Adenovirus helper plasmid

Abstract

The present disclosure provides improved adenovirus helper plasmids for the production of recombinant adeno-associated viruses. In some embodiments, the present disclosure provides adenovirus helper plasmids that are reduced in size compared to those known in the art. In some embodiments, the present disclosure provides adenovirus helper plasmids that include nucleotide sequences encoding E2a, VA RNA, E4, and L4 regions. In some embodiments, the adenovirus helper plasmids described herein include nucleotide sequences encoding proteins from other viruses.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application Nos. 63 / 426,028, filed November 16, 2022, and 63 / 596,201, filed November 3, 2023, the entire contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Adeno-associated virus (AAV) technology has quickly become a major form of gene therapy for genetic diseases. AAV can be produced on a large scale in a variety of host cell systems, including mammalian cells such as HEK293 cells. Traditionally, AAV production in mammalian cells involves the introduction of multiple plasmids into the host cell, such as plasmids encoding the human gene or genes of interest as well as various viral genes important for viral replication and packaging. Due to the number of genes required for proper replication, these are traditionally delivered on two or three separate plasmids.

[0003] One such plasmid, called the "adenovirus helper" plasmid, contains genes important for AAV production from host cells. Adenovirus helper plasmids containing the E2a, VA RNA, and E4 genes have been shown to be important for facilitating AAV production in mammalian host cell systems.

[0004] Despite significant advances over the past two decades, concerns about the cost and safety of AAV production continue to limit the therapeutic potential of AAV technology. These concerns are due, in part, to the large size of many helper plasmids, which provide multiple genes on a single helper plasmid to support AAV production. Safety concerns are also due, in part, to the production of low-level, but potentially cytotoxic and / or inflammatory, viral proteins that are not required for AAV replication. Summary of the Invention

[0005] In some embodiments, the present disclosure provides, among other things, adenovirus helper plasmids. In some embodiments, the present disclosure provides adenovirus helper plasmids that are reduced in size compared to those known in the art. In some embodiments, the present disclosure provides adenovirus helper plasmids that include nucleotide sequences encoding E2a, VA RNA, E4, and L4 regions. In some embodiments, the adenovirus helper plasmids described herein include nucleotide sequences encoding proteins from other viruses. In some embodiments, the adenovirus helper plasmids described herein include nucleotide sequences encoding proteins from other viruses, including HSV-1 UL30, HSV-1 UL42, and / or HSV-1 UL29.

[0006] In some embodiments, the present disclosure provides an adenovirus helper plasmid that does not include one or more nucleotide sequences encoding one or more of the following: fiber protein; L1-52 / 55K (packaging protein 3), peripentonal hexon-associated protein, and L4 region. In some embodiments, the present disclosure provides an adenovirus helper plasmid that includes a fragment, portion, or partial form of E2a protein, VA RNA, E4, and the L4 region. In some embodiments, the present disclosure provides an adenovirus helper plasmid that does not include one or more nucleotide sequences encoding one or more of hexon-associated precursor (L4 pVIII) protein, DNA-termination protein, and 23 kDa endoprotease. In some embodiments, the present disclosure provides an adenovirus helper plasmid that does not include one or more nucleotide sequences encoding one or more of E4orf1, E4orf2, E4orf3, and E4orf7. In some embodiments, the adenovirus helper plasmids provided herein include a kanamycin resistance gene.

[0007] In some embodiments, the present disclosure provides adenovirus helper plasmids in which expression of the E2a protein is under the control of one or more of the E2a promoter, the chicken β-actin promoter, and the SV40 promoter. In some embodiments, the present disclosure provides adenovirus helper plasmids in which expression of the E4 open reading frame (ORF) is under the control of one or more of the native E4 promoter and the SV40 promoter.

[0008] In some embodiments, the present disclosure provides an adenovirus helper plasmid comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 1-3, 5, 7, 9, 11-12, 14-20, 22, 24, 26-29, 31, 33, 35-37, 39-53, 56, 59, 62, 65, 68, or 80-105. In some embodiments, the present disclosure provides an adenovirus helper plasmid comprising a nucleotide sequence encoding an amino acid sequence at least 80% identical to SEQ ID NO: 4, 6, 8, 10, 13, 21, 23, 25, 30, 32, 34, 38, 55, 58, 61, 64, 67, or 69. In some embodiments, the present disclosure provides an adenovirus helper plasmid comprising a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 41-49, 70, or 80-105.

[0009] In some embodiments, the disclosure provides for providing an L4 region in trans to an adenovirus helper plasmid. In some embodiments, the disclosure provides an L4 trans-plasmid comprising a nucleotide sequence encoding an engineered L4 region. In some embodiments, the L4 trans-plasmid encodes an L4 region (e.g., L4 33K and L4 22K). In some embodiments, the L4 trans-plasmid encodes L4 33K. In some embodiments, the L4 trans-plasmid encodes L4 22K. In some embodiments, the disclosure provides a composition comprising an adenovirus helper plasmid described herein and an L4 trans-plasmid described herein.

[0010] In some embodiments, the present disclosure provides methods for producing recombinant adenovirus-associated viral vectors (rAAV). In some embodiments, the methods for producing rAAV include transfecting producer cells with an AAV vector plasmid and an adenovirus helper plasmid described herein. In some embodiments, the methods for producing rAAV include transfecting producer cells with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, and an adenovirus helper plasmid. In some embodiments, the methods for producing rAAV further include transfecting producer cells with an L4 transgene plasmid. In some embodiments, the producer cells stably express Rep-Cap. In some embodiments, the producer cells comprise a nucleotide sequence encoding the L4 region or a portion thereof (e.g., L4 33K and / or L4 22K). [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a plasmid map illustrating the adenovirus helper plasmid pEMBR-1.2.

[0012] [Figure 2]FIG. 2 shows the vector yields obtained using pEMBR-1.2 and the commercially available pX80 as adenovirus helper plasmids.

[0013] [Figure 3] FIG. 3 shows the vector transgene purity and vector capsid purity obtained using pEMBR-1.2 or the commercially available pX80 as the adenovirus helper plasmid.

[0014] [Figure 4] FIG. 4 shows a comparison between the GFP expression levels obtained after transformation of HEK293 cells with recombinant AAV RH.10, the ssCMV-GFP transgene, and either the pX80 or pEMBR helper plasmid.

[0015] [Figure 5] FIG. 5 shows a plasmid map showing the adenovirus helper plasmids pEMBR-1.3 and pEMBR-1.3B.

[0016] [Figure 6] FIG. 6 shows a plasmid map showing the adenovirus helper plasmids pEMBR-1.4 and pEMBR-1.4B.

[0017] [Figure 7] FIG. 7 shows a plasmid map illustrating the adenovirus helper plasmid pEMBR-1.5.

[0018] [Figure 8] FIG. 8 shows a plasmid map illustrating the adenovirus helper plasmid pEMBR-1.5A.

[0019] [Figure 9-1]Figure 9 shows an exemplary sequence map of the native adenovirus type 5 genome, including, among other things, the VA RNA, E2a, and E4 regions (Panel A). Panels B, C, and D show the approximate sizes of our VA RNA region (Panel B), E2a region (Panel C), and E4 region (Panel D) from pEMBR-1.2B2 compared to the regions from Matsushita 1998, which annotated the native adenovirus sequence. [Figure 9-2] Figure 9 shows an exemplary sequence map of the native adenovirus type 5 genome, including, among other things, the VA RNA, E2a, and E4 regions (Panel A). Panels B, C, and D show the approximate sizes of our VA RNA region (Panel B), E2a region (Panel C), and E4 region (Panel D) from pEMBR-1.2B2 compared to the regions from Matsushita 1998, which annotated the native adenovirus sequence. [Figure 9-3] Figure 9 shows an exemplary sequence map of the native adenovirus type 5 genome, including, among other things, the VA RNA, E2a, and E4 regions (Panel A). Panels B, C, and D show the approximate sizes of our VA RNA region (Panel B), E2a region (Panel C), and E4 region (Panel D) from pEMBR-1.2B2 compared to the regions from Matsushita 1998, which annotated the native adenovirus sequence. [Figure 9-4] Figure 9 shows an exemplary sequence map of the native adenovirus type 5 genome, including, among other things, the VA RNA, E2a, and E4 regions (Panel A). Panels B, C, and D show the approximate sizes of our VA RNA region (Panel B), E2a region (Panel C), and E4 region (Panel D) from pEMBR-1.2B2 compared to the regions from Matsushita 1998, which annotated the native adenovirus sequence.

[0020] [Figure 10] FIG. 10 shows an exemplary plasmid map containing the VA RNA, E2a, and E4 regions, defined by restriction enzyme sites, for example, as found in the plasmids described herein.

[0021] [Figure 11] FIG. 11 shows a sequence map showing the E2a region of pEMBR-1.2B2.

[0022] [Figure 12] FIG. 12 shows a sequence map of the various promoters upstream of the E2a region in the various pEMBR plasmids described herein.

[0023] [Figure 13] FIG. 13 shows sequence maps for various truncations to the NotI end of the pEMBR1.2B2 E2a region, with one truncation construct having the addition of a promoter, as described herein.

[0024] [Figure 14] FIG. 14 shows a table summarizing the plasmid size (bp), VA RNA region modifications, E2a region modifications, and E4 region modifications for the various pEMBR plasmids described herein.

[0025] [Figure 15-1] Figure 15 shows the mean AAV9:eGFP titers (VG / mL) measured by qPCR (Panel A) and ddPCR (Panel B) using various pEMBR plasmids described herein, as well as protein expression of Rep and E2a proteins measured by Western blot (Panel C). [Figure 15-2] Figure 15 shows the mean AAV9:eGFP titers (VG / mL) measured by qPCR (Panel A) and ddPCR (Panel B) using various pEMBR plasmids described herein, as well as protein expression of Rep and E2a proteins measured by Western blot (Panel C).

[0026] [Figure 16]Figure 16 shows the mean AAV9:eGFP titers (VG / mL) measured by qPCR using various pEMBR plasmids described herein.

[0027] [Figure 17] FIG. 17 shows a table summarizing the inclusion of JEP, GEP, and native L4 (33K and 22K) in various pEMBR plasmids described herein, as well as the expression of E2a protein and AAV production.

[0028] [Figure 18] FIG. 18 shows a table summarizing the plasmid size (bp), VA RNA region modifications, E2a region modifications, and E4 region modifications for the various pEMBR plasmids described herein.

[0029] [Figure 19] FIG. 19 shows a table summarizing the plasmid size (bp), VA RNA region modifications, E2a region modifications, E4 region modifications, and AAV production compared to pEMBR-1.2.B2 for the various pEMBR plasmids described herein.

[0030] [Figure 20] FIG. 20 shows sequence maps of the native E4 region (panel A) and the SV40 promoter-driven E4 ORF4 and E4 ORF6 with the SV40 poly(A) signal (panel B) (eg, present in pEMBR-1.7B2).

[0031] [Figure 21] Figure 21 shows a sequence map showing the E2a region of pEMBR-1.2B2. The native L4 region (circled) shows the sequences of the 33kJ and 22kJ proteins and contains 50 bp upstream of the native promoter and the likely native poly(A) signal sequence. The L4 expression construct shown below retains all the same characteristics as the native L4 region, except that the native poly(A) signal has been replaced with a synthetic 49 bp poly(A) signal sequence.

[0032] [Figure 22] Figure 22 shows the mean AAV9:eGFP titers (VG / mL) using various pEMBR plasmids described herein.

[0033] [Figure 23] Figure 23 shows the pEMBR-1.2B2 titer and average AAV titer compared to plasmid size (bp) using various pEMBR plasmids described herein.

[0034] [Figure 24] FIG. 24 shows a plasmid map showing the adenovirus helper plasmid pEMBR-1.37B2+L4-minKanR-v2.

[0035] [Figure 25] FIG. 25 shows a sequence map of an exemplary E2a region that has been engineered to reduce plasmid size while maintaining AAV production.

[0036] [Figure 26] FIG. 26 shows the location of the sequences that reduce the E2a region compared to the native adenovirus type 5 genome.

[0037] [Figure 27] Figure 27 shows the wild-type AAV E2a region with additional depiction of the L4 region ORFs of the 22K and 33K proteins. To draw attention to how this native E2a promoter sequence can be added to the plasmids described herein when the L4 33K protein is added, a partial JEP sequence is shown below the JEP with a minimal open arrow.

[0038] [Figure 28]Figures 28A and 28B show the rescue of AAV titer by the addition of the L4 region. Figure 28A shows the average rAAV titer (VG / mL) from triplicate 75 mL quadruple-plasmid transfected cultures determined by qPCR of pEMBR-1.3 plasmids containing specific L4 constructs plus pUC57 vectors, compared to wild-type + empty vector (pEMBR-1.2B2 + pUC57) production. The partial JEP (PJ) E2a construct is shown as a reference. Figure 28B shows Rep and E2a Western blots of RIPA lysates from the transfected cultures in Figure 28A. β-Actin is shown as a loading control. Adenovirus type 5-infected HEK293 / HEK293T cells are shown as a positive control, and uninfected / untransfected HEK293 cells are shown as a negative control.

[0039] [Figure 29A] Figures 29A, 29B and 29C show the increase in AAV titer by providing additional copies of L4 and L33K. Figure 29A shows the E2a region of the helper plasmid used in quadruple plasmid transfection. [Figure 29B-C] Figures 29A, 29B, and 29C show the increase in AAV titer by providing additional copies of L4 and L33K. Figure 29B shows the average rAAV titer (VG / mL) from triplicate 75 mL quadruple plasmid-transfected cultures determined by qPCR of pUC57 vectors containing specific L4 constructs in addition to the E2a constructs shown in this figure, comparing L4 gene dosages of 1x L4 gene, 2x L4 gene, 1x 33K + 2x 22K, and 2x 33K + 1x 22K to control conditions (wild-type + empty vector; 1.2B2 + pUC57). Figure 29C shows Rep and E2a Western blots of RIPA lysates from the transfected cultures in Figure 29A. β-actin is shown as a loading control. Adenovirus type 5-infected HEK293 / HEK293T cells are shown as a positive control, and uninfected / untransfected HEK293 cells are shown as a negative control.

[0040] definition Agent: Generally, as used herein, the term "agent" is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof (e.g., a cell, tissue, organism)) or a phenomenon (e.g., heat, an electric current or electric field, a magnetic force or field, etc.). Under appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively, or additionally, as the context will make clear, the term may be used to refer to a natural product in that it is found in nature and / or obtained from nature. In some cases, again as will be clear from the context, the term may be used to refer to one or more entities that are artificial in that they are designed, manipulated, and / or produced through the action of the human hand and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or pure form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as a collection or library that can be screened, for example, to identify or characterize active agents therein. In some cases, the term "agent" may refer to a compound or entity that is or includes a polymer, and in some cases, the term may refer to a compound or entity that includes one or more polymer 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 one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymer moieties.

[0041] Approximately / About: As used herein, the term "approximately" or "about," when 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," unless otherwise stated or clear from the context, refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (above or below) of the stated reference value (except when such number exceeds 100% of the possible values).

[0042] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but are sufficiently identical to permit comparisons between them, so that one of skill in the art will understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few variable characteristics. One of skill in the art will understand, depending on the context, what degree of identity is required for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered equivalent in any given situation. For example, one of skill in the art will understand that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics 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 manifested by variations in these varying characteristics.

[0043] Corresponding to: As used herein, the term "corresponding to" may be used to indicate the location / identity of a structural element in a compound or composition through comparison to an appropriate reference compound or composition. For example, in some embodiments, a monomer 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, for purposes of simplicity, residues in a polypeptide are often designated using a regular numbering system based on the reference related polypeptide, so that one of skill in the art will understand that, for example, an amino acid "corresponding to" a residue at position 190 need not actually be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at 190 in the reference polypeptide, and one of skill in the art will readily understand how to identify a "corresponding" amino acid. For example, one of skill in the art will recognize 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, which can be utilized to identify "corresponding" residues in polypeptides and / or nucleic acids, for example, in accordance with the present disclosure.

[0044] 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 during RNA transcription that 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.

[0045] 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, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring 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, the 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 and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 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-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) that are modified compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is 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), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the 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, 110, 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 in length. In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one factor that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0046] Operably linked: As used herein, the term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A regulatory 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 conditions compatible with the regulatory element. In some embodiments, an "operably linked" regulatory element is contiguous (e.g., covalently linked) with the coding element of interest, and in some embodiments, the regulatory element acts in trans with respect to the functional element of interest or at a distance from the functional element of interest.

[0047] Producer cell: As used herein, the term "producer cell" refers to any cell used to produce recombinant AAV (rAAV). In some embodiments, the producer cell is a mammalian cell. In some embodiments, the producer cell is a transformed mammalian cell. In some embodiments, the producer cell is a Vero, HeLa, HEK293, HEK293T cell, or derivatives thereof.

[0048] Transformation: As used herein, the term "transformation" refers to any process by which exogenous DNA is introduced into a host cell. Transformation can occur under natural or artificial conditions using a variety of methods well known in the art. Transformation can rely on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. In some embodiments, the particular transformation method is selected based on the host cell to be transformed and can include, but is not limited to, viral infection, electroporation, mating, and lipofection. In some embodiments, a "transformed" cell is stably transformed, in that the inserted DNA is capable of replicating either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, the transformed cell transiently expresses the introduced nucleic acid for a limited period of time.

[0049] 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 during RNA transcription that is near 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.

[0050] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule 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 can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host 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 operably linked to the vector. Such vectors are referred to herein as "expression vectors." Standard techniques of recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) can be used. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF THE INVENTION

[0051] The helper functions that adenovirus provides for AAV replication have been previously described. Without wishing to be bound by any particular hypothesis, the adenovirus E1A protein has been described to activate AAV gene expression by binding and activating the AAV P5 rep promoter. Similarly, another adenovirus protein, E2A, has been described to activate AAV P5 promoter transcription. E2A has also been described to cooperate with virus-associated RNA I (VA RNAI) to enhance AAV RNA translation. Adenovirus E4orf4 has been shown to induce cell cycle arrest at the G2 / M boundary and to support AAV production. Adenovirus E4orf6 has been described to enhance the conversion of single-stranded recombinant AAV genomes to double-stranded genomes, which is the rate-limiting step of viral DNA replication both in vitro and in vivo. VA RNAI has also been described to support AAV replication. VA RNAI has been described to physically interact with double-stranded RNA-activated protein kinase (PKR) and induce an antiviral immune response that inhibits viral protein production.

[0052] Previous studies have suggested that the minimal set of genes in trans for efficient recombinant AAV production in HEK293 cells providing the E1 gene are the E2a, E4orf6, and VA RNAI genes. A helper plasmid designated pXX6-80, which contains this gene set as well as additional genes that may not be required for production, is used for adenovirus-free recombinant AAV production.

[0053] One major ongoing challenge in the development and optimization of AAV vectors for clinical use is increasing the amount of virus produced. Due to their non-replicative nature, their production depends solely on the transfection efficiency of parvovirus genome components into packaging cell lines (e.g., human embryonic kidney cells, HEK293 or HEK293T, or insect cells, e.g., Sf9). Therefore, developing means to increase recombinant AAV (rAAV) production remains crucial.

[0054] Other major challenges associated with the production of rAAV for clinical use relate to the cost of producing such rAAV in large quantities and also to the safety of the final product itself. For example, commercially available helper plasmids such as pXX6-80 appear to transcribe low levels of Ad fiber protein. Importantly, fiber protein is not required for AAV production and can be immunogenic in humans. In addition, the size of pXX6-80 is somewhat large, exceeding 18 kb. This large plasmid size increases the difficulty and cost of its production, which can significantly impact the use of GMP plasmids for the production of clinical-grade AAV.

[0055] Different versions of adenovirus helper plasmids have been derived by others, including, for example, pFAdDeltaF6 (derived at the University of Pennsylvania) and pHelper (Agilent). The pFAdDeltaF6 plasmid is approximately 3 kb smaller than pXX6-80 but retains the fiber gene sequence. The pHelper plasmid, also available from Agilent, is smaller than pXX6-80, at approximately 11.6 kb. However, it contains an ampicillin resistance gene, which is generally not recommended for plasmids used in AAV production.

[0056] The present disclosure addresses the above technical problems by providing the compositions and methods described herein.

[0057] In some embodiments, the present disclosure relates to an adenovirus-derived helper plasmid (adenovirus helper plasmid) comprising an adenovirus DNA sequence encoding a viral helper protein. In some embodiments, the adenovirus helper plasmid of the present disclosure is used in a method for producing recombinant adeno-associated virus (rAAV). In some embodiments, the adenovirus helper plasmid of the present disclosure increases the production of rAAV.

[0058] In some embodiments, the present disclosure provides adenovirus helper plasmids comprising nucleotide sequences encoding proteins derived from a source other than adenovirus. In some embodiments, the present disclosure provides adenovirus helper plasmids comprising nucleotide sequences encoding proteins derived from a virus other than adenovirus. In some embodiments, the adenovirus helper plasmids comprise all or a portion of the adenovirus nucleotide sequences encoding the adenovirus proteins E2a and E4, the L4 region, and the non-coding RNA VA RNA. In some embodiments, the present disclosure describes improved adenovirus helper plasmids that are smaller than the leading commercially available adenovirus helper plasmids and enable safer and more cost-effective production of rAAV in producer cell expression systems.

[0059] In some embodiments, the present disclosure provides adenovirus helper plasmids that have a reduced overall size compared to currently available adenovirus helper plasmids (e.g., pXX6-80 at 18.932 kbp, pALD-X80 at 18.876 kbp, pHelper at 11.635 kbp, pFAdDeltaF6 at 15.420 kbp).

[0060] In some embodiments, the present disclosure provides adenoviral helper plasmids having smaller sizes. In some embodiments, the adenoviral helper plasmids of the present disclosure are approximately 6.5 kb to 15.5 kb. In some embodiments, the adenoviral helper plasmids of the present disclosure are approximately 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 16 kb in size. In some embodiments, the adenovirus helper plasmids of the present disclosure have a size of approximately 6-7 kb, 6.5-7.5 kb, 7-8 kb, 7.5-8.5 kb, 8-9 kb, 8.5-9.5 kb, 9-10 kb, 9.5-10.5 kb, 10-11 kb, 10.5-11.5 kb, 11-12 kb, 11.5-12.5 kb, 12-13 kb, 12.5-13.5 kb, 13-14 kb, 13.5-14.5 kb, 14-15 kb, 14.5-15.5 kb, or 15-16 kb. The smaller size of the adenovirus helper plasmids of the present disclosure allows for simpler and less costly production of AAV in the quantities required for large-scale AAV manufacturing. In some embodiments, removing genes and / or portions of genes makes the adenoviral helper plasmids of the present disclosure safer because the producer cells do not produce adenoviral structural proteins (e.g., fiber, 100K, and hexon assembly) that may be co-purified with AAV during downstream processing, and therefore present a lower risk of inadvertently introducing adenoviral structural proteins into a patient.

[0061] In some embodiments, removal of adenoviral helper genes, resulting in smaller adenoviral helper plasmids, allows for the addition of complementary genes to further improve AAV quality and yield. Although these complementary genes increase the size of the plasmid compared to the minimal versions, they allow for comparable or higher AAV productivity and are therefore worth the additional cost to produce. Importantly, these plasmids are still smaller than commercially available helper plasmids, such as pALD-X80.

[0062] Adenovirus helper plasmid Helper and resistance genes In some embodiments, the adenovirus helper plasmids of the present disclosure comprise one or more nucleotide sequence(s) encoding a protein selected from the group consisting of E2b, E2a, E4orf4, E1B55K, E1b19K, E1a, E4orf6, VA RNA, and combinations thereof.

[0063] In some embodiments, the adenovirus helper plasmid comprises a nucleotide sequence encoding an E4 region and a VA RNA region. In some embodiments, the E4 region comprises one or more of E4orf1, E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, and E4orf7. In some embodiments, E4orf1 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:53. In some embodiments, E4orf1 has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:55. In some embodiments, E4orf2 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:56. In some embodiments, E4orf2 has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 58. In some embodiments, E4orf3 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 59. In some embodiments, E4orf3 has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 61. In some embodiments, E4orf4 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 62. In some embodiments, E4orf4 has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64. In some embodiments, E4orf6 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, E4orf6 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 67. In some embodiments, E4orf7 has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 68.In some embodiments, E4orf7 has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:69. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf1. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf2. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence comprising E4orf1 and does not comprise a nucleotide sequence comprising E4orf2. In some embodiments, expression of the E4 region is under the control of an E4 minipromoter. In some embodiments, the E4 region is operably linked to the E4 minipromoter. In some embodiments, the E4 minipromoter has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:1. In some embodiments, the E4 region is operably linked to an SV40 promoter. In some embodiments, expression of the E4 region is under the control of an SV40 promoter. In some embodiments, the SV40 promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:2.

[0064] In some embodiments, the adenoviral helper plasmids of the invention comprise a resistance gene. In some embodiments, the adenoviral helper plasmids of the invention comprise an ampicillin resistance gene (e.g., a nucleotide sequence encoding a protein that confers resistance to ampicillin). In some embodiments, the adenoviral helper plasmids of the invention do not comprise an ampicillin resistance gene. In some embodiments, the adenoviral helper plasmids of the invention comprise a kanamycin resistance gene (e.g., a nucleotide sequence encoding a protein that confers resistance to kanamycin). In some embodiments, the adenoviral helper plasmids of the invention do not comprise a kanamycin resistance gene.

[0065] E2a area In some embodiments, the adenovirus helper plasmids of the present disclosure comprise an E2a region. In some embodiments, as used herein, the E2a region comprises one or more nucleotide sequences encoding an E2a protein and an engineered L4 region. In some embodiments, the adenovirus helper plasmids of the present disclosure comprise a modified E2a region. In some embodiments, the E2a region of the adenovirus helper plasmids of the present disclosure comprises a deletion to reduce plasmid size while maintaining production of the E2a protein and AAV.

[0066] In some embodiments, the E2a region is modified to contain a deletion while maintaining the JEP promoter sequence corresponding to that identified by Jing et al., 2001. Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus. Virology, 291(1), pp. 140-151, incorporated herein by reference for any purpose. In some embodiments, the E2a region is modified to contain a deletion while maintaining the GEP promoter sequence corresponding to that identified by Guilfoyle et al., 1985. Two functions encoded by adenovirus early region 1A are responsible for the activation and repression of the DNA-binding protein gene. The EMBO Journal, 4(3), pp. 707-713, incorporated herein by reference for any purpose.

[0067] In some embodiments, the E2a region is modified to contain a deletion while retaining a specific amount of nucleotides upstream of E2a (eg, 1 kbp or 50 bp upstream of E2a).

[0068] In some embodiments, the E2a region is modified to contain a truncation beginning at the NotI end of the adenoviral helper plasmid of the present disclosure.

[0069] In some embodiments, the E2a region is modified as shown in Figures 12-14 and / or Figures 18-19 and / or Figure 25, Figure 29A.

[0070] L4 area In some embodiments, the adenovirus helper plasmids of the present disclosure comprise an L4 region. In some embodiments, the adenovirus helper plasmids of the present disclosure comprise one or more nucleotide sequence(s) within the L4 region that encode one or more protein(s) (e.g., 33k, 22k, etc.).

[0071] In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a modified L4 region, ie, the L4 region of the adenoviral helper plasmids of the present disclosure comprises deletions to reduce plasmid size while maintaining production of the L4 33k and L422k proteins and AAV.

[0072] In some embodiments, the adenoviral helper plasmids of the present disclosure encode but do not express the L4 33k and L422k proteins, hi some embodiments, the L4 region of the adenoviral helper plasmids of the present disclosure comprises a modification that abolishes expression of the L4 33k and L422k proteins while maintaining the nucleotide sequence encoding L4 33k and L422k.

[0073] In some embodiments, the L4 region of the adenoviral helper plasmid of the present disclosure comprises a deletion that abolishes expression of L4 100K. In some embodiments, the L4 region of the adenoviral helper plasmid of the present disclosure comprises a deletion that removes the nucleotides encoding L4 100K, abolishing expression of L4 100K.

[0074] In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding L4 22K. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K, wherein the L4 33K has been modified so that it is not expressed. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K, wherein the L4 22K has been modified so that it is not expressed.

[0075] In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a native L4 region (approximately 1379 bp in length). In some embodiments, the adenoviral helper plasmids of the present disclosure comprise an L4 expression construct (approximately 1184 bp), designated +L4 in adenoviral helper plasmid designs (e.g., those described herein), in which the native polyA signal has been replaced with a 49 bp synthetic polyA signal (SEQ ID NO: 108). In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence encoding L4 33K. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence encoding L4 22K. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise nucleotide sequences encoding L4 33K and L4 22K. In some embodiments, the adenoviral helper plasmids of the present disclosure express the L4 genes 33K and L4 22K from the native L4 promoter. In some embodiments, the adenovirus helper plasmid of the present disclosure comprises a nucleotide sequence encoding the native L4 promoter, 50 bp nucleotides upstream of the native promoter, L4 33K, and L4 22K. In some embodiments, the 50 bp nucleotides upstream of the native promoter are determined by comparison with a reference adenovirus sequence. In some embodiments, the reference sequence is GenBank: M73260.1 (Ad5 sequence).

[0076] In some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise an L4 region. In some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise one or more nucleotide sequence(s) encoding one or more protein(s) (e.g., 33k, 22k, etc.) within the L4 region. In some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise a nucleotide sequence encoding L4 33K. In some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise a nucleotide sequence encoding L4 22K.

[0077] In some embodiments, the L4 region is provided in trans to the adenoviral helper plasmid. In some embodiments, the L4 region is provided to a producer cell. In some embodiments, the producer cell comprises a nucleotide sequence encoding the L4 region. In some embodiments, the producer cell comprises a nucleotide sequence encoding the L4 proteins L4 33K and L4 22K. In some embodiments, the producer cell comprises a nucleotide sequence encoding L4 33K. In some embodiments, the producer cell comprises a nucleotide sequence encoding L4 22K.

[0078] In some embodiments, the L4 region, or a portion thereof, is provided by a plasmid other than an adenovirus helper plasmid. In some embodiments, the present disclosure provides an L4 trans-plasmid. In some embodiments, the L4 trans-plasmid comprises an adenovirus L4 region. In some embodiments, the L4 trans-plasmid of the present disclosure comprises one or more nucleotide sequence(s) encoding one or more protein(s) (e.g., 33k, 22k, etc.) within the L4 region. In some embodiments, the L4 trans-plasmid of the present disclosure provides one or more nucleotide sequence(s) encoding one or more protein(s) (e.g., 33k, 22k, etc.) within the L4 region on a plasmid other than an adenovirus helper plasmid. In some embodiments, the one or more nucleotide sequence(s) within the L4 region encoding one or more protein(s) (e.g., 33k, 22k, etc.) are not provided on the same plasmid (e.g., in cis) as other adenovirus helper components, and the plasmid is referred to as an L4 trans-plasmid.

[0079] In some embodiments, an L4 transformer-plasmid of the present disclosure comprises one or more nucleotide sequence(s) within the L4 region that encode and express one or more protein(s) (e.g., 33k, 22k, etc.). In some embodiments, an L4 transformer-plasmid of the present disclosure comprises nucleotide sequences that encode and express the L4 33k and L4 22k proteins. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence that encodes and expresses the L4 33k protein. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence that encodes and expresses the L4 22k protein.

[0080] In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence encoding L4 22K. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K, wherein the L4 33K has been modified so that it is not expressed. In some embodiments, an L4 transformer-plasmid of the present disclosure comprises a nucleotide sequence encoding L4 33K and L4 22K, wherein the L4 22K has been modified so that it is not expressed.

[0081] In some embodiments, expression of a protein encoded by an L4 transformer-plasmid of the present disclosure is under the control of a promoter. In some embodiments, expression of a protein encoded by an L4 transformer-plasmid of the present disclosure is under the control of the EF1 alpha promoter.

[0082] In some embodiments, the adenoviral helper plasmids of the present disclosure comprise an E2a region modified as shown in FIG. 18 and / or FIG. 19 and / or FIG.

[0083] Fiber genes In some embodiments, the adenoviral helper plasmid of the present disclosure does not comprise a nucleotide sequence encoding an adenoviral fiber protein. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding a full-length adenoviral fiber protein. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence encoding a portion or fragment of an adenoviral fiber protein. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the nucleotide sequence of pXX6-80, excluding the nucleotide sequence encoding the adenoviral fiber protein.

[0084] L1-52 / 55K (packaging protein 3) gene In some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise a nucleotide sequence encoding the L1-52 / 55K (packaging protein 3) protein, hi some embodiments, the adenoviral helper plasmids of the present disclosure do not comprise a nucleotide sequence encoding a penton peripheral hexon associated gene.

[0085] L4 area In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a complete L4 (hexon assembly) gene. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence encoding a complete L4 (hexon assembly). In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:3. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:4. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a complete L4 (33 kDa Ex2) gene. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence encoding a complete L4 (33 kDa Ex2). In some embodiments, the adenoviral helper plasmids of the present disclosure comprise a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the adenoviral helper plasmids of the present disclosure comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 6.

[0086] In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a complete L4 encapsidation protein gene. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding the complete L4 encapsidation protein. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:7. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:8.

[0087] In some embodiments, the adenoviral helper plasmid of the present disclosure does not comprise an L4 (hexon assembly) gene. In some embodiments, the adenoviral helper plasmid does not comprise an L4 encapsidation protein gene. In some embodiments, the adenoviral helper plasmid does not comprise an L4 (hexon assembly) gene and does not comprise an L4 encapsidation protein gene. In some embodiments, the adenoviral helper plasmid of the present disclosure does not comprise a nucleotide sequence encoding L4 (hexon assembly). In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding an L4 encapsidation protein. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding an L4 (hexon assembly) and does not comprise a nucleotide sequence encoding an L4 encapsidation protein gene. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of the L4 33 kDa Ex2. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:9. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence encoding a fragment of L4 33 kDa Ex2. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:10. In some embodiments, the nucleotide sequence encoding a fragment of L4 33 kDa Ex2 comprises an E2a promoter region (see, e.g., Casper et al., "Identification of an adeno-associated viral Rep protein binding site in the adenovirus E2a promoter," Journal of Virology 79.1 (2005)).In some embodiments, the E2a promoter region has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the adenoviral helper plasmid does not include a nucleotide sequence encoding a fragment of the L4 33 kDa Ex2. In some embodiments, the adenoviral helper plasmid does not include an E2a promoter region.

[0088] In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of a hexon-related precursor (L4 pVIII). In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 12. In some embodiments, the adenoviral helper plasmid comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding a hexon-related precursor (L4 pVIII). In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding a partial hexon-related precursor (L4 pVIII) fragment.

[0089] VA RNA region In some embodiments, an adenoviral helper plasmid of the present disclosure comprises a VA RNA region having a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, an adenoviral helper plasmid comprises a VA RNA region having a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the VA RNA region comprises a VA RNAI gene having a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, the VA RNA region comprises a VA RNAI gene having a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the VA RNA region comprises a VA RNAII gene having a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the VA RNA region comprises a VA RNAII gene having a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 19.

[0090] In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding a fragment of a DNA-termination protein. In some embodiments, the nucleotide sequence encoding the fragment of the DNA-termination protein is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:20. In some embodiments, the fragment of the DNA-termination protein has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:21. In some embodiments, the adenoviral helper plasmid does not comprise a nucleotide sequence encoding a DNA-termination protein. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence encoding a fragment of a 23 kDa endoprotease. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:22. In some embodiments, the fragment of the 23 kDa endoprotease region has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the adenoviral helper plasmid does not include a nucleotide sequence encoding the 23 kDa endoprotease region. Introduction of genes encoding complementary traits

[0091] In some embodiments, the adenoviral helper plasmid of the present disclosure comprises an E2a gene. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence encoding E2a. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 24. In some embodiments, the adenoviral helper plasmid of the present disclosure comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 25. In some embodiments, the expression of E2a is under the control of a promoter. In some embodiments, the nucleotide sequence encoding E2a is operably linked to a promoter. In some embodiments, the promoter is, for example, a CMV promoter, a PGK promoter, an SV40 promoter, an EF-1α promoter, a Ubc promoter, a CAG promoter, or a β-actin promoter. In some embodiments, the nucleotide sequence encoding E2a is operably linked to a transcriptional enhancer. In some embodiments, the transcriptional enhancer is, for example, a CMV enhancer. In some embodiments, the nucleotide sequence encoding E2a is operably linked to a regulatory intron. In some embodiments, expression of E2a is under the control of a chicken β-actin promoter. In some embodiments, the nucleotide sequence encoding E2a is operably linked to a chicken β-actin promoter. In some embodiments, the chicken β-actin promoter has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, the chicken β-actin promoter is located upstream of the nucleotide sequence encoding E2a. In some embodiments, expression of E2a is under the control of an E2a promoter and a chicken β-actin promoter. In some embodiments, the nucleotide sequence encoding E2a is operably linked to an E2a promoter and a chicken β-actin promoter.In some embodiments, the chicken β-actin promoter is located upstream of the E2a promoter. In some embodiments, expression of E2a is under the control of the chicken β-actin promoter and the CMV enhancer. In some embodiments, the nucleotide sequence encoding E2a is operably linked to the chicken β-actin promoter and the CMV enhancer. In some embodiments, the chicken β-actin promoter and the CMV enhancer are located upstream of the E2a promoter. In some embodiments, the adenovirus helper plasmid comprises an E2a polyadenylation signal. In some embodiments, the E2a polyadenylation signal is located downstream of the nucleotide sequence encoding E2a. In some embodiments, the E2a polyadenylation signal has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:27. In some embodiments, the adenovirus helper plasmid comprises an SV40 polyadenylation signal. In some embodiments, the SV40 polyadenylation signal is located downstream of the nucleotide sequence encoding E2a. In some embodiments, the SV40 polyadenylation signal is located downstream of the E2a polyadenylation signal. In some embodiments, the SV40 polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:28.

[0092] In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence encoding UL30 from HSV-1. In some embodiments, the nucleotide sequence encoding UL30 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:29. In some embodiments, the amino acid sequence of UL30 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:30. In some embodiments, the adenoviral helper plasmid comprises a nucleotide sequence encoding UL42 from HSV-1. In some embodiments, the nucleotide sequence encoding UL42 has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:31. In some embodiments, the amino acid sequence of UL42 is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:32. In some embodiments, the adenovirus helper plasmid comprises a nucleotide sequence encoding UL30 derived from HSV-1 and a nucleotide sequence encoding UL42 derived from HSV-1. In some embodiments, the nucleotide sequence encoding UL30 and the nucleotide sequence encoding UL42 are separated by a P2a cleavage site. In some embodiments, the P2a cleavage site has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 33. In some embodiments, the P2a cleavage site has an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, expression of the UL30 and / or UL42 genes is under the control of the EF-1α promoter. In some embodiments, the nucleotide sequence encoding UL30 is operably linked to the promoter. In some embodiments, the nucleotide sequence encoding UL30 is operably linked to a CMV promoter, a PGK promoter, an SV40 promoter, an EF-1α promoter, a Ubc promoter, a CAG promoter, or a β-actin promoter.In some embodiments, the nucleotide sequence encoding UL30 is operably linked to a transcriptional enhancer. In some embodiments, the transcriptional enhancer is, for example, a CMV enhancer. In some embodiments, the nucleotide sequence encoding UL30 is operably linked to a regulatory intron. In some embodiments, the nucleotide sequence encoding UL42 and / or the nucleotide sequence encoding UL30 is operably linked to an EF-1α promoter. In some embodiments, the EF-1α promoter has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, expression of UL30 and / or UL42 is under the control of an SV40 promoter. In some embodiments, the nucleotide sequence encoding UL42 and / or the nucleotide sequence encoding UL30 is operably linked to an SV40 promoter. In some embodiments, the SV40 promoter has a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 51.

[0093] In some embodiments, the adenovirus helper plasmid comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is synthetic. In some embodiments, the synthetic polyadenylation signal is a 49 bp synthetic polyadenylation signal (SEQ ID NO: 108).

[0094] In some embodiments, the polyadenylation signal is a β-globin polyadenylation signal, an SV40 polyadenylation signal, or a bovine growth hormone (bGH) polyadenylation signal. In some embodiments, the adenoviral helper plasmid comprises a polyadenylation signal downstream of the nucleotide sequence encoding UL42. In some embodiments, the adenoviral helper plasmid comprises a β-globin polyadenylation signal downstream of the nucleotide sequence encoding UL42. In some embodiments, the β-globin polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 36. In some embodiments, the adenoviral helper plasmid comprises a bovine growth hormone (bGH) polyadenylation signal downstream of the nucleotide sequence encoding UL42. In some embodiments, the bGH polyadenylation signal has a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:52. Exemplary Adenovirus Helper Plasmids

[0095] In some embodiments, the adenoviral helper plasmids of the disclosure have a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, the adenoviral helper plasmids of the disclosure have the following components with nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO: 1), L4 (hexon assembly) (SEQ ID NO: 3; SEQ ID NO: 4), L4 (33 kDa Ex2) (SEQ ID NO: 5; SEQ ID NO: 6), L4 encapsidation protein (22 kDa) (SEQ ID NO: 7; SEQ ID NO: 8), L4 pVIII hexon-related precursor (SEQ ID NO: 12; SEQ ID NO: 13), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA It contains RNAII-A (SEQ ID NO: 18), partial DNA terminal protein (SEQ ID NO: 20; SEQ ID NO: 21), 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), but does not contain the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, and penton perihexon-associated gene.

[0096] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:42. In some embodiments, the adenovirus helper plasmids of the present disclosure contain the following components having nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO:1), L4 (33 kDa Ex2) (SEQ ID NO:9; SEQ ID NO:10), VA RNA region A (SEQ ID NO:14), VA RNAI-A (SEQ ID NO:16), VA RNAII-A (SEQ ID NO:18), partial DNA terminal protein (SEQ ID NO:20; SEQ ID NO:21), 23 kDa endoprotease fragment region (SEQ ID NO:22; SEQ ID NO:23), and E2a (SEQ ID NO:24; SEQ ID NO:25), and do not contain or encode the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, penton periphery hexon associated gene, full-length L4 (hexon assembly) gene, L4 encapsidation protein, and L4 pVIII hexon-associated precursor.

[0097] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:43. In some embodiments, the adenovirus helper plasmids of the present disclosure comprise the following components having nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 mini-promoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), and E2a (SEQ ID NO: 24; SEQ ID NO: 25), and do not contain or encode the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, penton periphery hexon-associated gene, full-length L4 (hexon assembly) gene, L4 encapsidation protein, L4 pVIII hexon-associated precursor, DNA termination protein, and 23 kDa endoprotease fragment region.

[0098] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:44. In some embodiments, the adenovirus helper plasmids of the present disclosure contain the following components having a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO:1), L4 (33 kDa Ex2) (SEQ ID NO:9; SEQ ID NO:10), VA RNA region A (SEQ ID NO:14), VA RNAI-A (SEQ ID NO:16), VA RNAII-A (SEQ ID NO:18), partial DNA terminal protein (SEQ ID NO:20; SEQ ID NO:21), 23 kDa endoprotease fragment region (SEQ ID NO:22; SEQ ID NO:23), E2a (SEQ ID NO:24; SEQ ID NO:25), and chicken β-actin promoter upstream of E2a, and do not contain the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, penton periphery hexon associated gene, full-length L4 (hexon assembly) gene, L4 encapsidation protein, and L4 pVIII hexon-associated precursor.

[0099] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:45. In some embodiments, the adenovirus helper plasmids of the present disclosure contain the following components having nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), and a chicken β-actin promoter upstream of E2a, and do not contain or encode the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, penton periphery hexon-associated gene, full-length L4 (hexon assembly) gene, L4 encapsidation protein, L4 pVIII hexon-associated precursor, DNA terminal protein, and 23 kDa endoprotease fragment region.

[0100] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:46. In some embodiments, the adenovirus helper plasmids of the present disclosure comprise the following components having nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), an SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), and a chicken β-actin promoter upstream of E2a, and the following components: a fiber gene, an L1-52 / 55K (packaging protein 3) gene, a penton periphery hexon associated gene, a full-length L4 (hexon assembly) gene, an L4 encapsidation protein, an L4 pVIII does not contain or encode the hexon-related precursor, DNA terminal protein, and 23 kDa endoprotease fragment regions.

[0101] In some embodiments, the adenoviral helper plasmids of the present disclosure have a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 47. In some embodiments, the adenoviral helper plasmids of the present disclosure have the following components with nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA RNAII-A (SEQ ID NO: 18), a partial DNA terminal protein (SEQ ID NO: 20; SEQ ID NO: 21), a 23 kDa endoprotease fragment region (SEQ ID NO: 22; SEQ ID NO: 23), E2a (SEQ ID NO: 24; SEQ ID NO: 25), a chicken β-actin promoter upstream of E2a, the UL30 gene from HSV-1 (SEQ ID NO: 29; SEQ ID NO: 30), the UL42 gene from HSV-1 (SEQ ID NO: 31; SEQ ID NO: 32), the EF-1α promoter upstream of UL30 (SEQ ID NO: 35), and a β-globin polyadenylation signal downstream of UL42 (SEQ ID NO: 36), and does not contain or encode the following components: a fiber gene, an L1-52 / 55K (packaging protein 3) gene, a penton periphery hexon-associated gene, a full-length L4 (hexon assembly) gene, an L4 encapsidation protein, and an L4 pVIII hexon-associated precursor.

[0102] In some embodiments, the adenoviral helper plasmids of the present disclosure have a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 48. In some embodiments, the adenoviral helper plasmids of the present disclosure have the following components with nucleotide sequences at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: E4 minipromoter (SEQ ID NO: 1), L4 (33 kDa Ex2) (SEQ ID NO: 9; SEQ ID NO: 10), VA RNA region A (SEQ ID NO: 14), VA RNAI-A (SEQ ID NO: 16), VA It contains RNAII-A (SEQ ID NO:18), a partial DNA terminal protein (SEQ ID NO:20; SEQ ID NO:21), a 23 kDa endoprotease fragment region (SEQ ID NO:22; SEQ ID NO:23), E2a (SEQ ID NO:24; SEQ ID NO:25), a chicken β-actin promoter upstream of E2a, the HSV-1-derived UL30 gene (SEQ ID NO:29; SEQ ID NO:30), the HSV-1-derived UL42 gene (SEQ ID NO:31; SEQ ID NO:32), an SV40 promoter upstream of UL30 (SEQ ID NO:51), and a bovine growth hormone (bGH) polyadenylation signal downstream of UL42 (SEQ ID NO:52), and does not contain or encode the following components: fiber gene, L1-52 / 55K (packaging protein 3) gene, penton periphery hexon-associated gene, full-length L4 (hexon assembly) gene, L4 encapsidation protein, and L4 pVIII hexon-associated precursor.

[0103] In some embodiments, the adenovirus helper plasmid of the present disclosure has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO:49. In some embodiments, the adenovirus helper plasmids of the present disclosure contain the following components having a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to the sequences shown: an SV40 promoter upstream of the E4 region (SEQ ID NO: 2), VA RNA region B (SEQ ID NO: 15), VA RNAI-B (SEQ ID NO: 17), VA RNAII-B (SEQ ID NO: 19), E2a (SEQ ID NO: 24; SEQ ID NO: 25), an SV40 polyadenylation signal downstream of E2a (SEQ ID NO: 28), an SV40 polyadenylation signal downstream of E4orf6 (SEQ ID NO: 50), and a chicken β-actin promoter upstream of E2a, and the following components: a fiber gene, an L1-52 / 55K (packaging protein 3) gene, a penton periphery hexon-associated gene, a full-length L4 (hexon assembly) gene, an L4 encapsidation protein, an L4 pVIII hexon-associated precursor, an L4 (33 kDa It does not contain or encode the DNA terminal protein and 23 kDa endoprotease fragment region, the E4 minipromoter upstream of the E4 region, the gene encoding E4orf1, the gene encoding E4orf2, and the gene encoding E4orf3.

[0104] In some embodiments, adenoviral helper plasmids of the present disclosure have a nucleotide sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 94. In some embodiments, adenoviral helper plasmids are designed to contain a "B2" design, which includes an SV40 polyA site, potentially increasing expression of E2A, and a synthetic sequence for a smaller VA region (containing Ad2 VA RNA I and VA RNA II) that does not contain adjacent Ad terminal protein or endoprotease gene sequences. In some embodiments, this region was synthesized flanked by StuI and BsrGI sites, and the insert was cloned into pEMBR-1.2 to generate pEMBR-1.2B2 (e.g., SEQ ID NO: 94).

[0105] In some embodiments, the adenoviral helper plasmids described herein are 11,000 bp or less. In some embodiments, the adenoviral helper plasmids described herein are 10,000 bp or less. In some embodiments, the adenoviral helper plasmids described herein are 9,500 bp or less. In some embodiments, the adenoviral helper plasmids described herein are 9,000 bp or less, and in some embodiments, the adenoviral helper plasmids described herein are 8,000 bp or less. In some embodiments, the adenoviral helper plasmids described herein are 7,500 bp or less. In some embodiments, the adenoviral helper plasmids described herein are 7,000 bp or less.

[0106] In some embodiments, the L4 trans-plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 115. In some embodiments, the L4 trans-plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 119. In some embodiments, the L4 trans-plasmid comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.

[0107] Production method In some embodiments, the adenovirus helper plasmids of the present disclosure are useful in methods of producing rAAV. In some embodiments, the adenovirus helper plasmids and L4 trans plasmids of the present disclosure are useful in methods of producing rAAV. In some embodiments, the rAAV is produced by transfection of producer cells. In some embodiments, the producer cells are mammalian cells. In some embodiments, the producer cells are transformed mammalian cells. In some embodiments, the producer cells are Vero, HeLa, HEK293, HEK293T cells, or derivatives thereof.

[0108] In some embodiments, the L4 region is provided in a producer cell. In some embodiments, the producer cell comprises a nucleotide sequence encoding the L4 region. In some embodiments, the producer cell comprises a nucleotide sequence encoding the L4 proteins L4 33K and L4 22K. In some embodiments, the producer cell comprises a nucleotide sequence encoding L4 33K. In some embodiments, the producer cell comprises a nucleotide sequence encoding L4 22K. In some embodiments, the method of producing rAAV comprises transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, and an adenovirus helper plasmid. In some embodiments, the method of producing rAAV comprises transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, an adenovirus helper plasmid, and an L4 transformer plasmid. In some embodiments, the method of producing rAAV comprises transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, an adenovirus helper plasmid comprising an L4 region, and an L4 transformer plasmid. In some embodiments, methods of producing rAAV involve transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, an adenoviral helper plasmid comprising an L4 region, and an L4 transformer plasmid comprising a nucleotide sequence encoding L4 22K. In some embodiments, methods of producing rAAV involve transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, an adenoviral helper plasmid comprising an L4 region, and an L4 transformer plasmid comprising a nucleotide sequence encoding L4 33K. In some embodiments, methods of producing rAAV involve transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, and an adenoviral helper plasmid comprising an L4 region, wherein the producer cell comprises a nucleotide sequence encoding the L4 region or a portion thereof (e.g., L33K and / or L22K).

[0109] In some embodiments, the AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, the adenovirus helper plasmid is any of the adenovirus helper plasmids described herein. In some embodiments, the L4 transfer plasmid is any of the L4 transfer plasmids described herein.

[0110] In some embodiments, the method of producing rAAV involves transfecting a producer cell that stably expresses Rep-Cap. In some embodiments, the method of producing rAAV involves transfecting a producer cell that stably expresses Rep-Cap with an AAV vector plasmid and an adenovirus helper plasmid. In some embodiments, the method of producing rAAV involves transfecting a producer cell that stably expresses Rep-Cap with an AAV vector plasmid, an adenovirus helper plasmid, and an L4 transformer plasmid. In some embodiments, the method of producing rAAV involves transfecting a producer cell that stably expresses Rep-Cap with an AAV vector plasmid, an adenovirus helper plasmid comprising an L4 region, and an L4 transformer plasmid. In some embodiments, the method of producing rAAV involves transfecting a producer cell that stably expresses Rep-Cap with an AAV vector plasmid, an adenovirus helper plasmid comprising an L4 region, and an L4 transformer plasmid comprising a nucleotide sequence encoding L4 22K. In some embodiments, methods of producing rAAV involve transfecting a producer cell that stably expresses Rep-Cap with an AAV vector plasmid, an adenoviral helper plasmid comprising the L4 region, and an L4 trans-plasmid comprising a nucleotide sequence encoding L4 33K. In some embodiments, methods of producing rAAV involve transfecting a producer cell that stably expresses Rep-Cap and comprises a nucleotide sequence encoding the L4 region or a portion thereof (e.g., L33K and / or L22K) with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, and an adenoviral helper plasmid comprising the L4 region.

[0111] In some embodiments, the AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest. In some embodiments, the adenovirus helper plasmid is any of the adenovirus helper plasmids described herein. In some embodiments, the L4 transfer plasmid is any of the L4 transfer plasmids described herein. [Example]

[0112] The primary goal of the work described in this disclosure is to develop novel adenoviral helper plasmids for rAAV production that are smaller, contain fewer unnecessary adenoviral genes, and perform as well as or better than the most commonly used adenoviral helper plasmids.

[0113] The plasmids provided in this disclosure were synthesized de novo, sequence verified, and scaled up for use in large-scale rAAV production. rAAV production studies were conducted to compare vector yields using the provided plasmids with those of other commercially available adenovirus helper plasmids. Additionally, vector quality and activity were assessed from rAAV produced using different adenovirus helper plasmids to confirm that rAAV produced using the provided plasmids was at least comparable, if not superior, in quality. Collectively, these following examples demonstrate that the provided adenovirus helper plasmids, with their potentially safer and more cost-effective design, produce high-yield, high-quality rAAV.

[0114] Example 1: Exemplary methods for the production of rAAV using the adenovirus helper plasmids described herein HEK293 cells were transfected with a control adenoviral helper plasmid (e.g., a commercially available plasmid such as pALD-X80 or the adenoviral helper plasmid described herein). To generate AAV9 / GFP, the adenoviral helper plasmid was co-transfected with pAAVrep2cap9 and pAAV-CMV-GFP or pAAV-CAG-GFP plasmids using PEI transfection. Four days after transfection, HEK293 cells were harvested via 0.5% Triton® X-100 lysis and nuclease addition (to degrade RNA, cellular genomic DNA, and residual plasmid DNA). After 3-4 hours of lysis / nuclease treatment, cell lysates were sampled and submitted for qPCR titer analysis. Samples were treated with additional nucleases, followed by EDTA and heat treatment, followed by qPCR of diluted samples to determine the vector genome copy number per sample. Fluorescence microscopy was used to quantitate GFP-positive cells as a measure of transfection efficiency.

[0115] Example 2: Adenovirus helper plasmid lacking fiber, L1-52 / 55K, and peripenton hexon-related genes and containing a partial L4 hexon-related precursor To reduce the size of the adenovirus helper plasmid, an adenovirus helper plasmid (pEMBR-1.2: SEQ ID NO:41) was designed to lack the fiber gene, the L1-52 / 55K (packaging protein 3) gene, and most of the hexon-associated precursor, as well as the penton periphery hexon-associated protein. These deletions were made in comparison to commercially available helper plasmids such as pXX6-80. The adenovirus helper genes were synthesized and assembled into a kanamycin-resistant plasmid backbone. The resulting plasmid is approximately 6.7 kb smaller than pXX6-80.

[0116] The adenovirus helper plasmids described above enabled the production of AAV in HEK293 cells. No significant differences in AAV vector yield were observed between cells transfected with pALD-X80 and pEMBR-1.2, as measured by qPCR (see Figure 2). rAAV vectors produced with pEMBR-1.2 produced normal vectors with the correct ratio of VP proteins, as observed when assessing vector capsid purity by SDS-PAGE (see Figure 3), and packaged transgenes of the correct size, as observed when assessing vector transgene purity by alkaline gel electrophoresis (see Figure 3). Furthermore, pEMBR-1.2 enabled the production of fully functional vectors capable of transfecting cells. No differences were observed in the transfection of HEK293 cells to generate AAVRH.10 / ssCMV-GFP produced with pALD-X80 or pEMBR-1.2 (see Figure 4).

[0117] Example 3: Modifications to the E2a region of adenovirus helper plasmids to reduce plasmid size while maintaining AAV production To further reduce the size of adenovirus helper plasmids (such as those described herein), adenovirus helper plasmids were designed with modifications to the E2a region. These plasmids were designed to contain the VA RNA region (SEQ ID NO: 70) from pEMBR-1.2B2 and the E4 region (SEQ ID NO: 41) from pEMBR-1.2, along with various modifications to the E2a region based on promoter regions identified by Guilfoyle (GEP) and Jing (JEP) (see Figure 14). The sequence modifications to the E2a region based on the GEP and JEP promoters are shown in Figure 12, and the sequence modifications to the E2a region based on truncations to the NotI end of the pEMBR1.2B2 E2a region are shown in Figure 13.

[0118] These plasmids were tested for their ability to produce AAV in 500 mL large-scale cultures (see Figures 15A-B). At larger scales, pEMR-1.2B2-GEP+1 kbp (SEQ ID NO: 81) outperformed pEMBR-1.2B2-GEP (SEQ ID NO: 80). Rep52 and E2a protein expression were also assessed with these plasmids (see Figure 15C). These data indicate that Rep52 was present at similar levels in all constructs tested, and that E2a protein expression likely correlates with AAV production. The JEP+1 kbp plasmid produced E2a protein at normal levels but lacks the sequences encoding the L4 (33K and 22K) regions.

[0119] Further studies were performed to analyze AAV production using various constructs in 75 mL cultures (see Figure 16). These data indicate that there may be little difference in AAV production between pEMBR-1.2B2 (SEQ ID NO: 70), pEMBR-1.2B2-GEP+50bp (SEQ ID NO: 82), and pEMBR-1.2B2-GEP+50bp+Kozak (SEQ ID NO: 83). JEP+NotIΔ1.5 kbp, which does not contain the GEP promoter or the L4 (33K and 22K) regions, produced AAV at low levels.

[0120] A table showing which elements are included in various plasmids (e.g., those described herein) and their ability to produce E2a protein and AAV is shown in Figure 17. Without wishing to be bound by any particular theory, these data indicate that the L4 region (containing the 33K and 22K ORFs), the JEP / GEP region requirement, and proper E2a production are important for AAV production.

[0121] Example 4: Exemplary engineered adenovirus helper plasmids to reduce plasmid size while maintaining AAV production To further reduce the size of adenovirus helper plasmids (e.g., those described herein), adenovirus helper plasmids were designed with modifications specifically to the L4 region. These plasmids were designed with various modifications to the L4 region (see Figures 18-19). The sequence modifications to the L4 region are shown in Figure 21. The ability of these plasmids to produce AAV from 75 mL cultures was evaluated (see Figure 22). Without wishing to be bound by any particular theory, these data indicate that only constructs containing the nucleotide sequences of E4 ORFs 4 and 6 may be required to sustain AAV production. Without wishing to be bound by any particular theory, these data also indicate that adding sequences from the L4 region (containing the 33K and 22K ORFs) with a synthetic polyA tail rescues AAV production in constructs that previously did not produce AAV.

[0122] Further studies were performed to analyze AAV production in various constructs with and without an L4 region (containing the 33K and 22K ORFs) with a synthetic polyA tail compared to AAV production using pEMBR-1.2B2, where the plasmid size is also shown (see Figure 23). Addition of an L4 region (containing the 33K and 22K ORFs) with a synthetic polyA tail to a plasmid that previously did not produce AAV resulted in rescue of AAV production. Exemplary adenovirus plasmid maps engineered to reduce plasmid size (e.g., using the modifications described herein) while maintaining AAV production are shown in Figure 24.

[0123] Additionally, this example describes adenovirus helper plasmids engineered to reduce plasmid size while maintaining AAV production. In the plasmids described in this example, the E2a region was engineered to contain the native E2a ORF and native E2a polyA tail, and the L4 region consisted of 50 bp upstream of the native L4 promoter, the native L4 promoter, and the 33 kDa and 22 kDa ORFs (see Figure 25). A fragment of the L4 33 kDa Ex 2 containing the E2a promoter region was repeated for E2a expression. In the plasmids described in this example, the E2a region was engineered to not contain the L3 23 kDa viral endoprotease, hexon assembly, L4 100K region, or L4 pVIII hexon-related precursor protein.

[0124] Example 5: Introduction of complementary accessory genes into modified adenoviral helper plasmids This example describes the removal of adenovirus helper genes, resulting in a smaller adenovirus helper plasmid and allowing the addition of complementary genes to further improve AAV quality and yield. Specifically, various pEMBR plasmids containing various complementary genes (e.g., UL30, UL42, etc.) of varying sizes are engineered from pEMBR backbone plasmids (e.g., those described herein) and tested for AAV production. The HSV-1 DNA polymerase genes (UL30 and UL42) are added back to the plasmid to support AAV transgene replication even when cells are not in S phase. The complementary genes are designed to be produced as a single transcript. Any number of promoters may be used, including CBA, CMV, PGK, etc., and any number of poly(A) sites may be used. Theoretically, the orientation in which the construct is cloned into the plasmid should not affect expression, since this region contains both a promoter and a poly(A) signal that drives expression of the complementary gene independently of the rest of the plasmid.

[0125] Vector yields of AAV (e.g., AAV9) in clarified lysates are measured by qPCR using various pEMBR plasmids designed to contain various complementary genes (e.g., UL30, UL42, etc.; see pEMBR-1.5A+L4 in Figure 23).

[0126] Example 6: Provision of L4 region sequences in trans rescues AAV production This example demonstrates that provision of L4 region sequences, e.g., L4 33K and / or L4 22K, in trans (i.e., not included in the adenoviral helper plasmid containing VA RNA, the E4 region, and the E2a region) can rescue and / or increase AAV production.

[0127] In this example, quadruple transfection was performed to add an L4 trans-plasmid encoding the L4 region to cells in trans. Addition of the L4 region in trans restored rAAV production to wild-type (pEMBR-1.2B2) levels, regardless of whether the native promoter-driven L4 (L4_pUC57) or exogenously driven (human elongation factor 1 alpha, Ef1αL4_pUC57) L4 was added (Figure 28A).

[0128] To determine which L4 protein is more essential, we also utilized plasmids carrying the native codon-optimized 22K or 33K (22K_pUC57 and 33K_pUC57). Our results indicated that 22K is more essential for rAAV production, but the effect of L4 proteins on production appeared to be additive (Figure 28A). Analysis of E2a protein by Western blot showed a slight decrease in E2a protein when 22K was present (Figure 28B).

[0129] To further test the rescue and / or increase of AAV production, L4 gene loading was performed by adding additional copies of the L4 region protein. This example used for rAAV production was achieved by quadruple transfection of producer cells. The E2a region of the helper plasmid used for quadruple transfection is shown in Figure 29A. Figure 29B shows the average rAAV titer (VG / mL) from triplicate 75 mL quadruple-plasmid-transfected cultures determined by qPCR of pUC57 vectors containing specific L4 constructs in addition to the indicated E2a constructs, comparing L4 gene loadings of 1x L4 gene, 2x L4 genes, 1x 33K + 2x 22K, and 2x 33K + 1x 22K with control conditions (wild-type + empty vector; 1.2B2 + pUC57). Rep and E2a expression levels resulting from the transfection described in Figure 29B are shown in Figure 29C. β-actin is shown as a loading control. Adenovirus type 5-infected HEK293 / HEK293T cells are shown as a positive control, and uninfected / untransfected HEK293 cells are shown as a negative control. Notably, under some conditions, providing additional copies of the L4 region (e.g., 1.2B2+L4_pUC57) resulted in increased rAAV titers. Furthermore, providing additional copies of 33K resulted in increased rAAV titers under several different conditions. See Figure 29B.

[0130] Example 7: Sequence Listing The following sequence listing lists and describes the various sequences discussed herein. Unless otherwise specified, all sequences are listed in the 5' to 3' direction of the positive strand of the plasmid. This direction is maintained regardless of the orientation of the gene or element described as being associated with the sequence. As used herein, an asterisk indicates a stop codon. [Table 1-1] [Table 1-2] [Table 1-3] Table 1-4 Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35 Table 1-36 Table 1-37 Table 1-38 Table 1-39 Table 1-40 Table 1-41 Table 1-42 Table 1-43 Table 1-44 Table 1-45 Table 1-46 Table 1-47 Table 1-48 Table 1-49 Table 1-50 Table 1-51 Table 1-52 Table 1-53 Table 1-54 Table 1-55 Table 1-56 Table 1-57 Table 1-58 Table 1-59 Table 1-60 Table 1-61 Table 1-62 Table 1-63 Table 1-64 Table 1-65 Table 1-66

Table 1-67

Table 1-69

Table 1-90

Table 1-97

Table 1-110

Table 1-120

Table 1-123

Table 1-204

Table 1-206

Table 1-209

Table 1-210

Table 1-234

Table 1-239

[0131] equivalent 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 is instead set forth in the claims that follow.

Claims

1. (a) E2a protein, (b) the E4 region, and (c) an adenovirus helper plasmid comprising a nucleotide sequence encoding a VA RNA region, The adenovirus helper plasmid fiber proteins or portions thereof, L1-52 / 55K (packaging protein 3), and The adenovirus helper plasmid does not contain a nucleotide sequence encoding one or more of: penton peripheral hexon associated proteins.

2. 2. The adenoviral plasmid of claim 1, further comprising an engineered L4 region.

3. 3. The adenoviral plasmid of claim 2, wherein the engineered L4 region does not express the L4 100K protein.

4. 3. The adenoviral plasmid of claim 2, wherein the engineered L4 region does not include the entire L4 100K-encoding nucleotide sequence.

5. 5. The adenoviral plasmid of claim 4, wherein the engineered L4 region does not include base pairs 24061 to 25961 compared to GenBank Accession No. M73260.

6. 3. The adenoviral plasmid of claim 2, wherein the engineered L4 region comprises at least one nucleotide sequence encoding L4-33K that is at least 80% identical to SEQ ID NO:

5.

7. 7. The adenoviral plasmid of claim 6, wherein the engineered L4 region comprises two nucleotide sequences encoding L4-33K that are at least 80% identical to SEQ ID NO:

5.

8. 3. The adenoviral plasmid of claim 2, wherein the engineered L4 region comprises a nucleotide sequence encoding L4-22K that is at least 80% identical to SEQ ID NO:

7.

9. 3. The adenoviral plasmid of claim 2, wherein the engineered L4 region comprises a nucleotide sequence encoding L4-33K and a nucleotide sequence encoding L4-22K.

10. 10. The adenoviral plasmid of claim 9, wherein the engineered L4 region comprises 50 nucleotides upstream of the L4-33K / 22K region.

11. 11. The adenoviral plasmid of claim 10, wherein the 50 nucleotides upstream of the L4-33K / 22K region are identified by comparison with the L4-33K / 22K region of GenBank Accession No. M73260.

12. The adenovirus plasmid of claim 11, further comprising a synthetic polyA signal downstream of the L4-33K / 22K region.

13. 2. The adenoviral plasmid of claim 1, wherein the adenoviral helper plasmid does not contain a nucleotide sequence encoding the L3 23 kDa viral endoprotease.

14. 2. The adenovirus plasmid of claim 1, wherein the adenovirus helper plasmid does not contain a nucleotide sequence encoding L4 pVIII.

15. 10. The adenovirus helper plasmid of claim 9, wherein the engineered L4 region comprises a nucleotide sequence comprising an E2a promoter region upstream of the nucleotide sequence encoding the L4-33K and the nucleotide sequence encoding the L4-22K.

16. 2. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid does not contain a nucleotide sequence encoding a DNA terminal protein.

17. 2. The adenovirus helper plasmid of claim 1, wherein the expression of the E2a protein is under the control of an E2a promoter.

18. 2. The adenovirus helper plasmid of claim 1, wherein the expression of the E2a protein is under the control of an E2a promoter and a chicken β-actin promoter, and the chicken β-actin promoter is upstream of the E2a promoter.

19. The adenovirus helper plasmid of claim 1, wherein the expression of the E2a protein is under the control of the chicken β-actin promoter.

20. The adenovirus helper plasmid of claim 18 or 19, wherein the chicken β-actin promoter has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

26.

21. The adenovirus helper plasmid of claim 1 , wherein the adenovirus helper plasmid comprises an E2a polyadenylation signal downstream of the E2a.

22. 2. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid contains an SV40 polyadenylation signal downstream of the E2a.

23. 21. The adenovirus helper plasmid of claim 20, wherein the SV40 polyadenylation signal is downstream of the E2a polyadenylation signal.

24. 21. The adenovirus helper plasmid of claim 20, wherein the SV40 polyadenylation signal has a sequence that is at least 80% identical to SEQ ID NO:

28.

25. the adenovirus helper plasmid further comprises a nucleotide sequence encoding HSV-1 UL30 and HSV-1 UL42; at least one of the nucleotide sequences is at least 80% identical to SEQ ID NO:29; at least one of the nucleotide sequences is at least 80% identical to SEQ ID NO: 31; 2. The adenovirus helper plasmid of claim 1, wherein the UL30 and the UL42 are separated by a P2A cleavage site encoded by a nucleic acid sequence that is at least 80% identical to SEQ ID NO:

33.

26. 2. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid further comprises a nucleotide sequence encoding HSV-1 UL29 / ICP8.

27. 20. The adenovirus helper plasmid of claim 19, wherein the HSV-1 UL30, the HSV-1 UL42, and the HSV-1 UL29 are separated by a P2A cleavage site encoded by a nucleic acid sequence at least 80% identical to SEQ ID NO:

139.

28. 2. The adenovirus helper plasmid of claim 1, wherein the E4 region does not include E4orf1 and the E4 region does not include E4orf2.

29. 2. The adenovirus helper plasmid of claim 1, wherein the E4 region is operably linked to the E4 mini-promoter, and the E4 mini-promoter has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

1.

30. 2. The adenovirus helper plasmid of claim 1, wherein the E4 region is operably linked to the SV40 promoter, and the SV40 promoter has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

2.

31. 10. The adenoviral helper plasmid of any one of the preceding claims, wherein the adenoviral helper plasmid comprises a resistance gene.

32. 32. The adenovirus helper plasmid of claim 31, wherein the resistance cassette is a kanamycin resistance gene.

33. An adenovirus helper plasmid having 80% sequence identity to any one of SEQ ID NOs: 80-105.

34. An adenovirus helper plasmid comprising an engineered E2A region comprising any one of nucleotides SEQ ID NO:106 or SEQ ID NO:

112.

35. 3. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid does not contain a nucleotide sequence encoding L4 33K.

36. 3. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid does not contain a nucleotide sequence encoding L4 22K.

37. 3. The adenovirus helper plasmid of claim 1, wherein the adenovirus helper plasmid does not contain a nucleotide sequence encoding L4 22K and does not contain a nucleotide sequence encoding L4 33K.

38. An L4 trans-plasmid comprising a nucleotide sequence encoding an engineered L4 region.

39. 39. The L4 trans-plasmid of claim 38, wherein the engineered L4 region comprises a nucleotide sequence encoding an L4 that is at least 80% identical to SEQ ID NO:

115.

40. 39. The L4 trans-plasmid of claim 38, wherein the engineered L4 region comprises a nucleotide sequence encoding L4-33K that is at least 80% identical to SEQ ID NO:

120.

41. 39. The L4 trans-plasmid of claim 38, wherein the engineered L4 region comprises a nucleotide sequence encoding L4-22K that is at least 80% identical to SEQ ID NO:

7.

42. an adenoviral helper plasmid according to any one of claims 35, 36, or 37; and A composition comprising an L4 transplasmid according to any one of claims 38 to 41.

43. 1. A method for producing a recombinant adenovirus-associated viral vector, comprising: The method comprises transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, and an adenovirus helper plasmid according to any one of claims 1 to 37.

44. 44. The method of claim 43, wherein the AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest.

45. 44. The method of claim 43, wherein the method further comprises transfecting a production cell with an L4 trans-plasmid according to any one of claims 38 to 41.

46. 46. ​​The method of any one of claims 43 to 45, wherein both the L4 trans-plasmid and the adenovirus helper plasmid comprise an engineered L4 region.

47. 47. The method of claim 46, wherein the engineered L4 region of the L4 trans-plasmid comprises a nucleotide sequence encoding L33K.

48. 1. A method for producing a recombinant adenovirus-associated viral vector, comprising:

38. A method comprising transfecting a producer cell with an AAV vector plasmid and the adenovirus helper plasmid of any one of claims 1 to 37, wherein the producer cell stably expresses Rep-Cap and comprises a nucleotide sequence encoding the L4 region or a portion thereof.

49. 49. The method of claim 48, wherein the AAV vector plasmid comprises AAV inverted terminal repeats (ITRs) and a transgene of interest.

50. 50. The method of claim 49, wherein the method further comprises transfecting a production cell with an L4 trans-plasmid according to any one of claims 38 to 41.

51. 1. A method for producing a recombinant adenovirus-associated viral vector, comprising: The method comprises transfecting a producer cell with an AAV vector plasmid, an AAV Rep-Cap expression plasmid, an adenovirus helper plasmid according to any one of claims 1 to 37, and an L4 trans plasmid.

52. 52. The method of claim 51 , wherein the L4 trans-plasmid comprises a nucleotide sequence encoding L33K.

53. 52. The method of claim 51, wherein the producing cell comprises a nucleotide sequence encoding the L4 region or a portion thereof.

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