WO2024254232A2 - Two-plasmid system for aav vector particle production - Google Patents

Abstract

The present invention relates to plasmid systems for production of AAV vector particles, and in particular to a two-plasmid system with Cap and helper functions on a first plasmid and Rep and the gene of interest on a second plasmid.

Description

Two-Plasmid System for AAV Vector Particle Production Cross-Reference to Related Applications The present application claims priority to U.S. Provisional Application No. 63/472,155, filed June 9, 2023, which is incorporated herein by reference in its entirety. Sequence Listing The text of the computer readable sequence listing filed herewith, titled “CATA_42089_601_SequenceListing.xml” created June 6, 2024, having a file size of 67,865 bytes, is hereby incorporated by reference in its entirety. Field The present invention relates to plasmid systems for production of AAV vector particles, and in particular to a two-plasmid system with Cap and helper functions on a first plasmid and Rep and the gene of interest on a second plasmid. Background Adeno-associated virus (AAV) is one of the most actively investigated gene therapy vehicles. It was initially discovered as a contaminant of adenovirus preparations. AAV is a protein shell surrounding and protecting a small, single-stranded DNA genome of approximately 4.8 kilobases (kb). AAV belongs to the parvovirus family and is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate. Initially distinguished serologically, molecular cloning of AAV genes has identified hundreds of unique AAV strains in numerous species. Its single-stranded genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication and packaging. The Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), which are required for viral genome replication and packaging, while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. It is estimated that the viral coat is comprised of 60 proteins arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3). The aap gene encodes the assembly-activating protein (AAP) in an alternate reading frame overlapping the cap gene. This nuclear protein is thought to provide a scaffolding function for capsid assembly. While AAP is essential for nucleolar localization of VP proteins and capsid assembly in AAV2, the subnuclear localization of AAP varies among 11 other serotypes recently examined, and is nonessential in AAV4, AAV5, and AAV11. Summary The present invention relates to plasmid systems for production of AAV vector particles, and in particular to a two-plasmid system with Cap and helper functions on a first plasmid and Rep and the gene of interest on a second plasmid. Accordingly, in some preferred embodiments, the present invention provides an engineered, non-naturally occurring system for producing AAV vector particles comprising: a first nucleic acid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a second nucleic acid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV ITR sequence. In some preferred embodiments, the functional AAV Cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes. In some preferred embodiments, the one or more sequences encoding functional AAV helper genes are a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein. In some preferred embodiments, the Rep gene comprises a gene encoding a functional Rep 52 protein, a gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein, and a gene encoding a functional Rep 78 protein. In some preferred embodiments, the at least one Rep gene does not comprise a functional internal p40 promoter. In some preferred embodiments, the AAV Rep gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Rep genes. In some preferred embodiments, the Rep gene is an AAV2 Rep gene. In some preferred embodiments, the Rep gene and the gene of interest are expressed from different promoters. In some preferred embodiments, the gene of interest is operably linked to a promoter selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter. In some preferred embodiments, the promoter is selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter. In some preferred embodiments, the gene of interest encodes a therapeutic gene product. In some preferred embodiments, the therapeutic gene product is selected from the group consisting of a polypeptide, peptide, protein, short interfering RNA (siRNA), miRNA and small hairpin RNA (shRNA). In some preferred embodiments, the first and second nucleic acids sequences are and not operably linked to one another, e.g., they are distinct nucleic acid molecules. In some preferred embodiments, the first nucleic acid sequence is in a first vector and the second nucleic acid sequence is in a second vector. In some preferred embodiments, the first nucleic acid sequence is in a first plasmid and the second nucleic acid sequence is in a second plasmid. In some preferred embodiments, the present invention provides a cell or population of cells comprising the system as described in any of the embodiments described above. In some preferred embodiments, the cell or population of cells are producer cells. In some preferred embodiments, the present invention provides an engineered, non-naturally occurring two-plasmid system for producing AAV vector particles comprising: a first plasmid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a second plasmid encoding an AAV Rep gene, and a gene of interest flanked on at one side by an AAV ITR sequence. In some preferred embodiments, the functional AAV Cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes. In some preferred embodiments, the one or more sequences encoding functional AAV helper genes are a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein. In some preferred embodiments, the Rep gene comprises a gene encoding a functional Rep 52 protein, a gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein, and a gene encoding a functional Rep 78 protein. In some preferred embodiments, the at least one Rep gene does not comprise a functional internal p40 promoter. In some preferred embodiments, the AAV Rep gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Rep genes. In some preferred embodiments, the Rep gene is an AAV2 Rep gene. In some preferred embodiments, the Rep gene and the gene of interest are expressed from different promoters. In some preferred embodiments, the gene of interest is operably linked to a promoter selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter. In some preferred embodiments, the promoter is selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter. In some preferred embodiments, the gene of interest encodes a therapeutic gene product. In some preferred embodiments, the therapeutic gene product is selected from the group consisting of a polypeptide, peptide, protein, short interfering RNA (siRNA), miRNA and small hairpin RNA (shRNA). In some preferred embodiments, the present invention provides a cell or population of cells comprising the plasmid system of any of the foregoing embodiments. In some preferred embodiments, the cell or population of cells are producer cells. In some preferred embodiments, the present invention provides a method for producing recombinant AAV vector particles comprising: (a) transfecting a host cell with the nucleic acid system or plasmid system as described in any of the embodiments above; and (b) culturing the host cell under conditions suitable for production of recombinant AAV vector particles. In some preferred embodiments, the methods further comprise the step of harvesting the recombinant AAV vector particles. In some preferred embodiments, the host cells are transfected with 2 plasmids simultaneously or with first plasmid at least 12 hours before the second plasmid. In some preferred embodiments, the present invention provides a plasmid system comprising: a set of first plasmid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes, wherein the set of first plasmids comprises plasmids comprising AAV Cap genes from at least two different AAV serotypes; and a second plasmid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV ITR sequence. In some preferred embodiments, the set of first plasmids comprises plasmids comprising AAV Cap genes from at least five different AAV serotypes. In some preferred embodiments, the set of first plasmids comprises plasmids comprising AAV Cap genes from at least ten different AAV serotypes. In some preferred embodiments, the AAV Cap genes are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes. Brief Description Of The Drawings FIG.1: SEQ ID NO:1, Cap5-Helper Plasmid Sequence. FIG.2: SEQ ID NO:2, Cap6-Helper Plasmid Sequence. FIG.3: SEQ ID NO:3, Rep-GOI Plasmid Sequence. Detailed Description The present invention relates to plasmid systems for production of AAV vector particles, and in particular to a two-plasmid system with Cap and helper functions on a first plasmid and Rep and the gene of interest on a second plasmid. It is contemplated that the system of the present invention provides advantages over other systems for production of AAV vector particles because a library of pre-made Cap/Helper plasmids with different Cap sequences can be used in conjunction with the Rep/GOI plasmid to provide flexibility for producing AAV vector particles with different serotypes. Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. As used herein, the term “genome” refers to the genetic material (e.g., chromosomes) of an organism. As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral particle. The viral vector can contain a nucleic acid (e.g., a payload) encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. [00123] As used herein, an "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wild- type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant AAV vector particle" or simply a "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector is contained within a rAAV particle. As used herein, the term “viral capsid polypeptide” refers to the proteinaceous shell or coat of a viral particle. At a minimum, a viral capsid polypeptide as described herein permits packaging or assembly of the capsid polypeptide into a viral particle that is competent for delivery of nucleic acid to the host cell. Capsids function to encapsidate, protect, transport, and release into a host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of a polypeptide of the viral capsid polypeptides. As used herein, the term “encapsidated” means enclosed within a viral capsid. As an example, the AAV genome comprises three overlapping sequences which encode capsid proteins, VP1, VP2 and VP3, which start from one promoter, p40. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of approximately 1 : 1 : 10. As used herein, "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. As used herein, “payload” refers to a nucleic acid which is encapsidated within a viral vector, e.g., an AAV vector. A payload nucleic acid can encode, e.g., a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA. As used herein, the term “producer cell” refers to any cell line suitable for the production of AAV vectors. The term " polypeptide " as used herein refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a "polypeptide." Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc. The term "polypeptide sequence" or "amino acid sequence" as used herein can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N- terminal to C- terminal direction unless otherwise indicated. A given amino acid can be replaced by a residue having similar physicochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. ligand-mediated receptor activity and specificity of a native or reference polypeptide is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into He or into Leu. As used herein, the term "DNA" is defined as deoxyribonucleic acid. The term "polynucleotide" is used herein interchangeably with "nucleic acid" to indicate a polymer of nucleosides. Typically, a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine) joined by phosphodiester bonds. However, the term encompasses molecules comprising nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications. Where this specification refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single- and double-stranded forms (and complements of each single-stranded molecule) are provided. "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and/or to the sequence information (i.e. the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated. As used herein, the term “corresponding to,” when used in reference to an amino acid or polynucleotide sequence means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has structural properties, functional properties, or both that are similar relative to an amino acid or polynucleotide sequence in a similar location in another polypeptide or polynucleotide molecule. Homologues of a given polypeptide in different species “correspond to” each other, as do regions or domains of homologous polypeptides from different species. Similarly, capsid polypeptides of different serotypes of viral vectors, including but not limited to adeno-associated virus (AAV) vectors, “correspond to” each other, as do regions of such polypeptides, defined, for example by alignment of their amino acid sequences. While other alignment parameters can be used to define such regions, for the avoidance of doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) using default parameters of version BLAST+ 2.8.0 released March 28, 2018. As used herein, a “transgene” is a polynucleotide that is delivered to a cell by a vector. A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product after being transcribed, and sometimes also translated. The term “gene” or “coding sequence” refers to a nucleotide sequence in vitro or in vivo that encodes a gene product. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product. In other instances, the gene comprises additional, non-coding, sequence. For example, the gene may or may not include regions preceding and following the coding region, e.g.5' untranslated (5' UTR) or “leader” sequences and 3' UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons). A “gene product” is a molecule resulting from expression of a particular gene. Gene products include, e.g., a polypeptide, an aptamer, an interfering RNA, an mRNA, and the like. In particular embodiments, a “gene product” is a polypeptide, peptide, protein, or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA). In particular embodiments, a gene product is a therapeutic gene product, e.g., a therapeutic protein. As used herein, a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed. Examples of beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic. Therapeutic genes include, but are not limited to, genes that correct a genetic deficiency in a cell or mammal. As used herein, the term "host cell" refers to any eukaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo. As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos. The term "gene of interest" refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, expression of a protein of interest in a host cell, expression of a ribozyme, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.). As used herein, the term “protein of interest” refers to a protein encoded by a gene of interest. As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA or RNA sequence thus codes for the amino acid sequence. The term "promoter," "promoter element," or "promoter sequence" as used herein, refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al., Science 236:1237 [1987]). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells, and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see, Voss et al., Trends Biochem. Sci., 11:287 [1986]; and Maniatis et al., supra). For example, the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema et al., EMBO J. 4:761 [1985]). Two other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]) and the long terminal repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (Boshart et al., Cell 41:521 [1985]). As used herein, the term "promoter/enhancer" denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions (i.e., the functions provided by a promoter element and an enhancer element, see above for a discussion of these functions). For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer/promoter is one that is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is one that is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques such as cloning and recombination) such that transcription of that gene is directed by the linked enhancer/promoter. As used herein, the terms "complementary" or "complementarity" are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-A-G-T-3'," is complementary to the sequence "3'-T-C-A-5'." Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. As used herein, the percentage of identity of an amino acid sequence or nucleic acid sequence, or the term "% sequence identity," is defined herein as the percentage of residues of the full length of an amino acid sequence or nucleic acid sequence that is identical with the residues in a reference amino acid sequence or nucleic acid sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. The percentage homology of an amino acid sequence or the term "% homology to" is defined herein as the percentage of amino acid residues in a particular sequence that are homologous with the amino acid residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. This method takes into account conservative amino acid substitutions. Conservative substitutions are substitutions of an amino acid to be substituted with a similar amino acid. Amino acids can be similar in several characteristics, for example, size, shape, hydrophobicity, hydrophilicity, charge, isoelectric point, polarity, aromaticity, etc. Preferably, a conservative substitution is an exchange of one amino acid within a group for another amino acid within the same group, whereby the groups are the following: (1) alanine, valine, leucine, isoleucine, methionine, and phenylalanine: (2) histidine, arginine, lysine, glutamine, and asparagine; (3) aspartate and glutamate; (4) serine, threonine, alanine, tyrosine, phenylalanine, tryptophan, and cysteine; and (5) glycine, proline, and alanine. Methods and computer programs for the alignment are well known in the art, for example "Align 2". Programs for determining nucleotide sequence identity are also well known in the art, for example, the BESTFIT, FASTA and GAP programs. These programs are readily utilized with the default parameters recommended by the manufacturer. The terms "in operable combination," "in operable order," and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. 2. AAV Plasmid Systems The present invention relates to plasmid systems for production of AAV vector particles, and in particular to a two-plasmid system with Cap and helper functions on a first plasmid the Cap-Helper plasmid) and Rep and the gene of interest on a second plasmid (the Rep-GOI plasmid). In some preferred embodiments, the viral particle produced by the two- plasmid system is replication-incompetent or replication defective. As used herein, the terms “replication-incompetent” and “replication defective” refer to a virus or viral particle that cannot independently replicate and package its genome. For example, when a cell is infected with the virus or viral particle, the heterologous gene is expressed in the infected cells, however, the virus or the viral particle is not able to replicate further. In preferred embodiments, the plasmid system is a two-plasmid system. It is contemplated that the two plasmid system is useful for producing recombinant AAV vector particles. The AAV vector particles find use in gene therapy, including providing a protein in a subject in need thereof and for gene editing in a subject in need thereof. When used for gene editing, it will be understood that certain components of the gene editing system (e.g., Cas9) are including on a first Rep-GOI plasmid and other components of the gene editing system (e.g., sgRNA and donor template RNA) are included a second Rep-GOI plasmid. In some preferred embodiments, the two-plasmid system can be used without the need for additional plasmids to produce AAV vector particles. In some preferred embodiments, the two-plasmid system can be used to produce rAAV without the need for helper virus such as adenovirus. In some preferred embodiments, the two-plasmid system can be used to produce rAAV without the need for genetic material originating from a host cell, with the exception of a gene encoding El A/B if that gene is present in the host cell. Accordingly, in some preferred embodiments, the two-plasmid system of the invention comprises all the necessary genetic information for the production of rAAV. For example, the two-plasmid system of the invention may comprise at least one rep gene, at least one cap gene and at least one helper gene. In some preferred embodiments, the two-plasmid system of the invention comprises all the necessary genetic information required for the production of AAV vector particles suitable for use in gene therapy. For example, the two-plasmid system of the invention may comprise at least one rep gene, at least one cap gene, at least one helper gene and an expression cassette comprising a transgene operably linked to at least one regulatory control element. In some preferred embodiments, the Cap-Helper plasmid of the two-plasmid system comprises Cap and helper functions. For example, the Cap-Helper plasmid may comprise: - a Cap gene encoding at least one functional Cap protein - a promoter operably linked to the Cap gene - at least one helper virus gene - a promoter operably linked to the at least one helper virus gene. The REP-GOI plasmid may comprise: - a Rep gene encoding at least one functional Rep protein - a promoter operably linked to the Rep gene - an expression cassette flanked on at least one side by an ITR. Exemplary complete plasmid sequences are provided for Cap-Helper plasmids with AAV5 capsid proteins (SEQ ID NO:1) and AAV6 capsid protein (SEQ ID NO:2) and a REP-GOI plasmid with AAV2 Rep (SEQ ID NO:3). It will be understood that various components of these plasmids may be substituted with equivalent elements as described in more detail below. For example, the Cap 5 and 6 gene sequences may be substituted with Cap gene sequences from any suitable AAV serotype, e.g., AAV1, 2, 3, 4, 7, 8, 9, 10, 11, 12, and 13 Cap gene sequences and the AAV Rep2 gene sequence may be substituted with Rep gene sequences from any suitable AAV serotype, e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 Rep gene sequences. Exemplary sequences are provided are provided in Table 1 below. Table 1 Selected component sequences from Cap5-Helper and Cap6-Helper plasmids A ATT ATTATT A TA TTATTAATA TAAT AA

Catcaagatcaagcccgtggtcttgtgctgcctggt Tataactatctcggacccggaaacggtctcgatcga

Gtcaaccgcgccagtgtcagcgccttcgccacgacc Aataggatggagctcgagggcgcgagttaccaggtg

Taatgcctcaggaaattggcattgcgattccacat Ggctgggcgacagagtcatcaccaccagcacccga Acatgggccttgcccacctataacaaccacctcta

CCGTACCTTAATCAAACTCACAGAACCCTAGTATTC AACCTGCCACCTCCCTCCCAACACACAGAGTACACA GTCCTTTCTCCCCGGCTGGCCTTAAAAAGCATCATA

ACACTGATTATGACACGCATACTCGGAGCTATGCTA ACCAGCGTAGCCCCTATGTAAGCTTGTTGCATGGGC GGCGATATAAAATGCAAGGTGCTGCTCAAAAAATCA

CCTCCTCCTCGTCCAAAACCTCCTCTGCCTGACTGT CCCAGTATTCCTCCTCGTCCGTGGGTGGCGGCGGCG GCAGCTGCAGCTTCTTTTTGGGTGCCATCCTGGGAA

TCCCCCCGCCCGACTTGTTCCTCGTTTGCCTCTGCG TCGTCCTGGTCTTGCTTTTTATCCTCTGTTGGTACT GAGCGATCCTCGTCGTCTTCGCTTACAAAACCTGGG

AGTGACAAAAAGAAGTGGCGCTCCTAATCTGCGCAC TGTGGCTGCGGAAGTAGGGCGAGTGGCGCTCCAGGA AGCTGTAGAGCTGTTCCTGGTTGCGACGCAGGGTGG

Ccggtcggcaccagttgcgtgagcggaaagatggccg Cttcccggccctgctgcagggagctcaaaatggagga

TCCTCTTGCCGAACTTTTTCGTGGCCCATCCCAGAAAG ACGGAAGCCGCATATTGGGGATCGTACCCGTTTAGTTC

Additional AAV CAP and REP sequences suitable for use in the expression systems of the invention are available in public databases and may be obtained from the annotated complete genome sequences for

ccor ng y, n some pre erre em o ments, t e present nvent on prov es an engineered, non-naturally occurring system for producing AAV vector particles comprising a first nucleic acid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a second nucleic acid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV IRT sequence. In other preferred embodiments, the present invention provides An engineered, non-naturally occurring two-plasmid system for producing AAV vector particles comprising: a Cap-Helper plasmid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a Rep-GOI plasmid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV IRT sequence. In some preferred embodiments, the functional AAV Cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes. Exemplary Cap genes are provided in Table 1 above. In some preferred embodiments, the functional AAV Cap gene is at least 80%, 90%, 95%, 98% or 100% identical to one of SEQ ID NOs: 5 and 6. In preferred embodiments, the Cap gene encodes a functional Cap protein. The cap gene may encode a functional set of Cap proteins. A functional set of Cap proteins is one which allows for encapsidation of AAV. AAV generally comprise three Cap proteins, VP1, VP2 and VP3. These three proteins form a capsid into which the AAV genome is inserted, and allow the transfer of the AAV genome into a host cell. All of VP1, VP2 and VP3 are encoded in native AAV by a single gene, the cap gene. The amino acid sequence of VP1 comprises the sequence of VP2. The portion of VP1 which does not form part of VP2 is referred to as VPlunique or VPIU. The amino acid sequence of VP2 comprises the sequence of VP3. The portion of VP2 which does not form part of VP3 is referred to as VP2unique or VP2U. In preferred embodiments, the vector plasmid comprises a cap gene that encodes a VPl, a VP2 and a VP3 protein. Different serotypes of AAV have Cap proteins having different amino acid sequences. A cap gene encoding any (set of) Cap protein(s) is suitable for use in connection with the present invention. The Cap protein can be a native Cap protein expressed in AAV of a certain serotype. Alternatively, the Cap protein can be a non-natural, for example an engineered, Cap protein, which is designed to comprise a sequence different to that of a native AAV Cap protein. In some preferred embodiments, the functional AAV Cap gene encodes a Cap protein that is at least 80%, 90%, 95%, 98% or 100% identical to a Cap protein encoded by one of SEQ ID NOs: 5 and 6. The native Cap gene (i.e., the Cap gene of a wild type AAV) is operably linked to a p40 promoter, a p5 promoter and a pl9 promoter. In contrast, in the plasmid of the instant invention, the Cap gene is preferably operably linked to an exogenous promoter, and most preferably an exogenous strong promoter. Suitable strong promoters include, but are not limited, the hybrid promoter comprising a cytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter (SEQ ID NO:4: CAG promoter); CBA promoter (chimeric CMV chicken beta-actin promoter), the EF-1α promoter, SEQ ID NO:11, the SV-40 promoter and the CMV promoter, a synthetic or engineered strong promoter or an inducible promoter. In particularly preferred embodiments, the promoter is a CAG promoter. In some embodiments, the Cap-Helper plasmids of the invention comprise at least one helper virus gene. Preferably, the Cap-Helper plasmids of the invention comprise sufficient helper genes to allow for AAV replication and packaging. AAV is only able to propagate in the presence of a helper virus. Examples of helper viruses include adenoviruses and herpes viruses. In preferred embodiments, the at least one helper virus gene is an adenovirus gene. In some preferred embodiments, the at least one helper virus gene is an Adenovirus 5 gene or an Adenovirus 2 gene. The helper genes of adenoviruses encode the ElA, E1B, E4, and E2A proteins as well as VA RNA I and II. Some host cell lines express ElA and E1B constitutively, thus plasmid systems for use in those cell lines do require these genes. In some preferred embodiments, the one or more sequences encoding functional AAV helper genes are a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein. A functional VA RNA I and II, E2A protein or E4 protein is able to facilitate production of AAV vector particles. In some preferred embodiments, the VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein are at least 80%, 90%, 95%, 98% or 100% identical to portions of SEQ ID NOs: 7-8 encoding the E2A and E4 proteins, respectively. In preferred embodiments, expression of the E4 gene is driven by an E4 promoter, although exogenous promoters may also be utilized. In preferred embodiments, expression of the E2A gene is driven by the E2A promoter, although exogenous promoters may also be utilized. The sequences listed above include the E4 and E2A promoters. In some preferred embodiments, the Rep-GOI plasmid comprises at least one rep gene encoding at least one functional Rep protein. A functional Rep protein is one which allows for production of AAV vector particles. AAV comprises a rep gene region which encodes four Rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40). The gene region is under the control of the p5 and pl9 promoters. When the p5 promoter is used, a gene that encodes Rep 78 and Rep 68 is transcribed. Rep 78 and Rep 68 are two alternative splice variants (Rep 78 comprises an intron that is excised in Rep 68). Similarly, when the pl9 promoter is used, a gene that encodes Rep 52 and Rep 40 is transcribed. Rep 52 and Rep 40 are alternative splice variants (Rep 52 comprises an intron that is excised in Rep 40). The four Rep proteins are known to be involved in replication and packaging of the viral genome, and are, therefore, useful in AAV vector particle production. It is not necessary for all four Rep proteins to be present. Optionally, however, the at least one rep gene encodes a large Rep protein (Rep 78 or Rep 68) and a small Rep protein (Rep 52 or Rep 40). Rep 78 can be toxic to cells, and Rep 78 does not need to be present in order for AAV replication to take place. Accordingly, in some preferred embodiments, the helper plasmid may comprise at least one rep gene encoding: a functional Rep 52 protein; a functional Rep 40 protein; and/or a functional Rep 68 protein. In some preferred embodiments, the Rep gene comprises a gene encoding a functional Rep 52 protein, a gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein. In some preferred embodiments, the AAV Rep gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Rep genes. In some preferred embodiments, the Rep gene is an AAV2 Rep gene (i.e., Rep2). In some preferred embodiments, the functional AAV Rep gene is at least 80%, 90%, 95%, 98% or 100% identical to SEQ ID NO:12. In some preferred embodiments, the functional AAV Rep gene encodes a protein that is at least 80%, 90%, 95%, 98% or 100% identical to the protein encoded by SEQ ID NO:12. In some preferred embodiments, the expression of the one or more Rep genes is driven by native P5 and P19 promoters. In some preferred embodiments, the Rep-GOI plasmid further comprises an expression cassette that facilitates expression of a transgene or gene of interest (GOI) that may, for example, be useful for gene therapy. An expression cassette refers to a sequence of nucleic acids comprising a transgene or GOI and a promoter operably linked to the transgene. Preferred promoters include, but are not limited to, the EF1α promoter (e.g., SEQ ID NO: 11), CMV promoter, SV40 promoter and CAG promoter, a synthetic or engineered promoter, an inducible promoter, or a tissue-specific promoter. In some particularly preferred embodiments, an EF1α promoter is utilized. Optionally, the cassette further comprises additional transcription regulatory elements, such as enhancers, introns, untranslated regions, transcriptional terminators, etc. In some preferred embodiments, the expression cassette comprises at least one ITR. In some further preferred embodiments, the expression cassette comprises two ITRs (generally with one either end of the expression cassette, i.e. one at the 5’ end and one at the 3’ end). There may be intervening sequences between the expression cassette and one or more of the ITRs. The expression cassette may be incorporated into a viral particle located between two regular ITRs or located on either side of an ITR engineered with two D regions. In some embodiments, the vector plasmid comprises ITR sequences which are derived from AAV1, AAV2, AAV4 and/or AAV6. Preferably the ITR sequences are AAV2 ITR sequences. Exemplary ITR sequences include those that are at least 80%, 90%, 95%, 98%, 99% or 100% identical to one or both of SEQ ID NOs:9 and 10. The transgene may be any suitable gene. If the vector plasmid is for use in gene therapy, the transgene may be any gene that comprises or encodes a protein or nucleotide sequence that can be used to treat a disease. For example, the transgene may encode an enzyme, a metabolic protein, a signaling protein, an antibody, an antibody fragment, an antibody like protein, an antigen, or a non-translated RNA such as an miRNA, siRNA, snRNA, or antisense RNA. In some embodiments, the GOI of interest may encode all or a portion of a gene editing system, such as Cas9, an sgRNA and donor template DNA. In some preferred embodiments, a dual AAV system is utilized for CRISPR gene editing where CAS9 is included as the GOI in one Rep-GOI plasmid and sgRNA and donor template DNA as the GOI in a second Rep-GOI plasmid. In preparing the viral vector particles, any host cells for producing recombinant viral particles can be employed, including, for example, mammalian cells, insect cells, plant cells, microorganisms, and yeast. Exemplary packaging and producer cells include but are not limited to HeLa cell, COS cell, COS-1 cell, COS-7 cell, HEK293 cell, A549 cell, BHK cell, BSC-1 cell, BSC-40 cell, Vero cell, Sfc9 cell, Sf -21 cell, Tn-368 cell, BTI-Tn-5B1- 4 (High- Five) cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, WEHI cell, 3T3 cell, 10T1/2 cell, MDCK cell, BMT-10 cell, WI38 cell, or primary fibroblast, hepatocyte or myoblast cell derived from mammals. Also provided herein is a cell comprising the two-plasmid system described herein or a polynucleotide encoding the two plasmid-system described herein. The cell can be prokaryotic cell or eukaryotic cell. For example, the cell can be a mammalian cell, insect cell, plant cell, bacterial cell, or yeast cell. Some exemplary cells include, but are not limited to, alveolar cells, basophils, cardiac smooth muscle cells), cardiomyocyte, collecting duct intercalated cells, collecting duct principal cells, ectodermal cells, endocardial cells, endoderm cells, eosinophils, epithelial cells, hepatic stellate cells, interstitial kidney cells, intrahepatic lymphocytes, kidney distal tubule cells, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, lung epithelial cells, lung smooth muscle cells, lymphocytes, monocytes, muscle cells, neutrophils, non-parenchymal cells, parenchymal cells, phagocytic Kupffer cells, platelets, red blood cells, sinusoidal endothelial cells, splenic endothelial cells, splenic fibroblasts, splenocytes, and thick ascending limb cells. In some embodiments, the cell can be a cell used for producing a viral particle, e.g., a producer cell. In some embodiments, the cell can be a cell which has been transduced, infected, transfected or transformed with a viral vector described herein. Typically, a cell is referred to as “transduced”, “infected”; “transfected” or “transformed” dependent on the means used for administration, introduction, or insertion of heterologous DNA (i.e., the viral vector) into the cell. Also provided herein are pharmaceutical compositions comprising a two-plasmid system described herein or a polynucleotide encoding same and one or more pharmaceutically acceptable diluent, carrier, or excipient. For example, the composition can comprise the plasmids described herein. In some embodiments, the composition can comprise a cell, wherein the cell comprises the plasmids described herein. For example, the plasmids described herein (as is or as encompassed in a viral particle or cell) can be combined with pharmaceutically- acceptable carriers, diluents, and reagents useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for primate use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In particular embodiments, the pharmaceutical compositions are sterile. For instances in which ocular cells are to be contacted in vivo, the subject polynucleotide cassettes or gene delivery vectors comprising the subject polynucleotide cassette can be treated as appropriate for delivery to the eye. Pharmaceutical compositions can further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be fluid to the extent that easy syringability exists. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Provided herein is a method of delivering a payload to a cell comprising contacting a cell with an AAV virus or viral particle produced via the two-plasmid system described herein. In one embodiment, the contacting occurs in vitro. In one embodiment, the contacting occurs ex vivo. The term “contacting” or “contact” as used herein in connection with contacting a cell with an AAV virus or viral particle includes subjecting the cell to an appropriate culture medium which comprises the AAV virus or viral particle. Where the cell is in vivo, “contacting” or “contact” includes administering the AAV virus or viral particle in a pharmaceutical composition to a subject via an appropriate administration route such that the virus or viral particle contacts the cell in vivo. Further provided herein is a method of delivering a payload in vivo to a target cell, comprising administering an AAV virus or viral particle produced via the two-plasmid system described herein to a subject. In one embodiment, in vivo is systemic delivery. Exemplary target or host cells include blood cells (e.g., red blood cells, platelets, neutrophils, eosinophils, basophils, lymphocytes, or monocytes); heart cells (e.g., cardiomyocyte, endocardial cells, or cardiac smooth muscle cells); muscle cells; epithelial cells; endoderm cells; ectodermal cells; kidney cells (e.g., kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct intercalated cells, and interstitial kidney cells); liver cells (e.g., parenchymal cells, non-parenchymal cells, sinusoidal endothelial cells, phagocytic Kupffer cells, hepatic stellate cells, and intrahepatic lymphocytes); lung cells (e.g., lung epithelial cells, lung smooth muscle cells, and alveolar cells); and spleen cells (e.g., splenocytes, splenic endothelial cells and splenic fibroblasts). In vivo delivery of the AAV virus or viral particle can be, for example, by injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, intrasternal injection and infusion. The AAV virus or viral can be administered as a single bolus or multiple boluses, as a continuous infusion, or a combination thereof. In some embodiments, the AAV virus or viral particle can be administration into the blood stream of the subject. The dose of AAV virions or viral particles required to achieve a particular "therapeutic effect," e.g., the units of dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: the route of AAV virus or viral particle administration, the level of gene expression required to achieve a therapeutic effect, the specific disease or disorder being treated, a host immune response to the AAV virus or viral particle, a host immune response to the gene expression product, and the stability of the gene product. One of skill in the art can readily determine a AAV virus or viral particle dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. Generally speaking, by "therapeutic effect" is meant a level of expression of one or more transgenes in the AAV virus or viral particle sufficient to alter a component of a disease (or disorder) toward a desired outcome or clinical endpoint, such that a patient's disease or disorder shows clinical improvement, often reflected by the amelioration of a clinical sign or symptom relating to the disease or disorder. Exemplary doses for achieving therapeutic effects are AAV virus or viral particle titers of at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶ transducing units or more. For example, from about 10⁸to about 10¹³ transducing units. It is noted that more than one administration (e.g., two, three, four, or more administrations) can be employed to achieve desired (e.g. therapeutic) levels of gene expression. Examples Host cells were transfected with two-plasmid systems for production of AAV5 or AAV6. The Cap-helper plasmids for AAV5 and AAV6 are described in FIGs.1 and 2 (SEQ ID NOs:1 and 2) while the Rep-GOI plasmid used with the Cap-helper plasmids is described in FIG.3. On the day before transfection, 1.3E⁶ cells/ml clone of host cell line and host cell line 2 were seeded in a 200ml culture in a 1L flask and allowed to grow overnight. Cells were counted the next day. For both cell lines, the cell density at the time of transfection was 2.6 + 0.2 x 10⁶ vc/mL. The following parameters were used for transfection. Transfection Reagent : PEIpro, Complexation Buffer: Opti-PRO SFM. Complexation time: 20 + 2 min. Overall amount of DNA -1.0 mg/L*100 mL = 0.1 mg PEIpro to DNA ratio: 2:1. AAV particles were harvested on day 3 post transfection by treatment with 10% v/v lysis buffer in presence of 100 U/mL of Benzonase, followed by a 30-min treatment with 0.5M NaCl. The titer and percent full particles were determined by ddPCR and ELISA. The results are provided in Table 2 below. As can be seen, transfection of host cells with the 2-plasmid systems resulted in the production of AAV5 and AAV6 viral particles. Table 2 – Result Summary, ddPCR/ELISA Sample ddPCR ELISA ddPCR/ELISA (% full)

The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

Claims What is claimed is: 1. An engineered, non-naturally occurring system for producing AAV vector particles comprising: a first nucleic acid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a second nucleic acid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV ITR sequence.

2. The system of claim 1, wherein the functional AAV Cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes.

3. The system of any one of claims 1 or 2, wherein the one or more sequences encoding functional AAV helper genes are a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein.

4. The system of any one of claims 1 to 3, wherein the Rep gene comprises a gene encoding a functional Rep 52 protein, a gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein, and a gene encoding a functional Rep 78 protein.

5. The system of any one of claims 1 to 4, wherein the at least one Rep gene does not comprise a functional internal p40 promoter.

6. The system of any one of claims 1 to 5, wherein the AAV Rep gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Rep genes.

7. The system of claim 6, wherein the Rep gene is an AAV2 Rep gene.

8. The system of any one of claims 1 to 7, wherein the Rep gene and the gene of interest are expressed from different promoters.

9. The system of claim 8, wherein the gene of interest is operably linked to a promoter selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter.

10. The system of any one of claims 1 to 9, wherein the gene of interest encodes a therapeutic gene product.

11. The system of claim 10, wherein the therapeutic gene product is selected from the group consisting of a polypeptide, peptide, protein, short interfering RNA (siRNA), miRNA and small hairpin RNA (shRNA).

12. The system of any one of claims 1 to 11, wherein the first nucleic acid sequence is in a first plasmid and the second nucleic acid sequence is in a second plasmid.

13. A cell or population of cells comprising the system of any one of claims 1 to 12.

14. The cell or population of cells of claim 13, wherein the cell or population of cells are producer cells.

15. An engineered, non-naturally occurring two-plasmid system for producing AAV vector particles comprising: a first plasmid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes; and a second plasmid encoding an AAV Rep gene, and a gene of interest flanked on at one side by an AAV ITR sequence.

16. The system of claim 15, wherein the functional AAV Cap gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes.

17. The system of any one of claims 15 or 16, wherein the one or more sequences encoding functional AAV helper genes are a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein and an E4 gene encoding a functional E4 protein.

18. The system of any one of claims 15 to 17, wherein the Rep gene comprises a gene encoding a functional Rep 52 protein, a gene encoding a functional Rep 40 protein, and a gene encoding a functional Rep 68 protein, and a gene encoding a functional Rep 78 protein.

19. The system of any one of claims 15 to 18, wherein the at least one Rep gene does not comprise a functional internal p40 promoter.

20. The system of any one of claims 15 to 19, wherein the AAV Rep gene is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Rep genes.

21. The system of claim 20, wherein the Rep gene is an AAV2 Rep gene.

22. The system of any one of claims 15 to 21, wherein the Rep gene and the gene of interest are expressed from different promoters.

23. The system of claim 22, wherein the gene of interest is operably linked to a promoter selected from the group consisting of an EF1α promoter, a CMV promoter, chimeric CMV chicken ß–actin promoter (CBA), a P5 promoter, and a P19 promoter.

24. The system of any one of claims 15 to 23, wherein the gene of interest encodes a therapeutic gene product.

25. The system of claim 24, wherein the therapeutic gene product is selected from the group consisting of a polypeptide, peptide, protein, short interfering RNA (siRNA), miRNA and small hairpin RNA (shRNA).

26. A cell or population of cells comprising the system of any one of claims 15 to 25.

27. The cell or population of cells of claim 26, wherein the cell or population of cells are producer cells.

28. A method for producing recombinant AAV vector particles comprising: (a) transfecting a host cell with the system of any of claims 1 to 12 or 15 to 25; and (b ) culturing the host cell under conditions suitable for production of recombinant AAV vector particles.

29. The method of claim 20, further comprising a step of harvesting the recombinant AAV vector particles.

30. The method of any one of claims 28 to 29, wherein the host cells are transfected with 2 plasmids simultaneously or with first plasmid at least 12 hours before the second plasmid.

31. A plasmid system comprising: a set of first plasmid comprising a sequence encoding a functional AAV Cap gene and one or more sequences encoding functional AAV helper genes, wherein the set of first plasmids comprises plasmids comprising AAV Cap genes from at least two different AAV serotypes; and a second plasmid encoding an AAV Rep gene and a gene of interest flanked on at one side by an AAV ITR sequence.

32. The plasmid system of claim 31, wherein the set of first plasmids comprises plasmids comprising AAV Cap genes from at least five different AAV serotypes.

33. The plasmid system of any one of claims 31 to 32, wherein the set of first plasmids comprises plasmids comprising AAV Cap genes from at least ten different AAV serotypes.

34. The plasmid system of any one of claims 31 to 33, wherein the AAV Cap genes are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8 and AAVrh.10 Cap genes.