Engineered producer cell and methods of producing and using the same

Abstract

An engineered producer cell comprising an inactivating mutation in one or more endogenous REP binding sites is provided, as are methods for producing the engineered producer cell and using the engineered producer cell to produce a recombinant viral vector and reduce producer cell genomic DNA contamination of a recombinant adeno-associated virus vector preparation.

Description

ENGINEERED PRODUCER CELL AND METHODS OF PRODUCING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit from U . S . Patent Application Serial No . 63 / 678 , 152 , filed August 1 , 2024 , the content of which is incorporated herein by reference in its entirety .STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing in XML format, entitled 3 J0117WO_ST26 . xml , 23 , 583 bytes in size, generated on July 29 , 2025, is filed herewith . This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures .BACKGROUND

[0003] The relative ease and versatility of recombinant adeno- associated virus (rAAV) production, coupled with its robust transduction of non-dividing cells , has made AAV a popular gene delivery tool . Clinical successes in the treatment of hemophilia and other monogenic disorders have helped to increase interest in AAV-mediated gene transfer as a treatment strategy . Although rAAV therapies are providing significant clinical benefit , improving their safety remains a top priority .

[0004] The manufacture of viral vectors for human gene therapy and vaccination is well-documented . Methods of viral vector manufacture include the transfection of primary cells or mammalian / insect cell lines with vector DNA components, followed by a limited incubation period and then harvest of crude vector from culture media and / or cells . Despite having the best safety profile of any current clinical viral vector,it is known that AAV preparations contain contaminating sequences that are packaged alongside the expression cassette at a low rate . These sequences can originate from production plasmid DNA or chromosomal DNA from producer cell lines . It is therefore desirable to minimize DNA contamination to preclude the potential for unnecessary and potentially harmful functional gene sequences from being integrated into a patient' s cell during vector delivery .

[0005] Therefore, there is a need for methods for recombinantly producing AAV with reduced host genomic DNA contaminants , which are suitable for use as a medicament or for gene therapy .SUMMARY OF THE INVENTION

[0006] In one aspect , an engineered producer cell is provided, which comprises an inactivating mutation in one or more endogenous AAV REP binding sites .

[0007] Another aspect provides a method of producing an engineered vector producer cell by introducing into a producer cell an inactivating mutation in one or more endogenous AAV REP binding sites .

[0008] A further aspect relates to a method of producing a recombinant viral vector by (a) providing to the engineered producer cell described herein a viral expression system comprising an AAV REP protein; (b) culturing the engineered producer cell under conditions in which viral vector is produced; and ( c) optionally isolating the viral vector .

[0009] In another aspect is provided an improved method of producing a viral vector by introducing an inactivating mutation into one or more endogenous AAV REP binding sites of the genome of a producer cell thereby producing an engineered producer cell and preparing a rAAV vector with the engineered producer cell .

[0010] In a still further aspect is provided a method for determining the purity of a rAAV vector preparation prepared via a producer cell by detecting in the rAAV vector preparation the presence of producer cell genomic DNA contamination derived from a sequence proximal to one or more endogenous AAV REP binding sites of the genome of the producer cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGS. 1A-1D show scatter plot results from deep sequencing of AAV preparations of human genomic loci. Peaks indicate signal above background. Those coordinates are more likely to be packaged.

[0012] FIGS. 2A-2B show the strategy for elective deletion of the AAVS1 REP Binding Site (RBS) / Terminal Resolution Sequence (TRS) .

[0013] FIG. 3 shows gel electrophoresis analysis of amplicons of AAVS1 loci, including AAVS1 deletion clones 4A4 and 6G12 of HEK293T cells.

[0014] FIG. 4 shows the location and sequence of the 155 bp AAVS1 deletion in 4A4 and 6G12 clones of HEK293T cells. The two gRNA targets are bridged together with an ssODN indicated in the figure. The ssODN is a synthetic single stranded oligonucleotide donor.

[0015] FIG. 5 shows viral titer of AAV8 packaged by wild-type (WT) HEK293T cells or AAVS1 deletion mutants (HEK 4A4 and HEK 6G12) of HEK293T.

[0016] FIGS. 6A-6B show that deletion of AAVS1 in HEK293T cells reduces packaging of nearby DNA as determined by qPCR using primers designed to genomic sequences located on either side of the deletion locus (P1358-60, FIG. 6A and P1361-3, FIG. 6B) . lelOvg diluted to 100 pL was used for testing of genomic coordinates with 5e8vg loaded per reaction.

[0017] FIGS . 7A-7C show that deletion of AAVS1 in HEK293T cells did not impact packaging of contaminant DNA from a region on chromosome 1 ( FIG . 7A) or two different regions of chromosome 21 ( FIG . 7B-7C) , as determined by qPCR.

[0018] FIG . 8 . Location of REP binding sites relative to contaminant incorporation for example sites - unidirectional where RBS found on positive strand .

[0019] FIG . 9. Location of REP binding sites relative to contaminant incorporation for example sites - unidirectional where RBS found on negative strand .DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention described herein is based, in part , on the identification of hotspots within the human genome that are preferentially incorporated from the production cell line into viral particles as unwanted / contaminating, i . e . , residual , nucleic acids during viral vector production . These host DNA sequences have nearby REP binding sites and nicking sites , and packaging of host DNA occurs in a directional manner similar to what has been found with plasmid contamination ( Brimble et al . (2022 ) Mol . Ther. Methods Clin . Dev. 24 : 280-291 ) . A strategy was devised using CRISPR to delete sequences in the host cell genome implicated in the packaging of genomic DNA. Recombinant AAV vector was produced with the engineered cell lines and a significant reduction in signal of nearby loci per vector genome produced was observed . This reduction was specific to the REP binding site deletion and did not affect contamination by DNA at other hotspots . Accordingly, provided herein is an improved producer cell , the genome of which has been mutated to inactivate one or more endogenous AAV REP binding sites therein . As a result , unwanted ( contaminating) , residual nucleic acid from the production cell genome otherwise associated with viral vector production is decreased therebyreducing burdensome upstream and / or downstream commercial nuclease enzymatic treatment steps, which can sometimes impair to some extent viral vector quality and quantity. See, e.g. , well-known upstream and downstream processing steps in Merten et al. (2014) Pharma ceu t . Bioprocess. 2:183-203; Merten et al. (2014) Pharmaceut. Bioprocess. 2:237-251; Gousseinov et al. (2014) BioProcess. International 12:59-68. Advantageously, removal of the AAV REP binding sites from the genome of the production cell reduces levels of residual nucleic acids and improves the safety of the viral vector for gene therapy applications .

[0021] As used herein, the term "producer / production cell" or "vector producing / production cell" refers to a cell which contains all the elements necessary for production of vector particles. The producer cell may be either a stable producer cell line or derived transiently or may be a stable packaging cell wherein the genome is transiently expressed. The producer cell may be a primary cell or a cell cultured in vitro such as a tissue culture cell line. In some embodiments, the producer cell is derived from a mammalian cell or mammalian cell line. In some embodiments, the producer cell is derived from a human cell or human cell line. In some embodiments, the producer cell is a human embryonic kidney (HEK) cell or cell line. In some embodiments, the producer cell is a HEK293 cell, or a derivative thereof. Examples of HEK293 derivatives include HEK293S, HEK293SG, HEK293SGGD, HEK293FTM and HEK293T. These derivatives may also be referred to as variants of HEK293. In some embodiments, the producer cell is a HeLa cell (a human cell line derived from cervical cancer cells) or A549 cell (a human cell line derived from lung carcinoma epithelial cells) . In some embodiments, the producer cell is derived from a non-human primate cell or non-human primate cell line. In some embodiments, the producer cell is derived from a rodent cellor rodent cell line ( e . g. , mouse , rat or hamster such as a Chinese hamster ) . In some embodiments , the producer cell is a Chinese hamster ovary (CHO) cell, or a derivative thereof . In some embodiments, the producer cell is an insect cell . In some embodiments , the producer cell is an Sf 9 insect cel l ( a clonal isolate derived from the parental Spodoptera frugiperda cell line IPLB-Sf -21-AE) .

[0022] In accordance with embodiments herein, the producer cell is engineered, i . e .zthe producer cell . includes one or more non-naturally occurring mutations in its genome that distinguish the producer cell from a parental or wild-type producer cell . A "non-naturally occurring" mutation is a mutation that has been synthesized, engineered, or edited to produce a sequence that is different from a known natural sequence . "Wild type, " "WT" or "wild-type , " when in reference to a sequence, cell , and / or virus , refers to the sequence , cell , and / or virus as it occurs in nature . Given that most or all genetic loci, cells or viruses exist in a variety of forms , which vary in frequency throughout the geographic range of a species , a "wild type" sequence, cell , and / or virus may refer to the sequence, cell , and / or virus that occurs at the highest frequency in nature .

[0023] In some embodiments , an engineered producer cell as described herein has an inactivating mutation in one or more endogenous AAV REP binding sites . The term "mutation" refers to an insertion, deletion, truncation and / or missense mutation . As used herein, the term "inactivating mutation" in one or more endogenous AAV REP binding sites refers to a mutation that results in the reduction or complete loss of the ability of the one or more endogenous AAV REP binding sites to bind AAV REP . In some embodiments, an endogenous AAV REP binding is inactivated without altering ( increasing or decreasing) the expression of a coding sequence that may overlap or be in closeproximity (e.g., within about 1 to about 200 bp, or any range or value therebetween) to the endogenous AAV REP binding site.

[0024] In some embodiments, an inactivating mutation of an endogenous AAV REP binding site may be a deletion mutant such that binding of AAV REP to the endogenous AAV REP binding site is reduced or abolished. In some embodiments, deletion of an endogenous AAV REP binding site may comprise, consist essentially of, or consist of a deletion of about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, 500, 550, 600, or 650 or more consecutive nucleotides, or any range or value therein. In some embodiments, deletion of an endogenous AAV REP binding site may comprise, consist essentially of, or consist of a deletion of about 5 to about 200 consecutive nucleotides, e.g. , about 5,10, 15, 20, 30, 40, 50 to about 100, 120, 140, 160, 180, 200 consecutive nucleotides, or any range or value therebetween.

[0025] In some embodiments, the inactivating mutation of an endogenous AAV REP binding site may be an insertion or substitution such that binding of AAV REP to the endogenous AAV REP binding site is reduced or abolished. As used herein, the terms "reduce," "reduced," "reducing," "reduction," "diminish," and "decrease" (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% as compared to a control. In some embodiments, binding of AAV REP to the endogenous AAV REP binding site is abolished (i.e. , 100% reduction) . A reduction or loss in binding of the MW REP to endogenous AAV REP binding site may be assessed by conventional protein-DNA binding assays (e.g.zunder stringent hybridization conditions) , ChlP-Seq, qPCR, or assays described herein, which measure genomic DMA contamination of a rAAV vector preparation.-1-

[0026] AAV REP proteins are non-structural proteins involved in AAV genome replication and / or 7XAV genome packaging. Given that the wild-type rep gene includes multiple promoters and an intron that can be alternatively spliced, multiple transcripts and REP proteins can be formed, including REP78, REP52, REP68 and REP40, which have overlapping sequences. The REP proteins of AAV, REP68 and REP78 in particular, are multifunctional proteins. Their activities include DNA binding, site- and strand-specific endonuclease, DNA-DNA helicase, RNA-DNA helicase, and ATPase activities. These proteins bind to the REP binding sites (RBS) , also known as REP Recognition Sequences (RRS) or REP Binding Elements (RBE) , in the AAV inverted terminal repeats (ITRs) , the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. The 16-mer core sequence of the RBS in the AAV ITR has the sequence: 5 ' -GAGCGAGCGAGCGCGC-31(SEQ ID NO:1) . The 16-mer core sequence of the RBS in AAV p5 promoter has the following sequence: 5'- GCCCGAGTGAGCACGC-3 ' (SEQ ID NO:2) . The 16-mer core sequence of the RBS in AAVS1 has the sequence: 5 ' -CAGCGAGCGAGCGAGC-3 ' (SEQ ID NO: 3) . In some aspects, the endogenous AAV REP binding sites inactivated in the producer cell comprise one or more a nucleotide sequences selected from the group of 5'- GAGCGAGCGAGCGCGC-3 ' (SEQ ID NO:!) , 5 ' -GCCCGAGTGAGCACGC-31(SEQ ID NO:2), 5' -CAGCGAGCGAGCGAGC-3' (SEQ ID NO:3) , CGAGCGAGC, and TGAGCGAGC .

[0027] In some embodiments, the producer cell has an inactivating mutation (e.g., substitution, insertion or deletion) in one or more endogenous AAV REP binding sites. As used herein, the term "endogenous" refers to a naturally occurring sequence (e.g. , AAV REP binding site), in the location in which it is naturally found, (e.g. , within the in the genome of a producer cell) .

[0028] Deep sequencing of viral vectors indicated that a number of REP binding sites may be present in whole human genome. In particular, 358 were found to be associated with preferential contamination in at least one rAAV preparation analyzed (Table 1), and 29 were observed in multiple preparations (Table 3 and Table 4) . While not wishing to be bound by theory, it is believed that this relates to whether the chromatin is in an open or closed confirmation combined with the availability of a nicking site proximal to the genomic REP binding site. Accordingly, in some embodiments, 358 or more AAV REP binding sites endogenous to the human genome may be inactivated in the producer cell described herein. In some embodiments, the producer cell has from 1 to 358 of the endogenous AAV REP binding sites inactivated (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350, 351, 352, 353, 354, 355, 356, 357, 358, or any range or value therein) . In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or all 29 of the REP binding sites found to be present in multiple (i.e., 2 or more, e.g., 2, 3, 4, 5, 6, or 7) recombinant viral preparations are inactivated .

[0029] In some embodiments, an endogenous 7XAV REP binding site comprises, consists essentially of, and / or consists of a nucleotide sequence of contiguous nucleotides that are identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the AAV REP binding site of an AAV ITR, AAV REP binding site of an AAV p5 promoter, or the AAV REP binding site of AAVS1. In some embodiments, the one or more endogenous AAV REP binding sites of the producer cell (parental or wild-type production cell) have a nucleotide sequence that shares at least 80% sequence identity (e.g. , 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity) to any one of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, CGAGCGAGC, or TGAGCGAGC. In some embodiments, the endogenous AAV REP binding sites inactivated in the producer cell have the core sequence 5 ' -SMSCGAGYGAGCRMGC-3 ' (SEQ ID NO: 4) or a 5'- GAGY-3' repeat sequence, e.g. , repeat having the sequence (GAGY)n, wherein n is at least 2 (e.g., 2, 3, 4, 5, 6 or more) .

[0030] In some embodiments, the one or more endogenous AAV REP binding sites is located in a nucleotide sequence of chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome 15, chromosome 16, chromosome 17, chromosome 18, chromosome 19, chromosome 20, chromosome 21, chromosome 22, chromosome 23, or X or Y chromosome. In some embodiments, the one or more endogenous AAV REP binding sites is located in a nucleotide sequence of chromosome 1, chromosome 3, chromosome 4, chromosome 5, chromosome 8, chromosome 9, chromosome 10, chromosome 11, chromosome 14, chromosome 16, chromosome 18, chromosome 19, chromosome 21, chromosome 22, or chromosome 23. Similarly, in embodiments where a non-human cell is used (e.g., SF9 lepodoptera-derived cells) , the one or more endogenous AAV REP binding sites is located in a nucleotide sequence of one or more the chromosomes of such cells.

[0031] In some embodiments, the one or more endogenous AAV REP binding sites is located between, or located within 1000 nucleotides upstream (5' ) or downstream (3' ) of, nucleotides: 6003380 and 6005107 of chromosome 1 (as set forth in GENBANK Accession No. NC_000001 .11 ) , 10796284 and 10797669 of chromosome 1 (as set forth in GENBANK Accession No. NC_000001 .11) , 44985851 and 44988210 of chromosome 1 (as set forth in GENBANK Accession No. NC_000001.11) , 61075662 and 61077300 of chromosome 1 (as set forth in GENBANK Accession No. NC 000001.11) , 151459046 and 151460524 of chromosome 1 (as set forth in GENBANK Accession No. NC 000001.11 ) , 183804446 and 183806827 of chromosome 1 (as set forth in GENBANK Accession No. NC_000001.11) , 10850196 and 10851915 of chromosome 3 (as set forth in GENBANK Accession No. NC 000003.12 ) , 46883151 and 46885332 of chromosome 3 (as set forth in GENBANK Accession No. NC_000003.12) , 30718138 and 30720073 of chromosome 4 (as set forth in GENBANK Accession No. NC_000004.12 ) , 30042612 and 30044361 of chromosome 5 (as set forth in GENBANK Accession No. NC_000005 .10 ) , 123512313 and 123513360 of chromosome 5 (as set forth in GENBANK Accession No. NC_000005.10 ) , 137498974 and 137500979 of chromosome 5 (as set forth in GENBANK Accession No. NC_000005.10 ) , 26207238 and 26208703 of chromosome 8 (as set forth in GENBANK Accession No. NC_000008.11 ) , 6207086 and 6209319 of chromosome 10 (as set forth in GENBANK Accession No. NC_000010 .11 ) , 5224551 and 5226324 of chromosome 11 (as set forth in GENBANK Accession No. NC_000011.10 ) , 61909029 and 61909029 of chromosome 11 (as set forth in GENBANK Accession No. NC_000011.10) , 2740788 and 27409167 of chromosome 14 (as set forth in GENBANK Accession No. NC_000014.9) , 95774695 and 95775629 of chromosome 14 (as set forth in GENBANK Accession No. NC_000014.9) , 30697077 and 30699022 of chromosome 16 (as set forth in GENBANK Accession No, NC_000016.10) , 89342145 and 89343777 of chromosome 16 (as set forth in GENBANK AccessionNo . NC_000016. 10 ) , 70629 and 71560 of chromosome 17 ( as set forth in GENBANK Accession No . NT 187615 . 1 ) , 107961 and 110340 of chromosome 18 (as set forth in GENBANK Accession No . NC_000018 . 10) , 55115533 and 55119207 of chromosome 19 (as set forth in GENBANK Accession No . NC_000019. 10 ) , 6557123 and 6559560 of chromosome 21 ( as set forth in GENBANK Accession No . NC_000021 . 9) , 43165383 and 43167617 of chromosome 21 (as set forth in GENBANK Accession No . NC_000021 . 9) , 18598982 and 18600792 of chromosome 22 (as set forth in GENBANK Accession No . NC__000022 . 11 ) , 21394536 and 21396314 of chromosome 22 (as set forth in GENBANK Accession No . NC_000022 . 11) , 21538348 and 21540375 of chromosome 22 (as set forth in GENBANK Accession No . NC_000022 . 11 ) , and / or 132949595 and 132951989 of chromosome 23 (as set forth in GENBANK Accession No . NC_000023 . 11 ) . In some embodiments , the one or more endogenous AAV REP binding sites is located within a nucleotide sequence of any one of SEQ ID NOs : 5-9 .

[0032] In some embodiments , a method of producing an engineered producer cell is provided that includes the step of introducing into a producer cell an inactivating mutation in one or more endogenous AAV REP binding sites . In another embodiment , a method of reducing producer cell genomic DNA contamination of a rAAV vector preparation is provided, wherein the method includes the steps of introducing an inactivating mutation into one or more endogenous AAV REP binding sites of the genome of a producer cell thereby producing an engineered producer cell , and producing a rAAV vector with the engineered producer cell .

[0033] Inactivating mutations ( e . g. , deletions , substitutions , or insertions) in the one or more endogenous AAV REP binding sites may be achieved using any established genome editing method in the art . "Genome editing" refers to the process of modifying the nucleotide sequence of a genome , e . g. , in a precise or pre-determined manner . Genome editing may includethe use of site-directed nucleases to cut DNA at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology directed repair (HDR) and non-homologous end joining (NHEJ) . NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in the target nucleic acid at the site of cleavage and can lead to disruption or alteration of a nucleic acid sequence. A third repair mechanism can be microhomology-mediated end joining (MMEJ) , also referred to as "Alternative NHEJ (ANHEJ)", in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. Each of these genome editing mechanisms can be used to create desired genomic alterations. Tools of use in genome editing include, but are not limited to, nucleases, e.g., CRISPR Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g. , Cas9 or Casl2a (Cpfl) ) and variants thereof, TALENs, ZFNs, meganucleases or recombinases, DNA-modifying enzymes, including base modifying enzymes such as cytidine deaminase enzymes, DNA-binding proteins, cr / tracr RNAs, guide RNAs, and the like, any and all of which may be used in the methods herein.

[0034] In some embodiments, an inactivating mutation in one or more endogenous AAV REP binding sites is introduced into a producer cell using a CRISPR nuclease and a genome-targeting nucleic acid. Five types of CRISPR systems (e.g. , Type I, Type II, Type III, Type U, and Type V) have been identified. In these systems, a genome-targeting nucleic acid directs the activities of the associated CRISPR nuclease to a specific target sequence. In some embodiments, the genome-targeting nucleic acid is an RNA. A genome-targeting RNA may be referredto as a "guide RNA" or "gRNA." A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest {e.g., a sequence at or proximal to an endogenous AAV REP binding site) , and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In some embodiments, a method of producing an engineered producer cell is provided that includes the step of (a) introducing into a producer cell a CRISPR-Cas system (e.g. , a CRISPR nuclease and a gRNA) that specifically introduces an inactivating mutation (e.g., a deletion) in an endogenous AAV REP binding site at a first site in the genome of the producer cell, and (b) optionally repeating step (a) between 1 and about 300 times to introduce an inactivating mutation into a second, third, fourth, fifth, sixth, etc. endogenous AAV REP binding site in the genome of the producer cell.

[0035] In some embodiments, a method of producing a recombinant viral vector is provided that includes the steps of (a) providing to an engineered producer cell described herein a viral expression system comprising an AAV REP protein; (b) culturing the cell under conditions in which viral vector is produced; and (c) optionally isolating the viral vector. As used herein, the term "viral vector, " "vector" or "gene delivery vector" refers to a virus (e.g, , AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA [vDNA] ) packaged within a virion. Alternatively, in some contexts, the term "vector" may be used to refer to the vector genome / vDNA alone.

[0036] A "viral expression system" is a system of one or more polynucleotides that are sufficient, when introduced into a suitable producer cell, to support production of a recombinant viral vector. In some embodiments, the viral expression system is for production of an AAV vector or bocavirus vector, e.g. ,where AAV Rep proteins are used in the production methods . In embodiments pertaining to an AAV expression system, such a system will typically include polynucleotides encoding AAV rep and cap, helper genes , and a rAAV genome . In some embodiments , the AAV expression system is a triple transfection system, which includes a recombinant AAV plasmid comprising a transgene , a packaging rep-cap-containing plasmid, and an adenovirus helper plasmid . In some instances , some of the necessary components for AAV production are stably expressed by the host cell . For example, a plasmid (or multiple plasmids ) including the P5 promoter, AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, may be integrated into the genome of the producer cell . The producer cell may then be co-infected with a helper virus ( e . g. , adenovirus providing the helper functions) and an AAV vector for expressing a heterologous gene of interest ( e . g. , transgene) . The advantages of this latter method are that the cells are selectable and are suitable for large-scale production of the rAAV . As another non-limiting example , adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into producer cells . As yet another non-limiting example, both the AAV vector for expressing a transgene and the rep-cap genes may be stably integrated into the DNA of producer cells , and the helper functions may be provided by a wild-type adenovirus to produce the rAAV .

[0037] The helper functions may be provided by one or more helper plasmids or helper viruses harboring adenoviral helper genes . Non-limiting examples of the adenoviral helper genes include E1A, E1B , E2A, E4 and VA, which can provide helper functions to AAV packaging . Helper viruses of AAV are known in the art and include , for example , viruses from the family Adenoviridae and the family Herpesviridae . Examples of helper viruses of AAV include , but are not limited to, SAdV-13 helpervirus and SAdV-13-like helper virus described in US 2011 / 0201088, and helper vectors pHELP (Applied Viromics) . A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[0038] In some embodiments, the producer cell is cultured under conditions in which viral vector is produced. In some embodiments, the producer cell is cultured in suspension. In some embodiments, the producer cell is cultured in animal component-free conditions. The animal component-free medium may be any animal component-free medium (e.g., serum-free medium) compatible with HEK293 cells. Examples include, without limitation, SFM4Transfx-293 (Hyclone) , Ex-Cell 293 (JRH Biosciences), LC-SFM (Invitrogen) , and Pro293-S (Lonza) .

[0039] A method of producing a recombinant viral vector may further include the step of collecting the viral vector from the culture. In one embodiment, the viral vector may be collected by lysing the cells, e.g., after removing the cells from the culture medium, e.g., by pelleting the cells. In another embodiment, the viral vector may be collected from the medium in which the cells are cultured, e.g., to isolate vector that is secreted from the cells. Some or all of the medium may be removed from the culture one time or more than one time, e.g., at regular intervals during the culturing step for collection of recombinant viral vector, e.g. , rAAV (such as every 12, 18, 24, or 36 hours, or longer extended time that is compatible with cell viability and vector production) , e.g., beginning about 48 hours post-transfection. After removal of the medium, fresh medium, with or without additional nutrient supplements, may be added to the culture. In one embodiment, the engineered producer cells may be cultured in a perfusion system such that medium constantly flows over the cells and is collected for isolation of secreted recombinant viral vector,e.g., rAAV. Collection of viral vector from the medium may continue for as long as the transfected cells remain viable, e.g. , 48, 72, 96, or 120 hours or longer post-transfection. In some embodiments, the collection of secreted viral vector is carried out with serotypes of AAV (such as AAV8 and AAV9) , which do not bind or only loosely bind to the producer cells. In other embodiments, the collection of secreted viral vector is carried out with heparin binding serotypes of AAV (e.g., AAV2) that have been modified so as to not bind to the cells in which they are produced. Examples of suitable modifications, as well as rAAV collection techniques, are disclosed in US 2009 / 0275107 .

[0040] In some embodiments, the engineered producer cell and methods described herein are of use in the production of all serotypes, chimeras, and hybrids of AAV , e.g. , AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7 , AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or synthetic variant, a chimeric or hybrid AAV thereof. In some aspects, the rAAV includes a transgene, e.g. , a reporter, therapeutic, immunogenic, or diagnostic gene.

[0041] An advantage of the engineered producer cell described herein is that rAAV may be produced with reduced contaminant DNA. Levels of contaminant DNA are a release criterion for AAV gene therapy trials, and reduction of contaminant DNA levels, would improve both the purity of the vector, and the likelihood of a vector being cleared for trial. Accordingly, in some embodiments, a recombinant viral vector composition produced by a method described here is provided, wherein said recombinant viral vector composition comprises a reduction in residual DNA or production cell DNA (gDNA) contaminants as compared to a recombinant viral vector composition produced under the same conditions by a parental or wild-type producer cell, e.g. , a production cell that does not include aninactivating mutation in one or more endogenous AAV REP binding sites .

[0042] A rAAV vector produced in accordance with the methods described herein finds particular use in gene therapy and / or for the preparation of a medicament suitable for gene therapy . By "gene therapy" is meant the administration of a nucleic acid to an individual, for treating and / or preventing and / or reducing the li kelihood of the occurrence of a disease . Several approaches have been proposed in the art . In view of the above, rAAV particles are used as vehicles for delivering said nucleic acid to the individual to be treated . One may replace a mutated gene that causes disease with a healthy copy of the gene . One may inactivate (" knocking-out" ) a mutated gene that is functioning improperly . One may introduce a new gene into the body for treating and / or preventing and / or reducing the li kelihood of the occurrence of a disease . The individual to be treated includes humans and non-human mammals .

[0043] In some embodiments , a rAAV vector being used in a gene therapy may be further isolated and / or purified . Methods to purify recombinant viral vectors are well-known in the art . For example, the recombinant viral vector may be isolated and / or purified from production cells and / or the supernatant of the production cells . In some embodiments, the recombinant viral vector is purified by a separation method such as differential centrifugation, e . g. , density gradients such as cesium chloride (CsCl ) gradient centrifugation; iodixanol gradient centrifugation; sucrose gradient centrifugation . In other embodiments , the recombinant viral vector is purified as described in US 2002 / 0136710 using a solid support that includes a matrix to which an artificial receptor or receptorlike molecule that mediates AAV attachment is immobilized. In some embodiments , the method may further include an additional step of tangential flow filtration .

[0044] The purity of a recombinant viral vector preparation may be assessed by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis (SDS-PAGE) . Bands corresponding to the viral structural (capsid) proteins may be visualized after staining and their size and relative intensity assessed with respect to contaminating proteins. In some embodiments, production cell genomic DNA contaminants or residual DNA testing may be assessed by quantitative PCR, e.g. , as described herein. Residual DNA may be expressed in ng per mL, or alternatively in ng per dose. In some embodiments, the rAAV has a purity equal to or superior to 90%, and preferably superior to 99%; or even 100%. In some embodiments, the rAAV has an amount of residual cellular DNA equal to or less than 50 ng per dose, e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 ng per dose.

[0045] Compositions comprising a rAAV particle described herein may be provided in the form of a pharmaceutical composition wherein the rAAV particle is in admixture with a pharmaceutically acceptable excipient, diluent or carrier suitable for administration to a subject in need of treatment. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buf fered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions and the like. Such carriers can be formulated by conventional methods and are administered to the subject at a therapeutically effective amount.

[0046] Screening assays are regularly employed for analyzing the purity of clinical grade AAV for known AAV DNA contaminants such as Ela, T-antigen, REP, CAP and the like. Accordingly, in some embodiments a method for determining the purity of a rAAV vector preparation prepared via a producer cell is alsoprovided herein, which includes the step of detecting in the rAAV vector preparation the presence of producer cell genomic DNA contamination derived from a sequence proximal to one or more endogenous AAV REP binding sites of the genome of the producer cell. As used herein, a sequence that is "proximal" to one or more endogenous AAV REP binding sites refers to a sequence that is within about 2000 base pairs of an endogenous AAV REP binding site, e.g.fabout 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 10 base pairs upstream (5' ) or downstream (3') of at least one endogenous AAV REP binding site. In some embodiments, the producer cell genomic DNA contamination is proximal to an AAV REP binding site having a nucleotide sequence of SEQ ID NO: 4 or (GAGY)n, wherein n is at least 2, and / or a nucleotide sequence sharing at least 80% sequence identity to any one of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, CGAGCGAGC, or TGAGCGAGC.

[0047] As used herein, "producer cell genomic DNA contamination derived from a sequence proximal to one or more endogenous AAV REP binding sites" means that the contaminant DNA is not endogenous to the rAAV vector and is incorporated into the rAAV vector by AAV REP protein-mediated binding and incorporation of the producer cell genomic DNA into the rAAV vector. In some embodiments, the producer cell is a human cell or cell line, e.g. , a HEK cell, HeLa cell, or A549 cell or variants thereof. In some embodiments, the genomic DNA contaminant is from a human cell, wherein said genomic DNA contaminant is a sequence located within any one of the 358 chromosomal regions identified in Table 1. In some embodiments, the genomic DNA contaminant is from a human cell, said genomic DNA contaminant being a sequence located within any one of the 29 chromosomal regions identified in Table 3 or Table 4.

[0048] Producer cell genomic DNA contamination may be assessed by any conventional technique including, but not limited to, NGS, PCR ( e. g. , qPCR or digital droplet PCR (ddPCR) ) , microarray analysis . In some embodiments , detection may include the use of primers that amplify all or a portion of a chromosomal region identified in Table 1 , Table 3 or Table 4 , wherein the presence of an amplicon indicates that the chromosomal region is present in the rAAV vector , i . e . , the rAAV vector is contaminated with human genomic DNA . In some embodiments , detection may include the use of a probe that hybridi zes to all or a portion of a chromosomal region identified in Table 1 , Table 3 or Table 4 , wherein hybridi zation of the probe to the rAAV vector indicates that the chromosomal region is present in the rAAV vector, i . e . , the rAAV vector is contaminated with human genomic DNA. In some embodiments , multiplex detection of producer cell genomic DNA contaminants is carried out to detect the presence of between 2 and 358 of the chromosomal regions identified in Table 1 , e . g. , between 2 and 300, 10 and 200 , 50 and 100, 30 and 50 , or any range of value therebetween .

[0049] In addition to AAV RBS sequences in host cells , the methods described herein may be used to modify cells and detect genomic DNA contamination from cells used in the production of lentivirus, adenovirus and other recombinant vectors with well- established packaging signals .

[0050] The following non-limiting examples are provided to further illustrate the present invention .EXAMPLESExample 1 : Identification of Preferentially Packaged Host Genomic Sequences

[0051] Deep sequencing of AAV preparations (n = 7 ) identified human genomic loci packaged above background frequency ( FIGS .1A-1D) . Peaks across the genome above background indicated that the genomic loci were preferentially packaged, indicative of sequence specificity . Notably, patterns of encapsidation were similar to what has been observed with plasmid contaminants ( Brimble et al . ( 2022 ) Mol . Ther . Methods Clin . Dev. 24 : 280- 291 ) , wherein proximity of a DNA sequence to a REP binding site correlated with the likelihood of the DNA sequence being packaged into rAAV.

[0052] Based upon sequence analysis, it was determined that the human genome has 37046 REP binding site regions , 358 of which were detected proximal to the human DNA contamination peak regions (Table 1) , i . e . , 358 regions were preferentially packaged . Notably, the REP binding sites were not necessarily contained within the sequences of Table 1 ; the parameters used included the identification of sequences within a radius of 1 kb from the incorporation peak locus . Because the REP binding site is the signal to package, the sequence at the terminus of the DNA molecule may not always be recoverable in the sequencing .TABLE 1

[0053] However , the detection of a REP binding site did not mean that the genomic sequence was necessarily packaged as not all sequences near an RBS were packaged . From the analysis performed, packaging at 358 of the 37 , 046 detected sites in the human genome was detected, or approximately 1% of the REP binding sites . The REP binding sites within the human genome are distributed across all chromosomes (Table 2 ) .TABLE 2

[0054] Examples of peak regions of packaged genomic DNA had the following sequences .NC_000001 . 11 [ 183804795 . . 183806953 ] :AAAATAGATAATAGGCAGATAATGGAAGTTTTTTGGACTTTCTCCTTCCATTGTTTTAGCG AAGGAAGAAGACACACACAAATCCTCCATCCCACACTCAGTTCTATTTTGGCACCTGGTGACCGTCAGGAAAACCTGATAAACTTGAAAGTGTTGTTTCCCAATCACCTTCAGAACTGGAGA AACGCTTTCATGCTGACTTTATCCATGGGACTGAAGAAAGGGAGAGTGTGCTCACAAGCACAAACAGGAAAGCCTTACATCACGGAGTGGCGTGGGGAGGAGTCTGCTCCTCGCTTGCTCGC TCGCTCGCCGCGCTCCCTTTGTGGCCCGAGTCGCGCGCACCGGCGGCGGCGGGGGCAGCGC GGCGCGTGTCTGTGCGCTGCGGTCGCTCGGGACCGGGACCGGGGCGAGGCGCCGCGGGGCT GAGCCCAGCAGACATTGCGTTGGCCTCCGAGCAGGGCGCATCATGCAGCGTTCGCGCACCG GAGAGAAAACTGAGAATGAAATTGCTTTGGCAAGCTAAAATGGTAACGAGAGCTCTCTGCC TTCTCCCGAGGCTTCTCTGGTGGGGAATCTTCTCCCGCTTTCGCTGGGCGTGTTTTGGACT CCCTCGGAGTGAACGGAGCAATCCGATTCCATACTTGTGAGCTAAAGCTCTCTCACTCCGC TTTGTATTCCTCTCTCTCTGCGTGTCTTCTCCCTTTCAACCGTACCTACTTTCTCAGCAAT TGCCTGTGGTGTGTCTGGGGCAGATGCATGTCAGGAGGGGAGAACTTTGAAGGAAAAAGAA CTGTTAGTGTGGGTGAACCGCGGAGCGCTTCTGCGTCCCAGCGGCTCTTTAACACAAACCT CAGGTTTAGCTGGGGACATGGAAATCCAGGGGGTCGCTGCACAGATTTTAGTAGAAGCTGC CTGATGATGTGTTGCTTTTTCTGCACTTGCCAGATTATTTTACGGAAATAAAAACTTTCCA CCCCCGCTAACTTTGTGGGCTCCAAAACAATGAGGAGAAAAGACAAGCAAACCACTTCACT TCCAAAGGAGTGAGGACTCTTGGCGTTTTAAACACTTGTTTGTGTCTGAGTTTAAAATCTG GTTTCTTCAATGACCGAGCCACTCTAAAAAGTACAGACTTATTCTTTTCTTTTTTTCTTTTTCTTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACA AAGAATTATTTTTCCTTTCACTTAAGGGGGACAAGTTAATCAGGACTAATCTAGTTTCTTT GTAAAGAAAAAGAAAGACTTCAAAACAAAACTATTTAAGATATGTGAGCAAGTCTAGGAGA AATAACTAAATGGAAAAGTCAGGGGATCCTGATACCACAGGAAGGGACTGCAAGCCAAGAA AATCTCAGCATTCCCTCTATCAAAATGTGTCTTGTATTGCGGATTGTAATATTTATTTTGA ACAGCCCATCTTGTGCCCTGAGTGCTGTTGTAAGCAGCAAGGCAGCTGGGATAGATTGTGG TTAGCAGAGAGGAAAAAAATACTCTTTTTCTTTCTCTTTATCCCGTCCCTGGCAGAGCTCG ATTCAGGACTGGGGTGAAGAGGTAGAGGAAGGAGCTGTTTACCATGTCACCCTCAAAAGAG TCCAGATTCAACAGGCTGCCAATAAAGGAGCAAGATGGCTAGGGGTGAGTAAAGCTGGCGA GATGGTGAACTTTGGAATTTCAGTGTCTGTCGTTCTGTGAATATCATTATTATTTAACAGA GGCAGGCTTTTTTTTTTTTCCTC'TTTTCGCTTTAAAAAGGAAAATGCTAATGACCAAGTGT TTGCACTAGCTGAACAGGTCTCTTTTTGTATTTAATGTTTTCAAAAGCATCTTCCCTAGGT TTGTCAATAATTCAGAACCTCTTAGAAACATCTTGAGGTCTTTGGCAAATAGATTGGGGGT GGAGTGGCAGAAAATGACACACAGAATACAGGCCCAGAGAGCAGCACTCTCTTAGACAAGA GCCATAAGCAGCTTTGCCCAGAAGCCACCCTTGTCTCTCTCTGCCCTCCCCCAATTGATGC TGTCAGCGTCTGCCTTGCTCCTATATCTCTAGGAGGTACCCAGATTGCTCAGGAGACAGAG CATTCCAAGTCTGCATCCTTTGAG ( SEQ ID NO : 5 )NC_000005 . 10 ( 137499323 . . 1347500826 ] :ATGTGCTCTGCTCGCTCGCTGGCTCGGGCGCGGCGTCCGCGGGCGGAGGAGGCCGAGCGCG CGAGCCAAGGCTCTGGCGCCCCCTGGTGGCCGCGCGCTGCCTGCCCGACAGCCCGGCCGCC CCAGCCCGGAACGTGGTCATTCACATCTTTTTTTTTTTTTTTTTTTTTTTTAATTTACTTC TATTTTTTTTTTTAATTATACTTTAAGTTTTAGGGTACATGTGCACATTGTGCAGGTTAGT TACATATGTATACATGTGCCATGCTGGTGCGCTGCACCCACTAACTCGTCATCTAGCATTA GGTATATCTCCCAATGCTATCCCTCCCCACTCCCCCCACCCCACCACAGTCCCCAGAGTGT GATATTCCCCTTCCTGTGTCCATGTGATCTCATTGTTCAATTCCCACCTATGAGTGAGAAT ATGCGGTGTTTGGTTTTTTGTTCTTGCGATAGTTTACTGAGAATGATGGTTTCCAATTTCA TCCACGTCCCTACAAAGGACATGAACTCATCCTTTTTTATGGCTGCATAGTATTCCATGGT GTATATGTGCCACATTTTCTTAATCCAGTCTATCATTGATGGACATTTGGGTTGGTTCCAA GTCTTTGCTATTGTGAATAATGCCGCAATACACATACGTGTGCATGTGGCTTTATAGCAGC ATGATTTATAGTCCTTTGGGTATATACCCAGTAATGGGATGGCTGGGTCAAATGGTATTTC TAGTTCTAGATCCCTGAGGAATCGCCACACTGACTTCCAC1VATGGTTGAACTAGTTTACAG TCCCACCAACAGTGTAAAAGTGTTCCTATTTCTCCACATCCTCTCCAGCACCTGTTGTTTC CTGACTTTTTAATGATTGCCATTCTAACTGGTGTGAGATGATATCTCATAGTGGTTTTGAT TTGCATTGCTCTGATGGCCAGTGATGATGAGCATTTTTTCATGTATTTTTTGGCTGCATAA ATGTCTTCTTTTGAGAAGTGTCTGTTCATGTCCTTCGCCCACTTTTTGATGGGGTTGTTTG TTTTTTTCTTGTAAATTTGTTTGAGTTCATTGTAGATTCTGGATATTAGCCCTTTGTCAGA TGAGTAGGTTGCGAAAATTTTCTCCCATGTTGTAGGTTGCCTGTTCACTCTGATGGTAGTT TCTTTTGCTGTGCAGAAGCTCTTTAGTTT7\ATTAGATCCCATTTGTCAATTTTGGCTTTTG TTGCCATTGCTTTTGGTGTTTTGGACATGAAGTCCTTGCCCACGCCTATGTCCTGGGTCAT TCACATCTTAATGCAGGGCTAAAATCAGCTTTCAGGCCCAGGCTGGTGCGAGACAGAAGAG GGGACTGAGGATATGAGGCCAAGACGCAGGCCAGCATGGAGGCATTCTCAAAAGGAAGGCC GTCGGGTTATCTAGATCAAAATCACCTGGGTGGAGAGAGAAGCGAACTTCCTAAAAATGAT TCCTGCCCCACCGCCCCACTTGCTGAGTCAGCATTCCTTA ( SEQ ID NO : 6 )NC 000019 . 10 [ 55115550 . . 55118000 ] :CCCCCTGCCAAGCTCTCCCTCCCAGGATCCTCTCTGGCTCCATCGTAAGCAAACCTTAGAG GTTCTGGCAAGGAGAGAGATGGCTCCAGGAAATGGGGGTGTGTCACCAGATAAGGAATCTGCCTAACAGGAGGTGGGGGTTAGACCCAATATCAGGAGACTAGGAAGGAGGAGGCCTAAGGA TGGGGCTTTTCTGTCACCAATCCTGTCCCTAGTGGCCCCACTGTGGGGTGGAGGGGACAGA TAAAAGTACCCAGAACCAGAGCCACATTAACCGGCCCTGGGAATATAAGGTGGTCCCAGCT CGGGGACACAGGATCCCTGGAGGCAGCAAACATGCTGTCCTGAAGTGGACATAGGGGCCCG GGTTGGAGGAAGAAGACTAGCTGAGCTCTCGGACCCCTGGAAGATGCCATGACAGGGGGCT GGAAGAGCTAGCACAGACTAGAGAGGTAAGGGGGGTAGGGGAGCTGCCCAAATGAAAGGAG TGAGAGGTGACCCGAATCCACAGGAGAACGGGGTGTCCAGGCAAAGAAAGCAAGAGGATGG AGAGGTGGCTAAAGCCAGGGAGACGGGGTACTTTGGGGTTGTCCAGAAAAACGGTGATGAT GCAGGCCTACAAGAAGGGGAGGCGGGACGCAAGGGAGACATCCGTCGGAGAAGGCCATCCT AAGAAACGAGAGATGGCACAGGCCCCAGAAGGAGAAGGAAAAGGGAACCCAGCGAGTGAAG ACGGCATGGGGTTGGGTGAGGGAGGAGAGATGCCCGGAGAGGACCCAGACACGGGGAGGAT CCGCTCAGAGGACATCACGTGGTGCAGCGCCGAGAAGGAAGTGCTCCGGAAAGAGCATCCT TGGGCAGCAACACAGCAGAGAGCAAGGGGAAGAGGGAGTGGAGGAAGACGGAACCTGAAGG AGGCGGCAGGGAAGGATCTGGGCCAGCCGTAGAGGTGACCCAGGCCACAAGCTGCAGACAG AAAGCGGCACAGGCCCAGGGGAGAGAATGCAGGTCAGAGAAAGCAGGACCTGCCTGGGAAG GGGAAACAGTGGGCCAGAGGCGGCGCAGAAGCCAGTAGAGCTCAAAGTGGTCCGGACTCAG GAGAGAGACGGCAGCGTTAGAGGGCAGAGTTCCGGCGGCACAGCAAGGGCACTCGGGGGCG AGAGGAGGGCAGCGCAAAGTGACAATGGCCAGGGCCAGGCAGATAGACCAGACTGAGCTAT GGGAGCTGGCTCAGGTTCAGGAGAGGGCAGGGCAGGGAAGGAGACAAAGTCCAGGACCGGC TGGAGGGGCTCAACATCGGAAGAGGGGAAGTCGAGGGAGGGATGGTAAGGAGGACTGCATG GGTCAGCACAGGCTGCCAAAGCCAGGGCCAGT'l’AAAGCGACTCCAATGCGGAAGAGAGTAG GTCGAAGGGGAATGGTAAGGAGGCCTGGGGCAGAGTGGTCAGCACAGAGTGGCTAAGCCCA GGGCCAGTTGAAGCGGCTCCAATTCGGAAGTGGGGTGGTCGAAGGGGAATGGTAAGGGGGA CTGGGACGGGGTGTCAGCATAGGGTGGCAAAGCCCAGGGCCAGGAACGACGGGGCGGATCG AGACTGGCAACGGGGAAGGAGGATGCCCCAGGTGGCGCAGCAGAGGGTGGACCTGGCCCCG GGAGACGCCGGGCGGGGGGCGCTGACCTGGTGCAGGGCGCTGATACCGTCGGCGTTGGTGG AGTCCAGCACGGCGCGGGCGGGCGGCGGCGCGGCGGGGTCGAGCTCGGCGCCGGGGCCAGG GTCGGCGGCGCGCAGCATCAGACGCGCCTCGTCCAGGTCGCCGCCCGCACAGGCCGCCAGG AACTCGGCGGCGCGCTCGAAGCGGACGGTGCGGGCGCGGCGCTCTCCGGGGCCAGGCTCGG CGCCCGCCCGCGGCCCCCACTGCCGCAGCTGCTCCCGTCGCCGCTCCCGGGCAGCCGCCGC CGCCGCCCCCGGGCCAGCCGCCGGGCCATCCTCTCCGGACATCGCACCGCCCGCCCGCCCA GCGAGCGAGCGAGCGCCGAGCCCCAACCGCCGCCACCACCCGCCCGCCCGCCCGCCCCGGG GGCCGCCGGGAACTGCCGCTGGCCCCCCACCGCCCCAAGGATCTCCCGGTCCCCGCCCGGC GTGCTGACGTCACGGCGCTGCCCCAGGGTGTGCTGGGCAGGTCGCGGGGAGCGCTGGG / kAA TGGAGTCCATTAGCAGAAGTGGCCCTTGGCCACTTCCAGGAGTCGCTGTGCCCCGATGCAC ACTGGGAAGTCCGCAGCTCCGAGGCGCCCAGTGGAAATCGCCAGATGAGGGCCTCCTCCGG GGAATGCTGGGAAATGGAGTCTACAGGCCGGAGGGGTGCCCCACGGCATACTAGGAAGTGT GTAGCACCGGGTAAAGGGGATGAATAGCAGACTGCCCCGGGGCAGTTAGGAATTCGACTGG ACAGCCGCGTG ( SEQ I D NO : 7 )NC_000021 . 9 [ 43165814 . . 43167705 ] :ATACAACACCTAAACTCGGGGTTTGAAAAGGCCCCTAGGGCCCTAGAGCCCTCCCTGCCCC CGGTCCAGGCCTTCCTGCTTCTGTTGTGCAGACTCAGGCGCTGGCTCACTCAGAAGCCCCC TGCAGGCCCGGCCAATCCTGTGGCAGAGCCTCGACGTCCCACGGCGGCCTCTGAGCCGCCA GGCCCTACAGCGTGTGGTGAGGGGCGCAAGAGGTGTGAGTGCCTGGACCTTGCTCTGCCCC GTGTCTGAGGACCTTGCCCTGCCCCGTGTCTGAGGACCTTGCCCCATCCTCGGCACCCAGA GCTGTGCAGTCCATAGGACCAGGCTGGTGCCCAGCCCCAGGTTGGTGCCTCAGCTGCCTCC TCAGCCTGCAGTCTCCACTGTTTGACTGACCATGTGCAGGGCCAGCTGTGAAGGACAGCACAGCTCCTCCCTCCCTGCCAAGTGTCAGGGGCACTAAAAACACTGCCATCAATGTTCTGATG TTTAAATCATGTTTTGATGCAATGTTTAAACAAATCAAAATGGGTCGGGCGCAGTGGCTCA CGCCTGTAATCCCATCACTCTGGGAGGCCGAAGCGAGTGGATCACCAGAGATCAGGAGTTC GAGACCAGCCTGGCCAACACGGCGAAACCTCATCTCTACTAAAAATATAAAAATTAGCTGG GTGTAGTGGTGTGTGCCTGTAATCTCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGTTTG AACCCGGGAGGCAGAGGTTGCAGTGAGCTGAGATTGTGCCACTGCACTCCAGCCTGGGCGA CAGAGCGAGACTCCATCTCAAAAATAAATAAATAAAATAAATAAGTAAATCAAAATGAATG CAAAAAATTTGTGGTGGACAAACTGTCAACATTTTACATAAAGCCAGGATGAGGACGACGG CCTTCTCCTTTTGCTTCAAGCTCCAGCATGGCTCTGCCCAGCACTGACCGTGCTCCTGGCC TGGCTCGACCCTCCCCAGGGCCAAGCTCCATGCTGGTTTCCATCCCAAAGGACTCTTCTTC CCCCTCCCTGTGCCCCGGGAATAGGCAGCCAGCACAGTGGGGCCGCCCGCTGCCTCACCCA GACCCTGCCACTGCTTGGATGTTGACCCCCAGACTGGAAAGGTGGCTTTAAACCCCCTTCC CTTCTTCCAAGCCAAGTGACCTGGGCGTGCGGCTTAACCTTCTAAGCTCCCATTTCCTGGT CTATGAAGCGGGAATGATGCTGGGACCTGCTTCACAGGGTGGCTCTGAGGACCCACAGGCA GAGAGGTCTGGTGTAGGCTTTCGGGGGAAGCACCCATCACTCTCCCAGCGTTTCCTATCTG CACCTTAATTTGGCACCCCAGGGAATCGCTCCTCTGGAATTGTAACCTTGCAGGGCAAAAG CTATCATTTCAAGATTTTTTGGGATCCTGTGTCCCCGGACAGTGCTGTAAATGTAGGAGGA AGGGTGCTGCTCATGGGTGCTGCTCATGATGGAGGTGCCCCGTGGAGCACGCCTCGGAGCC CCCCCTTTCATCCCTGATCCCATCTGGGATGTGTCAGGTCTGATCCGCATACACTGGGGCA TCTGACATTTCATGGCCTTCTGAGCCCATGGGCTGGAGATGAATTTGCAAAGCCCGGGAGT GTCCTGGGTGTGTGTTCACTCCACACGCACGAATCTGTTAGAGGCTGAGTAGGAACTAGGA GGCCAAAGAGAACCAGGCAACTTCCCACAGCCTGGACCGTACACTAGAAAAGCTAGCCTGG GAGAGGGGGCATGGCCTCTTCCCAGGCTGGCAGGGGGGATGTGGGGGAGGACCATGCTCCC CAGGGCCAGTTTACCAAAATATCAAAATACCAAAATATCAAAAGACTTAAAACTAGGAGTGA ( SEQ ID NO : 8 )NC_000022 . 11 [ 18598046 . . 18601312 ] :TTAGCTGGGCGCGGTGGTGTGTGCCTGTAATCCCAGCTACTCGGGAGGCTAAGGCAGGAGA ATCGCTTGAACCTGCAAGGTGAGGTTGCAGTAAGCTGAGATTGTGCCGTTGCACTCCAGCC CAGGCAACAAGAGTGAAACTCCATCTCCAAAAAATAAAAATAAAAATAAAAATAAAAAAGA AATAGGCCGGGTGCAGTGGCTCACGCCTATMTCCTAGCACTTTGGGAGGCCAAGGTGGGG GCGGGGTGGATCACTTGAGGTCAGAAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCA ATCTCCACTAAAAACACAAAAAATTAGCCGAGCATGGTGGTGGGCACCTGTAATCCCAGCT ACTCGAGAGGCTGAGGCAGGAGAATGGCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCA AGATCACCCCACTGTACTCCAGCCTGGGTGACAGAGTGTIAACTGTCTCZkAAAAATAAATAA ATAAATAAATA7KAACTTTTAAAAAGTAAGAAGAAGAAG7\AAA7\AAATATATGG7\AATTAAA AAACAAGAAAAAAATAATAGGCCAGGTGCAGTGGCTCATGCCTGTAATCCCAGCACTTTGA GAGGCCGAGGTGGGCGGATCATGATGTCAGGAGTTCAAGACCAGCCTGGCCAACATAGTGA GACCTCGTCGCTACTAAAAATAGAAAAAAATTAGCCAGGCGTGGTGGCGGGCACCTGTAAT CCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCCATGAGGCGGAGGTTGCAG TGAGCCGAGATTACGCCATTGCACTCCAGCCCGGGTGACAGTGCGAGACTCCATCAAAAAA AAAAATAAATAAAATAAATAAATATTAATAATAATTTTAAACAATTAAAAAATATGGGATT TTTTTGAGACAGAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAGTGGCATGATGTCAGCTC ACTGCAACCTCCGCCTCCTGGGTTCAAGTGATTCTTCTGCCTCAGCCTCCCAAGTAGCTGG GACTACAGGCACGCGCCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTT CACCATATTGGCCAAGCTGGTCTGGAACTCCTAACCTTGTGATCCACCCGCCTCGGCCTCC CAAAGTGCTGGAACTATAGGTGTGAGCCACTGCACCCGGCCAAAAAATATGTTTTTTAAAA TMTAGAGATGAGGCCGGGTGTGGTGGCTCACACCTGTAATCTCAGCACTTTGGGAGGCCA AGGTGGGTGGATCACTTGAGGTCAGGAGTTCGAGACCAGTCTGGGCAACATGGTGAAACCCTGCCTCTACTAAAAATACAAACCTGAGCTGGGCATGGTGACGCATGCCTCTAGTTCCAGCTACTCGAGAGGCTGAAGCAAGAGAATCGCTTGAACCCGGGAGGCGGAGACTGCAGTGAGCCAAGATAGCACCACTGCACTCCAGCCTGGGAGACAGAGCAAGACCCTGTCTCAAAAAATAAAATAAATAAAATAAAATAAAATAAAATAAAATAAAATAAAATAAAATAAAATAAAATAAAAAATAAAATAAAATAAAATAAAATAAAATAAATAAAATAGATGAAGGCCAGGTGCAGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCAGATCACCTGAGTTCAGGAGTTAGAGAGCATCCTGGCCAACATGGTGAAACCCTGTCTGTACTAAAAATACAAAAAAATTAGCCAGGCATTGTGGCGGGCGGCCAATAATCCTGGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCAAGATGGTACTACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTCAAAATAATAATAATAGGCCGGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCAGATCACAAGGTCAGGAGATCGAGACCATACTGGCTAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCGGGTGTGCTGGTGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGACAATGGCGTGAACCCGGTAGGCGGAGCTTGCAGTGAGCCAAGATCGTGCCATTGCACTCCAGCCTGGGCGACAGAGCGAAACTCCACCTCAAAAAATAATAATAAAATAAAATAAAAATAAAAATAATAATAATAGAGATGAGGTTTTGCCATGTTACCCATGCTGGTCTCCAACTCCTTGGCTCAAGTGATCCATCCACCTTGGCCTTCCAAAGTGCTGGAACTACAGGCTTGTGCCACCACCCTGGCCTTAAACAATTA'rTCATGAAAGGAAGGAAATTCTGACACATGCTGCAACATGGATGAAGCTCAAGGACATTATGCTAAGGGAAGGAAGCCAAAGACCTGCCAGCCGAGGTCCCAGTCCGTCAAGGGCTCCAGTGAGCGAGCAGGATTGAGCAGGTCCCTGGGCTGAGCAGTGGGAACTCTGCTTTGCTGTGAGTGTGGCACGGGTGGCGGCAGACTGTGGAGTGCAGGCTCTGTGGAGCAACTGGACACTCTGTTGAACGAAGTGCAAGGTGGTAGGTTTTTGCTTTTTTTTTTTTTTCTGAGACAGAGTCTCACTCTGTCTCCTAGGCTGGAGTGCAGTGGCATGATCTCGACTCATTGCAACCTCTGCCTCCCGGGTTCAAGCAATTCTCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGCGAGCCACCACACCCAGATAATTTTTAAAATATTTTTGGAAGAGACGGGGTTTTACCATCTTGGCCAGGCTGGTCTCGAACTCCTGACCTCGTGATCCACCTGCTTCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGCGCCCGGCCACAAGGTGGTAGTCTTTCTCAAGGACGCTGGGGCAACCCCATTGCGTCTTCACCAACACCCTGGTACAGATCCTGCAGCTGCAGTCTACCCCGCATACAACATGTTCAATGATCCCTAGCTAGACAAGGCATTGAAAACTATTCCATAGGCCGGGTGCAGTGGCTCCTACCTGAAATCCCAGCAGTTTTGGAGCAAGGCGGGTAGATCATCTGAGGTCAGGAGTTCGAGACCAGTCTGGCCAACATAGCGAAACACCGTCTCTTCTAAAATACAAAAAATTAGCTGGGTATGGTGGTGCATGCCTGTAATCCCAGCT (SEQ ID NO : 9)

[0055] A summary of genomic coordinates identified which were above background in multiple analyzed rAAV preparations is presented in Table 3 .TABLE 3

[0056] Upon further analysis of these sequences , a proportion of preferentially incorporated sites were identified to be sites that the wild-type AAV virus integrates (Table 4 ) . Notably, example REP binding sites in relation to the incorporation signals at chromosomes 19 and 22 were on the positive strand and the incorporation was observed to be 5' of that sequence ( FIG . 8 ) and example REP binding sites in relation to the incorporation signals at chromosomes 5 and 1 analyzed were on the negat ive strand ( FIG . 9) , wherein the phenotype of incorporation appeared to mirror that of shown in FIG . 8 . These analyses showed that incorporated host genomic DNA is associated with the presence of adj acent AAV REP binding sites and include but are not fully restricted to previously described sites of wildtype AAV integration into the human genome .TABLE 4Example 2: Genomic Engineering of HEK293T Cells to Delete Preferentially Packaged Host Genomic Sequences

[0057] To demonstrate that deletion of AAV REP binding sites in host genomic DNA can prevent packaging, CRISPR guides (CAGE3214. AAVS1.9. gl (gRNA 2) and CAGE3215. AAVS1.9. gl (gRNA 1) ) were designed to cut on both sides of the AAVS1 RBS / TRS, i.e., single guide RNA (sgRNA) were prepared and SpCas9 strategies were used to generate HEK293T cells engineered to delete the AAVS1 RBS / TRS (FIG. 2A) . Using this approach, the deleted sequence between gRNAs will not interfere with qPCR detection of nearby loci (FIG. 2A) . Primer sets were designed to bind to the NGS signal (Del PCR #1) , opposite sides of the REP binding element (RBE) (Del PCR #2) , and ALU repeats throughout the genome (Del PCR #3) (Table 5; FIG. 2B) .TABLE 5

[0058] The 155 bp deletion of hAAVSl in HEK293T cells was confirmed by PCR amplification using the primers in Table 5 and separation of amplicons by gel electrophoresis (FIG. 3) and NGS analysis (Table 6, FIG. 4) . All amplicons were 80% GC rich, therefore a GC-rich 7-deaza-2' dGTP PCR protocol was used. GC- Rich PCR conditions included denaturation for 10 seconds at 98°C; 35 cycles of 98°C for 10 seconds, 60°C for 5 seconds, 72°C for 1 second; incubation at 72°C for 5 minutes; and storage at 4°C.TABLE 6Example 3: Impact of AAVS1 RBS Knock Out on Packaging of Contaminant DNA

[0059] To analyze the impact of the AAVS1 RBS / TRS deletion on titer, an AAV8 vector was prepared using wild-type HEK293T cells and HEK293T mutants 4A4 and 6G12. The AAV8 vector included a small synthetic liver specific promoter (HLP) driving expression of plasma factor VIII (FVIII) variant V3, that contains a 17 amino acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain. The resultsof this analysis indicated that HEK293T mutants 4A4 and 6G12 produced vector, but not as efficiently as wild-type HEK293T cells ( FIG . 5 ) . The reduction in titer is likely due to clonal selection, and selection of more clones would likely result in isolation of an AAVS1 deletion cell line that produces vector efficiently . Notably, however, rAAV8 HLP FVIII V3 preparations showed that deletion of AAVS1 in HEK293T cells significantly reduced packaging of nearby DNA per vector genome produced ( FIGS . 6A-6B) .

[0060] The effect of AAVS1 deletion on other sequences was also determined . The results of this analysis indicated that other sequences were unperturbed ( FIGS . 7A-7C) .

Claims

What is claimed, is:

1. An engineered producer cell comprising an inactivating mutation in one or more endogenous adeno-associated virus (AAV) REP binding sites.

2. The engineered producer cell of claim 1, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID N0:l, SEQ ID NO: 2, SEQ ID NO: 3, CGAGCGAGC, or TGAGCGAGC.

3. The engineered producer cell of claim 1, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence of SEQ ID NO: 4 or (GAGY)n, wherein n is at least 2.

4. The engineered producer cell of claim 1, wherein the engineered producer cell is a human cell.

5. The engineered producer cell of claim 4, wherein the one or more endogenous AAV REP binding sites is located between nucleotides: 6003380 and 6005107 of chromosome 1, 10796284 and 10797669 of chromosome 1, 44985851 and 44988210 of chromosome1, 61075662 and 61077300 of chromosome 1, 151459046 and 151460524 of chromosome 1, 183804446 and 183806827 of chromosome 1, 10850196 and 10851915 of chromosome 3, 46883151 and 46885332 of chromosome 3, 30718138 and 30720073 of chromosome 4, 30042612 and 30044361 of chromosome 5, 123512313 and 123513360 of chromosome 5, 137498974 and 137500979 of chromosome 5, 26207238 and 26208703 of chromosome 8, 6207086 and 6209319 of chromosome 10, 5224551 and 5226324 of chromosomeII, 61909029 and 61909029 of chromosome 11, 2740788 and 27409167 of chromosome 14, 95774695 and 95775629 of chromosome 14, 30697077 and 30699022 of chromosome 16, 89342145 and89343777 of chromosome 16, 70629 and 71560 of chromosome 17, 107961 and 110340 of chromosome 18, 55115533 and 55119207 of chromosome 19, 6557123 and 6559560 of chromosome 21, 43165383 and 43167617 of chromosome 21, 18598982 and 18600792 of chromosome 22, 21394536 and 21396314 of chromosome 22, 21538348 and 21540375 of chromosome 22, and / or 132949595 and 132951989 of chromosome 23.

6. The engineered producer cell of claim 4, wherein said engineered producer cell is a human embryonic kidney (HEK) cell, HeLa cell, A549 cell, CHO cell or SF9 cell.

7. A method of producing an engineered producer cell comprising introducing into a producer cell an inactivating mutation in one or more endogenous adeno-associated virus (AAV) REP binding sites thereby producing the engineered producer cell.

8. The method of claim 7, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, CGAGCGAGC, or TGAGCGAGC.

9. The method of claim 7, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence of SEQ ID NO: 4 or (GAGY)n, wherein n is at least 2.

10. The method of claim 7, wherein the producer cell is a human cell.

11. The method of claim 10, wherein the one or more endogenous AAV REP binding sites is located between nucleotides: 6003380 and 6005107 of chromosome 1, 10796284 and10797669 of chromosome 1 , 44985851 and 44988210 of chromosomeI , 61075662 and 61077300 of chromosome 1, 151459046 and 151460524 of chromosome 1, 183804446 and 183806827 of chromosome 1 , 10850196 and 10851915 of chromosome 3 , 46883151 and 46885332 of chromosome 3 , 30718138 and 30720073 of chromosome 4 , 30042612 and 30044361 of chromosome 5, 123512313 and 123513360 of chromosome 5 , 137498974 and 137500979 of chromosome 5 , 26207238 and 26208703 of chromosome 8 , 6207086 and 6209319 of chromosome 10, 5224551 and 5226324 of chromosomeII , 61909029 and 61909029 of chromosome 11 , 2740788 and 27409167 of chromosome 14 , 95774695 and 95775629 of chromosome 14 , 30697077 and 30699022 of chromosome 16, 89342145 and 89343777 of chromosome 16, 70629 and 71560 of chromosome 17 , 107961 and 110340 of chromosome 18 , 55115533 and 55119207 of chromosome 19, 6557123 and 6559560 of chromosome 21 , 43165383 and 43167617 of chromosome 21, 18598982 and 18600792 of chromosome 22, 21394536 and 21396314 of chromosome 22 , 21538348 and 21540375 of chromosome 22 , and / or 132949595 and 132951989 of chromosome 23.

12. The method of claim 10, wherein said producer cell is a human embryonic kidney (HEK) cell, HeLa cell, A549 cell , CHO cell or SF9 cell .

13. A method of producing a recombinant viral vector, comprising :(a) providing to the engineered producer cell of claim 1 a viral expression system comprising an adeno-associated virus (AAV) REP protein;(b) culturing the cell under conditions in which viral vector is produced; and(c) optionally isolating the viral vector .

14. The method of claim 13, wherein the viral expression system is for production of an AAV vector or bocavirus vector.

15. The method of claim 14, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 , AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or synthetic variant, a chimeric or hybrid AAV thereof.

16. A recombinant viral vector produced by the method of claim 13, wherein said recombinant viral vector comprises a reduction in producer cell genomic DNA contaminants.

17. A method of reducing producer cell genomic DNA contamination of a recombinant adeno-associated virus (rAAV) vector preparation comprising introducing an inactivating mutation into one or more endogenous AAV REP binding sites of the genome of a producer cell thereby producing an engineered producer cell, and preparing a rAAV vector with the engineered producer cell, thereby reducing producer cell genomic DNA contamination of the rAAV vector preparation.

18. The method of claim 17, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID N0:l, SEQ ID NO: 2, SEQ ID NO: 3, CGAGCGAGC, or TGAGCGAGC.

19. The method of claim 17, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence of SEQ ID NO: 4 or (GAGY)n, wherein n is at least 2.

20. The method of claim 17, wherein said producer cell is a human cell.

21. The method of claim 20 , wherein the one or more endogenous AAV REP binding sites is located between nucleotides : 6003380 and 6005107 of chromosome 1, 10796284 and 10797669 of chromosome 1 , 44985851 and 44988210 of chromosomeI , 61075662 and 61077300 of chromosome 1 , 151459046 and 151460524 of chromosome 1 , 183804446 and 183806827 of chromosome 1 , 10850196 and 10851915 of chromosome 3 , 46883151 and 46885332 of chromosome 3, 30718138 and 30720073 of chromosome 4 , 30042612 and 30044361 of chromosome 5, 123512313 and 123513360 of chromosome 5 , 137498974 and 137500979 of chromosome 5, 26207238 and 26208703 of chromosome 8 , 6207086 and 6209319 of chromosome 10, 5224551 and 5226324 of chromosomeII , 61909029 and 61909029 of chromosome 11 , 2740788 and 27409167 of chromosome 14 , 95774695 and 95775629 of chromosome 14 , 30697077 and 30699022 of chromosome 16, 89342145 and 89343777 of chromosome 16, 70629 and 71560 of chromosome 17 , 107961 and 110340 of chromosome 18 , 55115533 and 55119207 of chromosome 19, 6557123 and 6559560 of chromosome 21, 43165383 and 43167617 of chromosome 21, 18598982 and 18600792 of chromosome 22 , 21394536 and 21396314 of chromosome 22 , 21538348 and 21540375 of chromosome 22 , and / or 132949595 and 132951989 of chromosome 23.

22. The method of claim 17 , wherein said producer cell is a human embryonic kidney (HEK) cell, HeLa cell, A549 cell, CHO cell or SF9 cell .

23. A method for determining the purity of a recombinant adeno-associated virus (rAAV) vector preparation prepared via a producer cell comprising detecting in the rAAV vector preparation the presence of producer cell genomic DIMA contamination derived from a sequence proximal to one or more endogenous AAV REP binding sites of the genome of the producercell thereby determining the purity of the rAAV vector preparation .24 . The method of claim 23, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO : 1 , SEQ ID NO : 2 , SEQ ID NO : 3 , CGAGCGAGC, or TGAGCGAGC .25 . The method of claim 23, wherein the one or more endogenous AAV REP binding sites comprise a nucleotide sequence of SEQ ID NO : 4 or (GAGY)n, wherein n is at least 2 .

26. The method of claim 23, wherein said producer cell is a human cell .27 . The method of claim 20, wherein the genomic DNA contamination comprises a sequence located between nucleotides : 6003380 and 6005107 of chromosome 1, 10796284 and 10797669 of chromosome 1 , 44985851 and 44988210 of chromosome 1 , 61075662 and 61077300 of chromosome 1 , 151459046 and 151460524 of chromosome 1, 183804446 and 183806827 of chromosome 1 , 10850196 and 10851915 of chromosome 3 , 46883151 and 46885332 of chromosome 3 , 30718138 and 30720073 of chromosome 4 , 30042612 and 30044361 of chromosome 5 , 123512313 and 123513360 of chromosome 5, 137498974 and 137500979 of chromosome 5 , 26207238 and 26208703 of chromosome 8 , 6207086 and 6209319 of chromosome 10, 5224551 and 5226324 of chromosome 11 , 61909029 and 61909029 of chromosome 11 , 2740788 and 27409167 of chromosome 14 , 95774695 and 95775629 of chromosome 14 , 30697077 and 30699022 of chromosome 16, 89342145 and 89343777 of chromosome 16, 70629 and 71560 of chromosome 17 , 107961 and 110340 of chromosome 18 , 55115533 and 55119207 of chromosome 19 , 6557123 and 6559560 of chromosome 21, 43165383 and 43167617 of chromosome 21 , 18598982and 18600792 of chromosome 22 , 21394536 and 21396314 of chromosome 22 , 21538348 and 21540375 of chromosome 22 , and / or 132949595 and 132951989 of chromosome 23.28 . The method of claim 23, wherein said producer cell is a human embryonic kidney (HEK) cell, HeLa cell, A549 cell , CHO cell or SF9 cell .

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