in a plasmid of the present disclosure is truncated by 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, or 330 nucleotides as compared to a canonical Rep78 encoding sequence. In some embodiments, a Rep78 protein expressed from an AAV Rep gene in a plasmid of the present disclosure is truncated 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 amino acids as compared to a canonical Rep78 protein (e.g., SEQ ID NO: 23).

[0080] In some embodiments a truncated Rep78 protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 24.Page 18 of 9213040886vlAttorney Docket No. 2013906-0060

[0081] Tn some embodiments, a Rep52 encoding sequence in a plasmid of the present disclosure is truncated by 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, or 330 nucleotides as compared to a canonical Rep52 encoding sequence. In some embodiments, a Rep52 protein expressed from an AAV Rep gene is truncated by 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 amino acids as compared to a canonical Rep52 protein (e.g., SEQ ID NO: 20).

[0082] In some embodiments, a truncated Rep52 protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 21.

[0083] In some embodiments, a first expression cassette described herein comprises a splicer donor sequence and a splicer acceptor sequence. In some embodiments, a splice donor described herein is or is a complement of SEQ ID NO: 1. In some embodiments, a splice donor site inactivated. In some embodiments, an inactivated splice donor site described herein is or is a compliment of SEQ ID NO: 4. In some embodiments, a first expression cassette described herein comprises at least one poly(A) sequence element. In some embodiments, a first expression cassette described herein comprises two poly(A) sequence elements. In some embodiments, a first expression cassette described herein comprises a p5 promoter. In some embodiments, a p5 promoter described herein is upstream of an AAV Rep gene. In some embodiments, a p5 promoter described herein is operably linked to an AAV Rep gene. In some embodiments, a first expression cassette described herein comprises a pl9 promoter. In some embodiments, a pl9Page 19 of 9213040886vlAttorney Docket No. 2013906-0060 promoter described herein is upstream of an AAV Rep gene. In some embodiments, a pl 9 promoter described herein is within an AAV Rep gene. In some embodiments, a first expression cassette described herein comprises a p40 promoter. In some embodiments, a p40 promoter described herein is downstream of a p!9 promoter disclosed herein and upstream of a splice donor sequence described herein.

[0084] In some embodiments, an AAV Rep-Cap plasmid described herein comprises a second cassette comprising an AAV Cap gene derived from a particular serotype. In some embodiments, an AAV Cap gene is derived from AAV serotype 9 (AAV9). In some embodiments, an AAV Rep- Cap plasmid described herein comprises modified an AAV9 Cap gene (e.g., modified to comprise one or more nucleic acid substitutions, deletions, or additions). In some embodiments, an AAV Cap gene is derived from AAV serotype rh.74 (AAVrh.74). In some embodiments, an AAV Rep-Cap plasmid comprises modified an AAVrh.74 Cap gene (e.g., modified to comprise one or more nucleic acid substitutions, deletions, or additions). In some embodiments, an AAV Cap gene is derived from AAV serotype rh.10 (AAVrh.10). In some embodiments, an AAV Rep- Cap plasmid comprises modified an AAVrh.10 Cap gene (e.g., modified to comprise one or more nucleic acid substitutions, deletions, or additions). In some embodiments, a second expression cassette described herein comprises a splicer donor sequence and a splicer acceptor sequence. In some embodiments, a splice donor sequence described herein is or is a complement of SEQ ID NO: 1. In some embodiments, a splice donor site described herein is modified. In some embodiments, a splice donor site sequence described herein is or is a complement of SEQ ID NO: 2. In some embodiments, a splice donor site sequence described herein is or is a complement of SEQ ID NO: 3. In some embodiments, a second expression cassette described herein comprises a p40 promoter. In some embodiments, a p40 promoter described herein is upstream of an AAV Cap gene. In some embodiments, a p40 promoter described herein is operably linked to an AAV Cap gene. In some embodiments, a p40 promoter described herein is upstream of a splice donor sequence described herein.Methods of AAV Production

[0085] Among other things, compositions and methods as described herein are useful in methods of producing AAV (e.g., rAAV). In some embodiments, rAAV is produced by transfection of aPage 20 of 9213040886vlAttorney Docket No. 2013906-0060 producer cell. In some embodiments, a producer cell is an insect cell. In some embodiments, the producer cell is Sf9 or a derivative cell line thereof. In some embodiments, a producer cell is a mammalian cell. In some embodiments, a producer cell is a transformed mammalian cell. In some embodiments, a producer cell is a Vero, HeLa, HEK293, HEK293T cell or derivative thereof.

[0086] In some embodiments, a method of producing rAAV comprises transfection of a producer cell with AAV Rep-Cap plasmid, an AAV vector plasmid, and an adenoviral helper plasmid. In some embodiments, an AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest.

[0087] Methods of generating and using an adenoviral helper plasmid are described in International Application WO2024 / 107985, the contents of which is hereby incorporated herein by reference in its entirety. In some embodiments, the present disclosure provides an adenoviral helper plasmid with reduced size relative to those known in the art. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising nucleotide sequences encoding E2a, VA RNA, E4; and an L4 region. In some embodiments, an adenoviral helper plasmid as described herein comprises nucleotide sequences encoding proteins from other viruses. In some embodiments, an adenoviral helper plasmid as described herein comprises nucleotide sequences encoding proteins from other viruses, including HSV-1 UL30, HSV-1 UL42, and / or HSV-1 UL29.

[0088] In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of fiber protein; Ll- 52 / 55K (Packaging Protein 3), peripentonal Hexon-Associated protein, and an L4 region. In some embodiments, the present disclosure provides an adenoviral helper plasmid comprising a fragment, portion, or partial form of E2a protein, VARNA, E4, and an L4 region. In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of Hexon Associated Precursor (L4 pVIII) protein, DNA Terminal Protein, and 23kDa endoprotease. In some embodiments, the present disclosure provides an adenoviral helper plasmid that does not comprise one or more nucleotide sequences encoding one or more of E4orfl, E4orf2, E4orf3,Page 21 of 9213040886vlAttorney Docket No. 2013906-0060 and E4orf7. In some embodiments, an adenoviral helper plasmid provides herein comprises a kanamycin resistance gene.

[0089] In some embodiments, the present disclosure provides an adenoviral helper plasmid in which expression of E2a protein is under the control of one or more of an E2a promoter, chicken -actin promoter, and SV40 promoter. In some embodiments, the present disclosure provides an adenoviral helper plasmid in which expression of E4 open reading frames (ORFs) is under the control of one or more of a native E4 promoter and SV40 promoter.

[0090] In some embodiments, an adenoviral helper plasmid described herein is pEMBR-2.0, as described in International Application WO2024 / 107985.

[0091] In some embodiments, an adenoviral helper plasmid described herein comprises a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100% identical to SEQ ID NO: 5.

[0092] One of skill in the art will appreciate that producer cells as described herein may be used to produce AAV of any serotype. In some embodiments, an AAV serotype useful in the present disclosure is selected from any of the following serotypes, and variants thereof, including, but not limited to: AAV1, AAV10, AAV106.1 / hu.37, AAV11, AAV114.3 / hu.4O, AAV 12, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.1 / hu.43, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV16.12 / hu. ll, AAV16.3, AAV16.8 / hu.lO, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2, AAV2.5T, AAV2- 15 / rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3 / rh.61, AAV24.1, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV27.3, AAV29.3 / bb. 1, AAV29.5 / bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3A, AAV3B, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-1 l / rh.53, AAV3-3, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu.l6, AAV3-9 / rh.52, AAV3a, AAV3b, AAV4, AAV4-19 / rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42- 15, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV4-8 / r 11.64, AAV4-8 / rh.64, AAV4-9 / rh.54, AAV5,Page 22 of 9213040886vlAttorney Docket No. 2013906-0060AAV52.1 / hu.2O, AAV52 / hu.l9, AAV5- 22 / rh.58, AAV5-3 / rh.57, AAV54.1 / hu.21, AAV54.2 / hu.22, AAV54.4R / hu.27, AAV54.5 / hu.23, AAV54.7 / hu.24, AAV58.2 / hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3 / hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9. 11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-l / hu.1, AAVH2, AAVH- 5 / hu.3, AAVH6, AAVhEl.l, AAVhER1.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.l, AAVhu.10, AAVhu. ll, AAVhu.12, AAVhu.13, AAVhu.14 / 9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.68, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.t 19, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVLG- 9 / hu.39, AAV-LKO1, AAV-LK02, AAVLKO3, AAV-LKO3, AAV-LK04, AAV-LKO5, AAV- LK06, AAV- LK07, AAV-LK08, AAV-LK09, AAV-LK1O, AAV-LK11, AAV-LK12, AAV- LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8 / rh.43, AAV-PAEC, AAV- PAEC11, AAV- PAEC12, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, 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.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl,Page 23 of 9213040886vlAttorney Docket No. 2013906-0060AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, B P61 AAV, B P62 AAV, B P63AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10- 2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV-PHP.B, AAV-PHP.N, AAV-PHP.S, AAVrh.74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV- CLg, and / or AAV CAP-B1 to AAVCAP-B25.

[0093] Without wishing to be bound by theory, in some embodiments, a bidirectional Rep-Cap plasmid produces a higher AAV yield compared to a canonical Rep-Cap plasmid. In some embodiments the impact of a bidirectional Rep-Cap plasmid on AAV yield is dependent on AAV serotype.EXEMPLIFICATIONExample 1 : Materials and Methods used in Examples 2-5 described hereinCell Lines and Plasmids

[0094] Ignition Cells™, a proprietary HEK293 suspension culture cell line, were transfected with the Ad helper plasmid(s), a Rep Cap plasmid, and a ITR-flanked GFP transgene plasmid using a PEI method. Triple transfected cells were transfected with 0.75 pg of DNA per cell utilizing linear PEI (Polysciences). 96 hours post transfection, ImL of culture was removed to generate RIPA whole cell lysate for western blotting or Protein Simple capillary western analysis. The remaining culture was triton lysed by 4-hours shaking incubation at 37°C. Lysate was centrifuged at 4000xG for 15 minutes at 4°C and 0.2pm filter.Titer determination

[0095] AAV titer was determined using quantitative real-time PCR (qPCR) with a QuantStudio™ 6 / 7. Negative and positive controls were used in each run. All diluted samples were tested in triplicate. Reaction mixtures contained: IX TaqMan Fast Advanced Master Mix (Applied Biosystems), 600nM of primers and 300nM of probe to eGFP (Table 1). The standard curve was generated using seven 1 : 10 serial dilutions of an eGFP DNA gblock (IDT).Page 24 of 9213040886vlAttorney Docket No. 2013906-0060Table 1: Primers and ProbesWestern blotting

[0096] RIPA whole cell lysate was prepared by RIPA buffer (ThermoFisher catalog#89900) lysis with Halt™ protease inhibitor (ThermoFisher catalog#1861278). Lysate supernatant was harvested after a 15-minute 18000xG centrifugation. The Therm oFisher / Invitrogen Bolt Bis-Tris Plus system was used to separate equal amounts of total protein (determined from a Bio-Rad DC protein assay, catalog#5000112) on a gradient 4-12% gel. Separated protein was transferred to a PVDF membrane via the Therm oFisher / Invitrogen iBlot2 dry blotting system at 15V for 7 minutes. Blocking occurred for 1 hour in TBST / PBST buffer with either 5% non-fat dry milk or BSA. Antibody diluent mirrored the blocking buffer. Primary antibodies for Rep (Progen mouse anti-Rep catalog#61069; 1 : 100), and P-actin (Abeam Rabbit Beta- Actin catalog#ab8227; 1 :2000) were all used overnight at 4°C. Secondary antibodies were HRP-conjugated goat anti-rabbit (Abeam catalog#ab205718; 1 :40000) and rabbit anti-mouse (Abeam catalog#ab97046; 1 : 10000) incubated for 1-hour at room temperature. Blots were developed using the ThermoFisher SuperSignal West Pico PLUS substrate (catalog#34580) and imaged by an Amersham ImageQuant800 system.Protein Simple Capillary Analysis

[0097] RIPA whole cell lysates analyzed on the Jess Simple Western™ instrument (ProteinSimple®, Bio-Techne, Minneapolis, MN, USA), were diluted to total protein concentrations of 0.2 mg / mL. The VP antibody (PROGEN catalog#: 690058) was used at 1 :50. Samples were diluted and plate was loaded according to manufacturer’s instructions withPage 25 of 9213040886vlAttorney Docket No. 2013906-0060 manufacturer’s reagents. The 12-230 kDa separation module (ProteinSimple®, Bio-Techne) was used for analysis.Example 2: Exemplary Bidirectional Rep-Cap Plasmid for AAV9 Production

[0098] The present Example describes generation and use of an exemplary bidirectional Rep- Cap plasmid for production of rAAV9 from HEK293 cells.

[0099] In a canonical Rep-Cap plasmid, Rep and Cap expression cassettes are overlapping. As shown in Figure 1A, bidirectional Rep-Cap plasmid separates expression cassettes while also introducing inverted repeats containing P40 promoter and partial Rep and Cap sequences.

[0100] AAV production was tested as described in Example 1 using either Rep-Cap plasmid pAAVrep2.1cap9 or AAVrep2cap9-insert#3C_pUC57-Kan (Figure IB, Figure 1C, SEQ ID NO: 6), each with helper plasmid pEMBR2.0 and GOI plasmid pTR130-CAG-eGFP- hBGpA+Sv40pA-3’CHIMin.s (Figure ID, SEQ ID NO: 17).

[0101] Comparison of a canonical Rep-Cap plasmid (pAAVrep2.1cap9) and bidirectional Rep- Cap plasmid (AAVrep2cap9-insert#3C_pUC57-Kan) for AAV9 indicated that a bidirectional Rep-Cap plasmid improved AAV yield about 4-fold (Figure IE).

[0102] A time course study using western blot analysis to measure Rep and Cap protein expression levels at 0 hr, 24 hr, 48 hr, 72 hr, and 96 hr revealed that bidirectional Rep-Cap plasmid AAVrep2cap9-insert#3C_pUC57-Kan produced slightly less Rep52 protein, slightly more Rep78 protein, and slightly more Cap (VP1, VP2, VP3) proteins (Figure IF). Western blot results also indicated that for bidirectional Rep-Cap plasmid Rep78:Rep52 ratio was 1: 1, and Rep40 protein was not expressed.

[0103] Use of bidirectional plasmid AAVrep2cap9-insert#3C_pUC57-Kan also slightly increased Cap (VP1, VP2, VP3) protein expression. Without wishing to be bound by any particular theory, in case of canonical Rep-Cap plasmid, Rep40 encoding mRNA precursor sequence (pre-mRNA) also includes Cap sequence that is not translated but may affect alternative splicing efficiencies. In bidirectional RepCap plasmid Cap sequence is separated from Rep. The bidirectional constructions and / or separation of Rep and Cap may affect the efficiency of alternative splicing donor sequences making one SA much more efficient than other. This can result in production ofPage 26 of 9213040886vlAttorney Docket No. 2013906-0060 a longer version of “Rep40” protein. Figures 2A-2B provide illustrations that may demonstrate how a bidirectional RepCap plasmid may result in these protein expression differences.

[0104] Together, these results demonstrated that a bidirectional Rep-Cap plasmid shows significantly increased AAV9 yield compared to a canonical Rep-Cap plasmid.Example 3: Exemplary Bidirectional Rep-Cap Plasmids with Modified Splice Donor Site Increase Cap Protein Expression and Improve AAV Yield

[0105] The present Example describes generation and use of exemplary bidirectional Rep-Cap plasmids with modified splice donor sites for production of rAAV9 from HEK293 cells.

[0106] To determine whether optimization of a splicing donor site donor site further improved Cap protein expression, two constructs, pInvRep2Cap9_B (Figure 3A, Figure 3B, SEQ ID NO: 8), and pInvRep2Cap9_C (Figure 3C, Figure 3D, SEQ ID NO: 9) were designed, each with a different splice donor sequence (Figure 3E). An additional construct in which one of Swal sites was inactivated by a single nucleotide mutation was also tested (Figure 3F, Figure 3G, SEQ ID NO: 7). A feature summary table of these constructs is shown in Figure 3H.

[0107] AAV production was tested as described in Example 1, using Rep-Cap plasmids: pAAVrep2.1cap9, AAVrep2cap9-insert#3c_puC57-Kan, pInvRep2Cap9_A, pInvRep2Cap9_B, or p!nvRep2Cap9_C, each with helper plasmid pEMBR2.0 and GOI plasmid pTR130-CAG-eGFP- hBGpA+Sv40pA-3’CHIMin.s (Figure 31).

[0108] pInvRep2Cap9_B and pInvRep2Cap9_C both resulted in enhanced Cap protein expression (Figure 3J). Both constructs had no impact on Rep protein expression. Removal of the Swal site in construct pInvRep2Cap9 A had no impact on AAV yield (Figure 3K).

[0109] Together, these data demonstrate that optimization of splicing donor sequence in a bidirectional Rep-Cap plasmid improves Cap protein production and increases AAV yield.Example 4: Exemplary Bidirectional Rep-Cap Plasmid for modified Rep Protein Expression

[0110] The present Example describes generation and use of exemplary bidirectional Rep-Cap plasmids for rAAV9 production in HEK293 cells.Page 27 of 9213040886vlAttorney Docket No. 2013906-0060

[0111] A bidirectional Rep-Cap plasmid with a truncated Rep expression cassette and modified splice donor site, p!nvRep2Cap9_F(G) (Figure 4A, Figure 4B, SEQ ID NO: 10), was designed to express truncated Rep78 and Rep52, wherein 100 amino acids were truncated from the C- terminal end of Rep78, and 100 amino acids were truncated from the C-terminal end of Rep52 by insertion of a stop codon in the Rep gene cassette (Figure 4C).

[0112] AAV production was tested as described in Example 1. As shown in Figure 4D, p!nvRep2Cap9_F(G) produced slightly lower, but not significantly different, AAV yield compared to bidirectional Rep-Cap plasmid AAVrep2cap9-insert#3C_pUC57-Kan, but still demonstrated a significant increase in AAV yield compared to a canonical Rep-Cap plasmid. Increased Cap protein expression was also observed (Figure 4E). As shown in Figure 4F, both Rep78 and Rep52 proteins were truncated.

[0113] Next, a bidirectional Rep-Cap plasmid, p!nvRep2Cap9_N was designed to express only Rep78 and Rep52 (Figure 5A, Figure 5B, Figure 5E, SEQ ID NO: 18). As shown in Figures 5C-5D, pInvRep2Cap9_N produced slightly higher Cap protein (Figure 5C) and increased AAV yield (Figure 5D).

[0114] Together, these results demonstrate that bidirectional Rep-Cap plasmid with truncated Rep78 and truncated Rep52 or expressing only Rep78 and Rep52 show similar or increased AAV yield compared to a canonical Rep-Cap plasmid.Example 5: Exemplary Bidirectional Rep-Cap Plasmids for Various AAV Serotypes

[0115] This Example describes an exemplary bidirectional Rep-Cap plasmids for production of AAVrh74, AAVrhlO, AAV2, AAV6, AAV2_7m8, and AAV3B from HEK293 cells.

[0116] AAVrh74 constructs were generated to determine whether a bidirectional Rep-Cap plasmid would improve AAVrh74 yield.

[0117] AAV production was performed as described in Example 1 using Rep-Cap plasmid pAAVrep2.1cap9, pAAVrep2.4capRh.74, pInvRep2Cap74_B (Figure 6A, SEQ ID NO: 11), each with helper plasmid pEMBR2.0 and GOI plasmid pTR130-CAG-eGFP-hBGpA+Sv40pA- 3’CHIMin.s.Page 28 of 9213040886vlAttorney Docket No. 2013906-0060

[0118] Bidirectional Rep-Cap plasmid p!nvRep2Cap74_B produced more than 3-fold increase in AAV yield compared to a canonical Rep-Cap plasmid pAAVrep2.4capRh.74_pUC57-Kan (Figure 6B).

[0119] Similarly, AAV2 and AAVrhlO bidirectional Rep-Cap plasmids were generated to determine whether a bidirectional Rep-Cap plasmid would improve AAV2 and / or AAVrhlO yield. AAV production was performed as described in Example 1 using pAAVrep2.2cap2, p!nvRep2Cap2 (Figure 7A, SEQ ID NO: 12), pAAVrep2. lcapRh.10, or pInvRep2CaplO_C (Figure 7B, SEQ ID NO: 15) Rep-Cap plasmid each with helper plasmid pEMBR2.0 and GOI plasmid pTR130-CAG-eGFP-hBGpA+Sv40pA-3’CHIMin.s. As shown in Figure 7A, pInvRep2Cap2 comprises a modified splice donor site (same as described for pInvRep2Cap9_C, SEQ ID NO: 3) and also comprises a MAAP sequence. As shown in Figure 7B, pInvRep2CaplO_C comprises a modified splice donor site (same as described for pInvRep2Cap9_C, SEQ ID NO: 3).

[0120] As shown in Figure 7C, bidirectional Rep-Cap plasmid pInvRep2Cap2 produced more than a 2-fold increase AAV2 yield compared to a canonical Rep-Cap plasmid pInv2Rep2Cap2 and bidirectional Rep-Cap plasmid pInvRep2CaplO_C produced more than a 2-fold increase AAVrhlO yield compared to a canonical Rep-Cap plasmid pAAVrep2. IcapRh.lO.

[0121] Next, a bidirectional plasmid pAAVrep2-cap6insert-#3Cv2 was generated to determine whether a bidirectional Rep-Cap plasmid would improve AAV6 yield compared to use of a canonical AAV6 Rep-Cap plasmid pAAV2.2cap6. pAAVrep2-cap6insert-3#CV2 contained a modified MAAP and optimized splicing donor sequence (Figure 8A, SEQ ID NO: 16).

[0122] As shown in Figure 8B, a bidirectional Rep-Cap AAV6 plasmid resulted in moderate increase in AAV production yield.

[0123] Similarly, AAV2_7m8 and AAV3B bidirectional Rep-Cap plasmids were generated to determine whether a bidirectional Rep-Cap plasmid would improve AAV2_7m8 and / or AAV3B yield. AAV production was performed as described in Example 1 using pAAVrep2.2Cap2, pInvRep2Cap2_7m8 (Figure 9A, SEQ ID NO: 13), pAAVrep2.2Cap3B (Figure 9B, SEQ ID NO: 14), or pInvRep2Cap3B each with helper plasmid pEMBR2.0 and GOI plasmid pTR130- CAG-eGFP-hBGpA+Sv40pA-3’CHIMin.s. As shown in Figure 9A, pInvRep2Cap2_7m8 has aPage 29 of 9213040886vlAttorney Docket No. 2013906-0060 modified splice donor site (same as described for p!nvRep2Cap9_C, SEQ ID NO: 3) and also comprises a modified MAAP sequence.

[0124] As shown in Figure 9C, bidirectional Rep-Cap plasmid pInvRep2Cap2 7m8 produced more than a 4-fold increase AAV2_7m8 yield compared to a canonical Rep-Cap plasmid pAAVrep2.2Cap2 and bidirectional Rep-Cap plasmid pInvRep2Cap3B produced more than a 2- fold increase AAV3B yield compared to a canonical Rep-Cap plasmid pAAVrep2.2Cap3B.

[0125] Bidirectional Rep-Cap plasmids increased AAV yield for AAV9, AAvrh74, AAV2, AAV2_7m8, AAV6, AAVrhlO, and AAV3B serotypes, while a bidirectional Rep-Cap plasmid did not increase AAV yield for AAV8, AAV1, AAV7 (data not shown). Bidirectional Rep-Cap plasmids increased AAV yield for certain transgenes compared to canonical Rep-Cap plasmids (Figure 10).

[0126] Without wishing to be bound by theory, increase of AAV yield by use of a bidirectional Rep-Cap plasmid may be serotype specific.

[0127] Together, these data demonstrate that a bidirectional Rep-Cap plasmid improves AAV9, Aavrh74, AAV2, AAV2_7m8, AAV6, AAVrhlO, and AAV3B serotypes.Example 6: Table of Sequences

[0128] Table 2 lists and describes the various sequences discussed herein. Unless stated otherwise, all sequences are recited with 5’ to 3’ directionality of the positive strand of a plasmid. This directionality is preserved irrespective of the orientation of a gene or element described to be associated with a sequence. Asterisks as used herein indicate a stop codon.

[0129] The following list provides exemplary amino acid and nucleic acid sequences as described herein.Table 2: Table of SequencesPage 30 of 9213040886vlAttorney Docket No. 2013906-0060Page 31 of 9213040886vlAttorney Docket No. 2013906-0060Page 32 of 9213040886vlAttorney Docket No. 2013906-0060Page 33 of 9213040886vlAttorney Docket No. 2013906-0060Page 34 of 9213040886vlAttorney Docket No. 2013906-0060Page 35 of 9213040886vlAttorney Docket No. 2013906-0060Page 36 of 9213040886vlAttorney Docket No. 2013906-0060Page 37 of 9213040886vlAttorney Docket No. 2013906-0060Page 38 of 9213040886vlAttorney Docket No. 2013906-0060Page 39 of 9213040886vlAttorney Docket No. 2013906-0060Page 40 of 9213040886vlAttorney Docket No. 2013906-0060Page 41 of 9213040886vlAttorney Docket No. 2013906-0060Page 42 of 9213040886vlAttorney Docket No. 2013906-0060Page 43 of 9213040886vlAttorney Docket No. 2013906-0060Page 44 of 9213040886vlAttorney Docket No. 2013906-0060Page 45 of 9213040886vlAttorney Docket No. 2013906-0060Page 46 of 9213040886vlAttorney Docket No. 2013906-0060Page 47 of 9213040886vlAttorney Docket No. 2013906-0060Page 48 of 9213040886vlAttorney Docket No. 2013906-0060Page 49 of 9213040886vlAttorney Docket No. 2013906-0060Page 50 of 9213040886vlAttorney Docket No. 2013906-0060Page 51 of 9213040886vlAttorney Docket No. 2013906-0060Page 52 of 9213040886vlAttorney Docket No. 2013906-0060Page 53 of 9213040886vlAttorney Docket No. 2013906-0060Page 54 of 9213040886vlAttorney Docket No. 2013906-0060Page 55 of 9213040886vlAttorney Docket No. 2013906-0060Page 56 of 9213040886vlAttorney Docket No. 2013906-0060Page 57 of 9213040886vlAttorney Docket No. 2013906-0060Page 58 of 9213040886vlAttorney Docket No. 2013906-0060Page 59 of 9213040886vlAttorney Docket No. 2013906-0060Page 60 of 9213040886vlAttorney Docket No. 2013906-0060Page 61 of 9213040886vlAttorney Docket No. 2013906-0060Page 62 of 9213040886vlAttorney Docket No. 2013906-0060Page 63 of 9213040886vlAttorney Docket No. 2013906-0060Page 64 of 9213040886vlAttorney Docket No. 2013906-0060Page 65 of 9213040886vlAttorney Docket No. 2013906-0060Page 66 of 9213040886vlAttorney Docket No. 2013906-0060Page 67 of 9213040886vlAttorney Docket No. 2013906-0060Page 68 of 9213040886vlAttorney Docket No. 2013906-0060Page 69 of 9213040886vlAttorney Docket No. 2013906-0060Page 70 of 9213040886vlAttorney Docket No. 2013906-0060Page 71 of 9213040886vlAttorney Docket No. 2013906-0060Page 72 of 9213040886vlAttorney Docket No. 2013906-0060Page 73 of 9213040886vlAttorney Docket No. 2013906-0060Page 74 of 9213040886vlAttorney Docket No. 2013906-0060Page 75 of 9213040886vlAttorney Docket No. 2013906-0060Page 76 of 9213040886vlAttorney Docket No. 2013906-0060Page 77 of 9213040886vlAttorney Docket No. 2013906-0060Page 78 of 9213040886vlAttorney Docket No. 2013906-0060Page 79 of 9213040886vlAttorney Docket No. 2013906-0060Page 80 of 9213040886vlAttorney Docket No. 2013906-0060Page 81 of 9213040886vlAttorney Docket No. 2013906-0060Page 82 of 9213040886vlAttorney Docket No. 2013906-0060EQUIVALENTS

[0130] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:Page 83 of 9213040886vl

Claims

Attorney Docket No. 2013906-0060CLAIMSWe claim:

1. A recombinant adenoviral packaging plasmid comprising:(i) a first expression cassette comprising a Rep gene; and(ii) a second expression cassette comprising a Cap gene, wherein the first and second expression cassette are in an opposite transcriptional orientation relative to one another.

2. The plasmid of claim 1, wherein the Rep gene is derived from AAV serotype 2 (AAV2).

3. The plasmid of claim 1 or 2, wherein the Rep gene comprises:(i) a Rep78 encoding sequence;(ii) a Rep68 encoding sequence;(iii) a Rep52 encoding sequence; and(iv) a Rep40 encoding sequence.

4. The plasmid of any one of the preceding claims, wherein the first expression cassette is truncated.

5. The plasmid of claim 4, wherein the Rep gene is truncated.Page 84 of 9213040886vlAttorney Docket No. 2013906-00606. The plasmid of claim 5, wherein the Rep78 encoding sequence is truncated by 300 nucleotides as compared to a canonical Rep78 encoding sequence.

7. The plasmid of claim 5, wherein the Rep52 encoding sequence is truncated by 300 nucleotides as compared to a canonical Rep52 encoding sequence.

8. The plasmid of any one of the preceding claims, wherein the first expression cassette encodes only Rep78 and Rep52.

9. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype 9 (AAV9).

10. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype rh.74 (AAVrh74).11 . The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype rh.10 (AAVrhlO).

12. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype 2 (AAV2).Page 85 of 9213040886vlAttorney Docket No. 2013906-006013. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype 2_7m8 (AAV2_7m8).

14. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype 3B (AAV3B).

15. The plasmid of any one of claims 1-8, wherein the Cap gene is derived from AAV serotype 6 (AAV6).

16. The plasmid of any one of the preceding claims, wherein the first expression cassette further comprises a splice donor sequence and one or two splice acceptor sequence(s).

17. The plasmid of claim 16, wherein the splice donor sequence is or is the complement of SEQ ID NO: 1.

18. The plasmid of 16, wherein the splice donor sequence is or is the complement of SEQ ID NO: 4.

19. The plasmid of any one of the preceding claims, wherein the second expression cassette further comprises a splice donor sequence and one or two splice acceptor sequence(s).Page 86 of 9213040886vlAttorney Docket No. 2013906-006020. The plasmid of claim 19, wherein the splice donor sequence is or is the complement of SEQ ID NO: 1.

21. The plasmid of claim 19, wherein the splice donor sequence is or is the complement of SEQ ID NO: 2.

22. The plasmid of claim 19, wherein the splice donor sequence is or is the complement of SEQ ID NO: 3.

23. The plasmid of any one of the preceding claims, wherein the first expression cassette further comprises at least one poly(A) sequence element.

24. The plasmid of claim 23, wherein the first expression cassette comprises two poly(A) sequence elements.

25. The plasmid of any one of the preceding claims, wherein the second expression cassette further comprises at least one poly(A) sequence element.

26. The plasmid of any one of the preceding claims, wherein the second expression cassette comprises two poly(A) sequence elements.Page 87 of 9213040886vlAttorney Docket No. 2013906-006027. The plasmid of any one of the preceding claims, wherein the first expression cassette comprises a p5 promoter.

28. The plasmid of claim 27, wherein the p5 promoter is upstream of the Rep gene.

29. The plasmid of claim 28, wherein the p5 promoter is operably linked to the Rep gene.

30. The plasmid of any one of the preceding claims, wherein the first expression cassette comprises a pl9 promoter.

31. The plasmid of claim 30, wherein the pl9 promoter is within the Rep gene.

32. The plasmid of any one of the preceding claims, wherein the first expression cassette comprises a p40 promoter.

33. The plasmid of claim 32, wherein the p40 promoter is downstream of the pl9 promoter and upstream of the splice donor sequence.

34. The plasmid of any one of the preceding claims, wherein the second expression cassette comprises a p40 promoter.Page 88 of 9213040886vlAttorney Docket No. 2013906-006035. The plasmid of claim 34, wherein the p40 promoter is upstream of the Cap gene.

36. The plasmid of claim 35, wherein the p40 promoter is operably linked to the Cap gene.

37. The plasmid of claim 35, wherein the p40 promoter is upstream of the splice donor sequence.

38. A method of producing recombinant AAV (rAAV) particles comprising transfecting a producer cell with:(i) the plasmid of any one of claims 1-37;(ii) an adenoviral helper plasmid;(iii) an AAV vector plasmid; or(iv) any combination of (i) - (iii).

39. The method of claim 38, wherein the producer cell is a mammalian cell.

40. The method of claim 39, wherein the producer cell is HEK293 or derivative cell line thereof.

41. The method of claim 38, wherein the adenoviral helper plasmid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5.Page 89 of 9213040886vlAttorney Docket No. 2013906-006042. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.

43. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7.

44. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.

45. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9.

46. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

47. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

48. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.

49. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13.Page 90 of 9213040886vlAttorney Docket No. 2013906-006050. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14.

51. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15.

52. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 16.

53. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 17.

54. A nucleic acid comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 18.Page 91 of 9213040886vl

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