EP4680626A1 - Engineered cells and cell lines for aav production and methods of making and using the same - Google Patents

Abstract

The present disclosure provides improved cells and cell lines useful in the production of recombinant adeno-associated viruses.

Description

ENGINEERED CELLS AND CELL LINES FOR AAV PRODUCTION AND METHODS OF MAKING AND USING THE SAME

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/451836 filed on March 13, 2023, and U.S. Provisional Patent Application No. 63/456338 filed on March 31, 2023 the entire contents each of which are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] Gene therapy is a promising cure for many diseases such as genetic disorders. Recombinant adeno-associated viral vectors (rAAV) are being developed as a gene delivery vehicle in the gene therapy field. Production of rAAV vectors relies on introduction of AAV genes (Rep and Cap), adenoviral helper genes and a transgene of interest flanked by AAV inverted terminal repeats (ITRs) into cells. Improvements to the cells and cell lines used in rAAV production can provide benefits for generating gene therapy vectors.

SUMMARY

[0003] In one aspect, the present disclosure provides an engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells in which (a) expression and/or activity of one or more genes and/or gene products in Tables 2-9 is reduced compared to control parental cells and/or (b) expression and/or activity of one or more genes and/or gene products in Table 10 or 11 is increased compared to control parental cells. In some embodiments, this involves reducing expression and/or activity of one or more genes and/or gene products in Tables 2-9. In some embodiments, this involves eliminating expression and/or activity of one or more genes and/or gene products in Tables 2-9. In some embodiments, this involves increasing expression and/or activity of one or more genes and/or gene products in Tables 10 or 11.

[0004] In one aspect, the present disclosure provides a lysate or a cell culture supernatant of an engineered cell or cell line.

[0005] In one aspect, the present disclosure provides methods of generating an engineered cell or cell line by modulating expression and/or activity of one or more genes and/or gene products in Tables 2-11 in a parental cell line. In some embodiments, this involves reducing expression and/or activity of one or more genes and/or gene products in Tables 2-9. In some embodiments, this involves eliminating expression and/or activity of one or more genes and/or gene products in Tables 2-9. In some embodiments, this involves increasing expression and/or activity of one or more genes and/or gene products in Tables 10 or 11.

[0006] In one aspect, the present disclosure provides methods of producing rAAV. In some embodiment, this involves transfecting an engineered cell or cell line with one or more plasmids. In some embodiments, the one or more plasmids comprise a packaging plasmid, an adenoviral helper plasmid, and/or a proviral plasmid. In some embodiments, this involves infecting an engineered cell or cell line with one or more helper viruses.

[0007] In one aspect the present disclosure provides an engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which have been engineered to (a) reduce or eliminate expression and/or activity of a gene product expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increase expression and/or activity of a gene product expressed from one or more genes in Tables 10 or 11 as compared to control parental cells. In some embodiments, the cell or cells have been engineered to reduce expression and/or activity of one or more genes and/or gene products in Tables 2-9. In some embodiments, the cell or cells have been engineered to eliminate expression and/or activity of one or more genes and/or gene products in Tables 2- 9. In some embodiments, the cell or cells have been engineered to increase expression and/or activity of one or more genes and/or gene products in Tables 10 or 11. [0008] In one aspect the present disclosure provides an engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which exhibit (a) reduced or eliminated expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increased expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 10 or 11 as compared to control parental cells. In some embodiments, the cell or cells exhibit reduced expression and/or activity of a protein or noncoding RNA expressed from one or more genes in Tables 2-9. In some embodiments, the cell or cells exhibit eliminated expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 2-9. In some embodiments, the cell or cells exhibit increased expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 10 or 11.

[0009] In one aspect the present disclosure provides a method of producing rAAV, the method comprising transducing or infecting an engineered cell or cell line that that has been engineered to reduce aggregation with one or more plasmids or helper viruses.

[0010] In one aspect the present disclosure provides a cell culture comprising an engineered cell or cell line that that has been engineered to reduce aggregation, wherein the engineered cell or cell line has been transduced of infected with one or more plasmids or helper viruses.

BRIEF DESCRIPTION OF THE DRAWING

[0011] Figure 1 shows a graphical overview of an exemplary method that was used to design a rAAV sgRNA library targeting genes in the human genome.

[0012] Figure 2 shows a graphical overview of an exemplary pooled CRISPR screening method that was used to identify genes that affect rAAV titer in HEK293 P1B2 suspension cells. [0013] Figure 3a shows RPM+1 of input libraries (x-axis) and libraries produced from one round of a pooled CRISPR screen (y-axis). As shown, there were 2 genome-wide libraries for CRISPRko (Al and Bl).

[0014] Figure 3b shows RPM+1 of input libraries (x-axis) and libraries produced from one round of a pooled CRISPR screen (y-axis). As shown, there were 2 genome-wide libraries for CRISPRa (Cl and DI).

[0015] Figure 4 shows a graphical overview of an exemplary arrayed CRISPR screening method that was used to identify genes that affect rAAV titer in HEK293T cells.

[0016] Figure 5 shows graphs of rAAV titer for HEK293T cells with different candidate gene knockouts.

[0017] Figure 6 shows a graph of rAAV titer for HEK293T cells with different candidate genes activated.

[0018] Figure 7 shows a graph of rAAV titer for HEK293 P1B2 cells with different candidate gene knockouts.

[0019] Figure 8a shows a graph of rAAV titer for HEK293 P1B2 cells with different candidate gene or gene combination knockouts.

[0020] Figure 8b shows a graph of rAAV titer for HEK293T cells with different candidate gene or gene combination knockouts.

[0021] Figure 9a shows the fold change in viable cell number of Accumax treated cells to untreated cells in either OCLN knockout or control cells.

[0022] Figure 9b shows images of HEK293 cell aggregation with or without OCLN knocked out and with or without Accumax exposure.

[0023] Figure 9c shows a graphic of the role OCLN plays in cell-to-cell adhesion. It also shows the role played by other tight junction molecules that could be targeted similarly to OCLN to mitigate cell aggregation. [0024] Figure 10a shows a graphic of titer vs. viable cell density (VCD) for wildtype HEK293 cells. Cell mass was enumerated via optical cell counting using a Vi-CELL™ XR cell counter.

[0025] Figure 10b shows a graphic of titer vs. LDH (Lactate dehydrogenase) concentration for wild- type HEK293 cells. Cell mass was evaluated using LDH measured on a CEDEX BioHT®, which is proportional to TCD (or total cell mass) present in a sample.

[0026] Figure Ila shows a graph of rAAV titer for a commercial HEK293T cell line and engineered HEK 293 P1B2-OCLN knockout pool.

[0027] Figure 11b shows a schematic overview of an engineered cell line evaluation workflow and a comparison in cell aggregation between engineered cell line HEK293 P1B2 and engineered HEK 293 P1B2-OCLN knockout pool.

[0028] Figure 12a shows graphs of rAAV titer for HEK293 cells with different candidate gene knockouts.

[0029] Figure 12b shows graphs of rAAV titer for engineered HEK293 cells with different candidate gene knockouts.

[0030] Figure 13a shows graphs of rAAV titer for HEK293 cells with different candidate gene knockouts.

[0031] Figure 13b shows graphs of rAAV titer for HEK293 cells with different candidate gene knockouts.

[0032] Figure 13c shows graphs of rAAV titer for engineered HEK293 cells with different candidate gene knockouts.

[0033] Figure 13d shows graphs of rAAV titer for engineered HEK293 cells with different candidate gene knockouts.

DEFINITIONS [0034] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended claims. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0035] As used herein, “modulation” or “modulate” refers to the alteration of the regulation, expression or activity of a gene and/or gene product, e.g., a protein or non-coding RNA. Modulation may be increasing, reducing (decreasing), or eliminating the expression and/or activity of one or more endogenous genes and/or gene product. In cases where multiple genes and/or gene products are modulated, all the expression and/or activity of genes and/or gene products may be increased, or all the expression and/or activity of genes and/or gene products may be decreased, or one or more of the genes and/or gene products may be increased while one or more of the other genes and/or gene products may be decreased.

[0036] As used herein, the term “cell” refers to any cell or cells capable of producing a recombinant adeno-associated virus (rAAV). In some embodiments, the cell is a mammalian cell, for example, a HeLa cell, a COS cell, a HEK293 cell, a A549 cell, a BHK cell, or a Vero cell. The term “cell line” refers to a clonal population of cells able to continue to divide and not undergo senescence. Unless otherwise indicated, the terms “cell” or “cell line” are understood to include modified or engineered variants of the indicated cell or cell line.

[0037] As used herein, the terms “engineered cells” or “engineered cell line” refer to cells or cell lines that have been modified by one or more means to modulate e.g., to reduce, eliminate or increase) the expression or other properties (e.g. , biological activity) of one or more endogenously expressed genes and/or gene products (e.g., those in Tables 2-9 or in Tables 10 or 11) so as to augment the production of rAAV.

[0038] As used herein, the term “control parental cells” refer to cells that have not been modified by one or more means to modulate the expression or other properties (e.g., biological activity) of one or more endogenously expressed genes and/or gene products (e.g. , those in Tables 2-9 or in Tables 10 or 11) so as to augment the production of rAAV. For example, in some embodiments, depending on the parental cell that was used to generate the engineered cell or cell line, a “control parental cell” can be an HEK293 cell, an HEK293T cell, an HEK293 P1B2 cell, a TFS HEK293 cell, etc. that has not been modified.

[0039] As used herein, the term “control parental cell line” refers to a clonal population of control parental cells able to continue to divide and not undergo senescence.

[0040] “Lysis” refers to the breaking down of the cell, often by viral, enzymatic, or osmotic mechanisms that compromise its integrity. A “lysed cell” is a cell that has undergone substantial lysis. As used herein, the term “lysate” refers to a fluid containing the contents of lysed cells.

[0041] As used herein, the term “higher titer” signifies an increased titer in comparison to titer produced by a control parental cell line and/or control parental cell.

[0042] As used herein, the term “cell culture supernatant” refers to the cell culture media in which cells are suspended and/or cultured.

[0043] As used herein, the term “gene” refers to a transcription unit and regulatory regions that are adjacent (e.g., located upstream and downstream), and operably linked, to the transcription unit. A transcription unit is a series of nucleotides that are transcribed into an RNA molecule. A transcription unit may include a coding region. A “coding region” is a nucleotide sequence that encodes an unprocessed preRNA (i.e., an RNA molecule that includes both exons and introns) that is subsequently processed to a messenger RNA (mRNA). A transcription unit may encode a non-coding RNA. A non-coding RNA is an RNA molecule that is not translated into a protein. Examples of non-coding RNAs include microRNA (miRNA). Proteins and non-coding RNA molecules that are encoded by a gene are examples of “gene products.” The boundaries of a transcription unit are generally determined by an initiation site at its 5’ end and a transcription terminator at its 3’ end. A “regulatory region” is a nucleotide sequence that regulates expression of a transcription unit to which it is operably linked. Non- limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, transcription terminators, and polyadenylation (polyA) signals. A regulatory region located upstream of a transcription unit may be referred to as a 5’ UTR, and a regulatory region located downstream of a transcription unit may be referred to as a 3’ UTR. A regulatory region may be transcribed and be part of an unprocessed preRNA.

[0044] In the context of this document, the term “target” or “target gene” refers to any gene, including protein-encoding genes and genes encoding non-coding RNAs (e.g., miRNA), that when modulated alters some aspect of rAAV production.

[0045] With regard to gene designations, single genes have often been denoted by multiple symbols. In the context of this document, gene symbols, whether they be human or non-human, may be designated by either upper-case or lower case letters. Neither the use of one particular symbol nor the adoption of lower or upper case symbols is intended to limit the scope of the gene in the context of these disclosures. All gene identification numbers identified herein (GenelD) are derived from the National Center for Biotechnology Information “Entrez Gene” web site unless identified otherwise.

[0046] The term “about” is used herein to mean approximately, in the region of, roughly or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending, within permissible value ranges, the boundaries above and/or below the numerical values set forth.

[0047] As used in the present disclosure, whether in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least.” When used in the context of a method, the term “comprising” means that the method includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components, but may also include additional features or components.

[0048] For the purposes of promoting an understanding of the embodiments described herein, reference made to preferred embodiments and specific language is used to describe the same. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure. As used throughout this disclosure, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents and other references mentioned herein are incorporated by reference.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0050] The present disclosure describes engineered cells and cell lines for producing recombinant adeno-associated virus (rAAV) in which expression and/or activity of one or more endogenous genes and/or proteins is modulated. In some embodiments, the modulation of gene expression and/or activity results in an increased rAAV titer yield compared to a cell line in which expression and/or activity of the one or more endogenous genes and/or proteins is not modulated. In some embodiments, the modulation of gene expression and/or activity results in an improved phenotype for rAAV production, e.g., reduced aggregation compared to a cell line in which expression and/or activity of the one or more endogenous genes and/or proteins is not modulated.

Adeno-Associated Virus (AAV)

[0051] AAV is a small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a lower immune response compared to other viruses. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. AAV includes numerous serologically distinguishable types including serotypes AAV1 to AAV12, as well as many more from nonhuman primates. AAV is non-autonomously replicating, and has a life cycle with a latent phase and an infectious phase. In the latent phase, after a cell is infected with an AAV, the AAV site-specifically integrates into the host’s genome as a provirus. The infectious phase does not occur unless the cell is also infected with a helper virus (for example, adenovirus or herpes simplex virus), which allows the AAV to replicate.

[0052] The wild-type AAV genome contains two inverted terminal repeats (ITRs), which contain signal sequences directing AAV replication, genome encapsidation and integration. In addition to the ITRs, promoters drive expression of two open reading frames encoding rep and cap genes. Two rep promoters, coupled with differential splicing of the single AAV intron, result in the production of four rep proteins (Rep78, Rep68, Rep52, and Rep40) from the rep gene. Rep proteins are responsible for genomic replication. The cap gene encodes three capsid proteins (VP1, VP2, and VP3) which are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0053] Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the internal genome may be replaced with foreign DNA, for example, an expression cassette for an exogenous protein or non-coding RNA of interest. In this case, the rep and cap proteins are provided in trans on, for example, a plasmid. In order to produce a recombinant AAV (rAAV) vector, a cell line permissive of AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions, for example adenoviral genes Ela, Elb, E2a, E4orf6, and VA RNA. Numerous mammalian cell types are suitable for producing rAAV vectors, including HeLa cells, COS cells, HEK293 cells, A549 cells, BHK cells, and Vero cells. rAAV vectors are typically produced in these cell types by transfecting the cells with one plasmid containing the ITR-flanked expression cassette (proviral plasmid), and one or more additional plasmids providing the additional AAV (packaging plasmid) and helper virus genes (adenoviral helper plasmid). Unlike wild-type AAV, rAAV are generally non-integrating and the recombinant AAV genome persists outside the host cell genome.

[0054] The genome of wild-type AAV is single- stranded DNA and is 4.7 kb. AAV vectors may have single-stranded genomes that are 4.7 kb in size, or are larger or smaller than 4.7 kb, including oversized genomes that are as large as 5.2 kb, or as small as 3.0 kb. Further, vector genomes may be substantially self-complementary, so that within the virus the genome is substantially double stranded. AAV vectors containing genomes of all types are suitable for use in the method of the instant disclosure.

[0055] As discussed above, AAV requires co-infection with a helper virus in order to enter the infectious phase of its life cycle. Helper viruses include any virus capable of creating and allowing AAV replication. Helper viruses include adenovirus and herpes simplex virus (HSV). Adenovirus is a nonenveloped nuclear DNA virus with a doublestranded DNA genome of approximately 36 kb. Adenovirus is capable of rescuing latent AAV provirus in a cell, by providing Ela, Elb55K, E2a, E4orf6, and VA genes, and allowing AAV replication and encapsidation. HSV is a family of viruses that have a relatively large double-stranded linear DNA genome encapsidated in an icosahedral capsid, which is wrapped in a lipid bilayer envelope. HSV are infectious and highly transmissible. The following HSV1 replication proteins were identified as necessary for AAV replication: the helicase/primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene, with other proteins enhancing the helper function.

Production of rAAV

[0056] In general terms, to allow for production of rAAV, the cell is provided with AAV ITRs flanking an expression cassette, AAV rep and cap gene functions, as well as additional helper functions. These may be provided to the cell using any number of appropriate plasmids or vectors. Additional helper functions can be provided by, for example, an adenovirus infection, by an adenoviral helper plasmid that carries all of the required adenoviral helper function genes, or by other viruses such as HSV. Any genes, gene functions, or genetic material necessary for rAAV production by the cell may transiently exist within the cell, or be stably inserted into the cell genome. It is to be understood that the engineered cells of the present disclosure may be used in any such method for production of rAAV.

[0057] In some embodiments, rAAV vectors are produced by transfection of an engineered cell or cell line of the present disclosure. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid. In some embodiments, the engineered cell or cell line is transfected with at least an adenoviral helper plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a proviral plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid and an adenoviral helper plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid and a proviral plasmid. In some embodiments, the engineered cell or cell line is transfected with at least an adenoviral helper plasmid and a proviral plasmid. In some embodiments, rAAV vectors are produced by transfection of an engineered cell or cell line of the present disclosure with a packaging plasmid, an adenoviral helper plasmid, and a proviral plasmid. It is to be understood that the present disclosure also encompasses methods where one or more of these plasmids are combined into a single plasmid, e.g. , where the packaging and adenoviral helper plasmids are combined into a single plasmid and a dual transfection is used instead of a traditional triple transfection.

[0058] In some embodiments, rAAV vectors are produced by infection of an engineered cell or cell line of the present disclosure. In some embodiments, the engineered cell or cell line are infected with a helper virus (e.g. , an adenovirus or herpes simplex virus), which allows the rAAV to replicate.

Packaging plasmid

[0059] In some embodiments, a packaging plasmid comprises nucleic acid sequences for AAV rep and cap genes. In some embodiments, a rep gene encodes for the Rep78, Rep68, Rep52, and Rep40 proteins. In some embodiments, a cap gene encodes for VP1, VP2, and VP3 proteins which form the capsid of the AAV particle. In some embodiments, an engineered cell used for rAAV production has nucleic acid sequences encoding AAV rep and cap proteins integrated in the cell genome and the method of rAAV production does not involve transfection with a packaging plasmid.

[0060] AAV of any serotype may be used in the present disclosure. AAV serotypes generally have different tropisms to infect different cells or tissues. In some embodiments, an AAV serotype is selected based on a tropism for a particular cell type or tissue type.

[0061] In some embodiments, an rAAV may comprise or be based on a serotype selected from any of the following serotypes, and variants thereof, including, but not limited to: AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.4O, AAV 12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.ll, AAV16.3, AAV16.8/hu.lO, AAV161.1O/hu.6O, AAV161.6/hu.61, AAVl-7/rh.48, AAVl-8/rh.49, AAV2, AAV2.5T, AAV2- 15/rh.62, AAV223. 1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2- 3/rh.61, AAV24.1, AAV2-4/rh.5O, AAV2-5/rh.51, AAV27.3, AAV29.3/bb. 1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3A, AAV3B, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-1 l/rh.53, AAV3-3, AAV33.12/hu.l7, AAV33.4/hu.l5, AAV33.8/hu.l6, AAV3-9/rh.52, AAV3a, AAV3b, AAV4, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42- 15, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42- 5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r 11.64, AAV4-8/rh.64, AAV4-9/rh.54, AAV5, AAV52.1/hu.2O, AAV52/hu.l9, AAV5- 22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2, AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.SRl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAV-h, AAVH-1/hu.l, AAVH2, AAVH- 5/hu.3, AAVH6, AAVhEl.l, AAVhER1.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.l, AAVhu.10, AAVhu.ll, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.6O, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.68, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLKO3, AAV-LKO3, AAV- LK04, AAV-LKO5, AAV- LK06, AAV-LK07, AAV-LKO8, AAV-LK09, AAV-LK1O, AAV- LK11, AAV-LK12, AAV- LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV- LK19, AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV- PAEC12, AAV-PAEC2, AAV- PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.l, AAVpi.2, AAVpi.3, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.6O, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, BAAV, B P61 AAV, B P62 AAV, B P63AAV, bovine AAV, caprine AAV, Japanese AAV10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK 16, AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV-PHP.B, AAV-PHP.N, AAV-PHP.S, AAVrh.74, AAV-HSC 1-17, AAV-CBr, AAV-CLv, AAV-CLg, and/or AAV.CAP-B1 to AAV.CAP-B25.

[0062] In certain embodiments, an rAAV comprises an AAV2 serotype or a variant thereof. In certain embodiments, an rAAV comprises an AAV5 serotype or a variant thereof. In certain embodiments, an rAAV comprises an AAV8 serotype or a variant thereof. In certain embodiments, an rAAV comprises an AAV9 serotype or a variant thereof. In certain embodiments, an rAAV comprises AAVhu.68 serotype or a variant thereof. In certain embodiments, an rAAV comprises AAVrh.lO serotype or a variant thereof.

[0063] As is well known in the art, a “variant” of an AAV serotype might include one or more point mutations relative to the wild-type sequence that modify the tropism and/or reduce the immunogenicity of the rAAV. Additionally or alternatively, a variant might include a peptide, e.g., a 7-10 amino acid peptide inserted within a hypervariable region and/or surface-exposed loop of the capsid protein. [0064] In some embodiments, a rep gene comprises any one of SEQ ID NOs: 1-2 and a cap gene comprises any one of SEQ ID NOs: 3-5 shown in Table 1. In some embodiments, a rep gene comprises any one of SEQ ID NOs: 1-2 and a cap gene comprises SEQ ID NO: 3. In some embodiments, a rep gene comprises any one of SEQ ID NOs: 1-2 and a cap gene comprises SEQ ID NO: 4. In some embodiments, a rep gene comprises any one of SEQ ID NOs: 1-2 and a cap gene comprises SEQ ID NO: 5.

Table 1 : Exemplary rep and cap sequences

Adenoviral helper plasmid

[0065] In some embodiments, an adenoviral helper plasmid comprises nucleic acid sequences encoding adenoviral helper functions. In some embodiments, an adenoviral helper plasmid comprises nucleic acid sequences encoding, for example, E2a, E4orf6, and/or VA RNA. In some embodiments, an adenoviral helper plasmid comprises nucleic acid sequences encoding, for example, Ela, Elb55K, E2a, E4orf6, and VA RNA. In some embodiments, an engineered cell used for rAAV production has nucleic acid sequences encoding adenoviral helper functions integrated in the cell genome and the method of rAAV production does not involve transfection with an adenoviral helper plasmid. It is to be understood that helper functions from any adenovirus type may be used, e.g., Ad2 or Ad5 and a person of skill in the art will be able to identify adenovirus types suitable for the production of their desired recombinant rAAV vector.

Proviral plasmid

[0066] In some embodiments, a proviral plasmid comprises nucleic acid sequences encoding a payload (e.g. , a cDNA expression cassette for a transgene of interest, DNA encoding a CRISPR sgRNA, etc.) flanked by AAV inverted terminal repeats (ITRs). In some embodiments, a proviral plasmid further comprises nucleic acid sequences encoding regulatory sequences, e.g., promoters, introns, enhancers, etc. to regulate expression of the payload in the cells or tissue of interest. In some embodiments, an engineered cell used for rAAV production has nucleic acid sequences encoding the payload flanked by AAV inverted terminal repeats (ITRs) integrated in the cell genome and the method of rAAV production does not involve transfection with a proviral plasmid.

Engineered cells and cell lines

[0067] In one aspect, the present disclosure provides engineered cells and cell lines comprising a cell or cells in which expression and/or activity of one or more endogenous genes and/or gene products is reduced compared to control parental cells. For example, expression and/or activity of one or more of the genes and/or gene products in Table 2 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 3 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 4 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 5 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 6 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 7 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 8 is reduced compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 9 is reduced compared to control parental cells. In some embodiments, expression and/or activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 genes and/or gene products in Tables 2, 3, 4, 5, 6, 7, 8 or 9 is reduced.

[0068] In one aspect, the present disclosure provides engineered cells and cell lines comprising a cell or cells in which expression and/or activity of one or more endogenous genes and/or gene products is eliminated compared to control parental cells. For example, expression and/or activity of one or more of the genes and/or gene products in Table 2 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 3 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 4 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 5 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 6 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 7 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 8 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes or gene products in Table 9 is eliminated compared to control parental cells. In some embodiments, expression and/or activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 genes and/or gene products in Tables 2, 3, 4, 5, 6, 7, 8 or 9 is eliminated.

Table 2: Exemplary target genes for reduced or eliminated expression

Table 3: Exemplary target genes for reduced or eliminated expression

Table 4: Exemplary target genes for reduced or eliminated expression

Table 5: Exemplary target genes for reduced or eliminated expression

Table 6: Exemplary target genes for reduced or eliminated expression

Table 7 : Exemplary target gene for reduced or eliminated expression Table 8: Exemplary target genes for reduced or eliminated expression

Table 9: Exemplary target genes for reduced or eliminated expression in cells with an OCLN knockout

[0069] In one aspect, the present disclosure provides engineered cells and cell lines comprising a cell or cells in which expression and/or activity of one or more endogenous genes and/or gene products is increased compared to control parental cells. For example, expression and/or activity of one or more of the genes and/or gene products in Table 10 is increased compared to control parental cells. In some embodiments, expression and/or activity of one or more of the genes and/or gene products in Table 11 is increased compared to control parental cells. In some embodiments, expression and/or activity of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 genes and/or gene products in Table 10 or 11 is increased.

Table 10: Exemplary target genes for increased expression

Table 11: Exemplary target genes for increased expression

[0070] In some embodiments, the engineered cells and cell lines of the present disclosure are a mammalian cell or cell line (e.g. , a HeLa, COS, HEK293, A549, BHK, or Vero cell or cell line). In some embodiments, the engineered cells and cell lines of the present disclosure are in an adherent form. In some embodiments, the engineered cells and cell lines of the present disclosure are in a suspension form.

[0071] In some embodiments, the method of rAAV production involves transfecting an engineered cell or cell line with an adenoviral helper plasmid, a packaging plasmid and a proviral plasmid.

[0072] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding AAV rep and cap proteins integrated in the cell genome. In some such embodiments, the method of rAAV production involves transfection with an adenoviral helper plasmid and a proviral plasmid. In some such embodiments, the method of rAAV production involves transfection with a proviral plasmid and infection with a helper virus, e.g., an adenovirus or herpes simplex virus (HSV).

[0073] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding adenoviral helper functions integrated in the cell genome. In some such embodiments, the method of rAAV production involves transfection with a packaging plasmid and a proviral plasmid.

[0074] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding the payload flanked by AAV inverted terminal repeats (ITRs) integrated in the cell genome. In some such embodiments, the method of rAAV production involves transfection with an adenoviral helper plasmid and a packaging plasmid. In some such embodiments, the method of rAAV production involves transfection with a packaging plasmid and infection with a helper virus, e.g., an adenovirus or herpes simplex virus (HSV).

[0075] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding AAV rep and cap proteins and adenoviral helper functions integrated in the cell genome. In some such embodiments, the method of rAAV production involves transfection with a proviral plasmid.

[0076] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding AAV rep and cap proteins and the payload flanked by AAV inverted terminal repeats (ITRs) integrated in the cell genome. In some such embodiments, the method of rAAV production involves transfection with an adenoviral helper plasmid or infection with a helper virus, e.g. , an adenovirus or herpes simplex virus (HSV).

[0077] In some embodiments, an engineered cell or cell line used for rAAV production has nucleic acid sequences encoding adenoviral helper functions and the payload flanked by AAV inverted terminal repeats (ITRs) integrated in the cell genome. In some embodiments, the method of rAAV production involves transfection with a packaging plasmid.

[0078] In certain embodiments, the present disclosure provides a method of producing engineered cells or cell lines to promote increased production of rAAV. In some embodiments, the rAAV titer is increased at least 1.5 fold (e.g., at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, or 20 fold) compared to the rAAV titer produced by a cell line without the modulation of expression and/or activity of the corresponding gene(s) and/or gene product(s). In some embodiments, the rAAV titer is increased by up to 20 fold (e.g., up to 15 fold, 10 fold, 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold, or 2 fold) compared to the rAAV titer produced by a cell line without the modulation of expression and/or activity of the corresponding gene(s) and/or gene product(s).

[0079] In some embodiments, the present disclosure provides a method of producing engineered cells, cell lines, and/or knock out pools to promote increased production of rAAV. In some embodiments an engineered cell line comprises dual-knockout pools. In some embodiments, an engineered cell line comprises an eliminated OCLN gene and candidate gene (e.g., dual knockout).

Methods of Modulating One or more Genes and/or Gene Products

[0080] Modulating (e.g., reducing, eliminating or increasing) the expression or activity of a gene and/or gene product can be achieved by different mechanisms, including, but not limited to, altering one or more of the following: 1) gene copy number, 2) transcription or translation of a gene, 3) transcript stability or longevity, 4) the number of copies of an mRNA or miRNA, 5) the availability of a non-coding RNA or non-coding RNA target site, 6) the position or degree of post-translational modifications on a protein, or 7) the activity of a protein. Tools that can be used to permanently or transiently modulate gene expression and/or activity include but are not limited to a nuclease, a transcriptional repressor, a transcriptional activator, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), an antisense RNA oligonucleotide (ASO), a gene disruption, or a partial or complete gene deletion. The described tools can be used to target coding or regulatory regions of the gene, e.g., promoters, protein-encoding regions (exons), introns, 5’ and 3’ UTRs, and more. In some embodiments the modulation is permanent. In some embodiments the modulation is transient. In some embodiments the modulation is inducible. In some embodiments the modulation is reversible.

Nucleases

[0081] In certain embodiments, gene modulation is achieved using zinc finger nucleases (ZFNs). Synthetic ZFNs are composed of a zinc finger binding domain fused with, e.g. , a FokI DNA cleavage domain. ZFNs can be designed for editing the genome of a cell, including to knockout gene expression and/or activity. Meganucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (e.g., Cas nucleases) can also be used for the same purpose. CRISPR-based systems are discussed in more detail in a later section.

Transcriptional activators and repressors

[0082] In certain embodiments, gene modulation is achieved using a transcriptional activator or repressor. Transcriptional activators or repressors can be composed of a DNA binding domain (e.g., a zinc finger binding domain, a transcription activator-like effector (TALE) or an inactivated or “dead” CRISPR associated (dCas) protein) that is fused or non- covalently associated with one or more transcriptional activation or repression domains. A non-limiting list of transcriptional activation domains includes fragments of transcription regulatory domains and fragments of domains having transcription regulation function of VP16, VP64, VP160, EBNA2, E1A, Gal4, Oafl, Leu3, Rtg3, Pho4, Gln3, Gcn4, Gli3, Pip2, Pdrl, Pdr3, Lac9, Teal, p53, p65, NFAT, Spl (e.g. , Spla), AP-2 (e.g., Ap-2a), Sox2, NF-KB, MLL/ALL, E2A, CREB, ATF, FOS/JUN, HSF1, KLF2, NF-1L6, ESX, Octi, Oct2, SMAD, CTF, HOX, Sox2, Sox4, Rta, VPR, RpoZ, or Nanog. In some embodiments, the transcriptional activator comprises the transcriptional activation domains in VPR, namely VP64, p65 and Rta. A non-limiting list of transcriptional repression domains includes fragments of transcription regulatory domains and fragments of domains having transcription regulation function of Mxil, Tbx3, KRAB (Kruppel-associated box), EnR, or SID, SID4X (a tandem repeat of four SID domains linked by short peptide linkers), PIE-1, and IAA28-RD. The described reagents can be used to target promoters, protein-encoding regions (exons), introns, 5’ and 3’ UTRs, and more. CRISPR-based systems are discussed in more detail in the next section. The described reagents can be transiently expressed in the engineered cell or cell line or can be integrated into the genome of the engineered cell or cell line. In some embodiments the reagents can be under the control of an inducible and/or reversible promoter so that expression can be controlled, e.g., at different points in the cell cycle.

CRISPR-based systems

[0083] In some embodiments, modulation of gene expression and/or activity in an engineered cell or cell line of the present disclosure is carried out using a CRISPR-based system. A CRISPR-based system typically comprises two distinct components: (1) a guide RNA (gRNA) and (2) a CRISPR associated (Cas) protein that can either be a nuclease e.g., Cas9 from S. pyogenes) or inactivated or “dead” as a result of mutations in the nuclease domain(s) of the Cas protein (e.g., dCas9 from .S’, pyogenes with D10A and H840A mutations). The guide RNA for certain CRISPR-based systems, e.g. , Cas9-based systems is composed of (1) a transactivating crRNA (tracrRNA) and (2) a crRNA where an about 20 nucleotide “guide” portion of the 5 ’ end of the crRNA is complementary to a target DNA sequence in the gene for modulation and there is a “duplex” region of complementarity between a 5’ portion of the crRNA and a 3’ portion of the tracrRNA. In some embodiments, the separate tracrRNA and crRNA components can be replaced by a single-guide RNA (sgRNA) that includes a covalent linkage between the tracrRNA and crRNA components, e.g. , an additional loop of nucleotides connecting the 3’ end of the crRNA to the 5’ end of the tracrRNA. The guide RNA for certain other CRISPR-based systems, e.g. , Casl2a-based systems is composed of a single crRNA component (i.e., no tracrRNA component) where an about 20 nucleotide “guide” portion of the 3 ’ end of the crRNA is complementary to a target DNA sequence in the gene for modulation.

[0084] The gRNA/Cas complex is recruited to the target sequence by base-pairing between the “guide” portion of the gRNA and the target DNA sequence in the gene for modulation. For successful binding of the Cas protein, the target DNA sequence typically needs to be adjacent to the correct Protospacer Adjacent Motif (PAM) sequence. The binding of the gRNA/Cas complex localizes the Cas protein to the target DNA sequence in the one or more endogenous genes of the present disclosure.

[0085] When the Cas protein is a nuclease (e.g., Cas9 or Casl2a) it can cut both strands of the DNA causing a double strand break. This can be repaired through one of two general repair pathways: (1) the non-homologous end joining DNA repair pathway or (2) the homology directed repair pathway. The non-homologous repair pathway can result in insertions/deletions at the double strand break that can lead to frameshifts and/or premature stop codons, effectively disrupting the open reading frame of the target gene. The homology directed repair pathway requires the presence of a repair template, which is used to repair the double strand break.

[0086] When the Cas protein is inactivated or “dead” (e.g., dCas9 or dCasl2a) it can bind but not cut the target DNA sequence in the gene for modulation. In some embodiments, the dCas protein is fused or non-covalently associated with one or more transcriptional activation or repression domains as described above and can thereby modulate (reduce when a transcriptional repression domain is present or increase when a transcriptional activation domain is present) the expression or activity of a gene and/or gene product.

[0087] Any appropriate gRNA may be used for a CRISPR -based system. gRNAs can be designed using known techniques and based on a knowledge of the sequence of the one or more endogenous genes to be modulated, typically using any publicly available appropriate computer program. Knockout engineered cells or cell lines may be generated using any appropriate technique, with standard techniques being known in the art and suitable kits being commercially available. [0088] gRNAs and Cas proteins can be delivered to an engineered cell or cell line of the disclosure by any appropriate means. Suitable techniques are known in the art and include the use of the gRNA/Cas complex (RNP) itself, vector/plasmid-based expression systems that encode the gRNA and/or Cas protein and lipid-based systems that encapsulate or conjugate the gRNA and/or mRNA encoding the Cas protein. In some embodiments, nucleic acid sequences encoding the gRNA and/or Cas protein can be integrated into the genome of the engineered cell or cell line.

[0089] In some embodiments, gRNAs may be modified to enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation. In some embodiments, these modifications may include replacing certain ribonucleotides with deoxyribonucleotides within the gRNA sequence, e.g., within the guide sequence. In some embodiments, these modifications may include using 2’-O-methyl (2’0Me) analogs and/or 5’ or 3’ phosphorothioate (PS) internucleotide linkages in the terminal one to three nucleotides on both 5’ and 3’ ends of the gRNA or within certain internal regions of the gRNA.

Double-stranded RNA (dsRNA) molecules

[0090] In certain embodiments, double-stranded RNA (dsRNA) molecules may be used to modulate expression and/or activity of one or more endogenous genes and/or gene products in an engineered cell or cell line of the present disclosure. dsRNA molecules can be designed to inhibit one or more endogenous genes by sequence homology-based targeting of the corresponding RNA sequence. Such dsRNAs can be small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), or micro-RNAs (miRNAs). The sequence of such dsRNAs will comprise a complementary portion of the mRNA encoding the one or more endogenous genes to be modulated. This portion can be 100% complementary to the target portion within the mRNA, but lower levels of complementarity (e.g., 90% or more or 95% or more) can also be used. Typically the percent complementarity is determined over a length of contiguous nucleic acid residues. A dsRNA molecule of the disclosure may, for example, have at least 80% complementarity to the target portion within the mRNA measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or more nucleotides. In some instances dsRNA molecule has at least 80% complementarity to the target portion of mRNA over the entire length of the dsRNA molecule.

[0091] One exemplary gene targeting reagent that uses RNA interference (RNAi) pathways are small hairpin RNAs, also referred to as shRNAs. shRNAs delivered to cells via, e.g. , expression constructs (e.g. , plasmids, lentiviruses) have the ability to provide long term reduction of gene expression in a constitutive or regulated manner, depending upon the type of promoter employed. In one embodiment, the genome of a lentiviral particle is modified to include one or more shRNA expression cassettes that target a gene (or genes) of interest. Such lentiviruses can infect a cell, stably integrate their viral genome into the host genome, and express a shRNA in a constitutive, regulated, or (in the case where multiple shRNAs are being expressed) constitutive and regulated fashion.

[0092] In embodiments described herein, gene targeting reagents including small interfering RNAs (siRNA) as well as microRNAs (miRNA) can also be used to modulate gene function. siRNAs and miRNAs can incorporate a wide range of chemical modifications, levels of complementarity to the target transcript of interest, and designs to enhance stability, cellular delivery, specificity, and functionality. In addition, such reagents can be designed to target diverse regions of a gene (including the 5 ’ UTR, the open reading frame, or the 3’ UTR of the mRNA), or (in some cases) the promoter/enhancer regions of the genomic DNA encoding the gene of interest. Gene modulation (e. ., reduction of gene expression and/or activity, knockdown) can be achieved by introducing into the engineered cell or cell line a single siRNA or miRNA or multiple siRNAs or miRNAs targeting different regions of the same mRNA transcript. Synthetic siRNA/miRNA delivery can be achieved by any number of methods including but not limited to self-delivery of the siRNA/miRNA itself, vector/plasmid-based expression systems that encode the siRNA/miRNA and lipid- based systems that encapsulate or conjugate the siRNA/miRNA. In some embodiments, nucleic acid sequences encoding the siRNA/miRNA can be integrated into the genome of the engineered cell or cell line.

Antisense RNA oligonucleotide (ASO)

[0093] Antisense RNA oligonucleotide (ASO), can be used to modulate expression and/or activity of one or more endogenous genes and/or gene products in an engineered cell or cell line of the present disclosure. Typically, ASOs are used to reduce expression and/or activity of one or more endogenous genes. Using known techniques and based on a knowledge of the sequence of the one or more endogenous genes to be modulated, ASO molecules can be designed to inhibit the one or more endogenous genes by sequence homology-based targeting of the corresponding RNA. The ASO sequence can comprise a nucleotide sequence that is complementary to a target portion of the mRNA or IncRNA produced from the one or more endogenous genes. This portion can be 100% complementary to the target portion within the mRNA or IncRNA but lower levels of complementarity (e.g., 90% or more or 95% or more) can also be used.

[0094] In some embodiments, the ASO can be an antisense RNA oligonucleotide wherein at least one nucleoside linkage of the sequence is a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, and an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, or a boranophosphate linkage. In a particular embodiment, at least one intemucleoside linkage of the antisense RNA oligonucleotide sequence is a phosphorothioate linkage. In some embodiments, all of the intemucleoside linkages of the antisense RNA oligonucleotide sequence are phosphorothioate linkages.

Modulation at protein level

[0095] In another embodiment, modulation of expression and/or activity of a gene and/or gene product takes place at the protein level. By way of example, reduction of gene function at the protein level can be achieved by methods including, but not limited to, targeting the protein with a small molecule, a peptide, an aptamer, destabilizing domains, or other methods that can, e.g. , down-regulate the activity or enhance the rate of degradation of the protein. Alternatively, the expressed protein may be modified to reduce or eliminate biological activity through site-directed mutagenesis and/or the incorporation of missense or nonsense mutations.

Modulation by delivering additional copies of the gene [0096] In another embodiment, expression and/or activity of a gene and/or gene product can be increased by integrating one or more exogenous copies of the gene into the genome of the engineered cell or cell line. In some embodiments, the one or more exogenous copies of the gene are integrated under the control of a strong promoter. In some embodiment the strong promoter is an endogenous promoter. In some embodiments the strong promoter is an exogenous promoter. In some embodiments the one or more exogenous copies of the gene can be under the control of an inducible and/or reversible promoter so that expression and/or activity can be controlled, e.g., at different points in the cell cycle.

[0097] In some embodiments, the one or more exogenous copies of the gene are integrated into the genome using a transposon system comprising a transposase and transposon donor vector. The transposase can be provided to the engineered cells or cell line via a vector/plasmid-based expression system or mRNA encoding the transposase. The transposon donor vector can be provided via a vector/plasmid comprising transposon terminal inverted repeats (TIRs). The one or more exogenous copies of the gene is cloned into the transposon donor vector between the TIRs. The engineered cells or cell line are cotransfected with these reagents and the one or more exogenous copies of the gene are excised from the transposon donor vector and integrated into the genome of the cells at a target transposon insertion site. Any suitable transposon system can be used including, without limitation, the piggyBac, Tol2, or Sleeping Beauty transposon systems.

[0098] In some embodiments, the one or more exogenous copies of the gene are integrated into the genome by homologous recombination using site-specific nucleases. For example, a construct can be integrated into a double-strand DNA break at the target locus by homology-directed repair. A DNA break may be created by a site-specific nuclease, such as, but not limited to zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (e.g. , Cas nucleases such as Cas9 or Casl2a). Any sitespecific nuclease that selectively cleaves a sequence at the target locus for integration of the construct may be used. The construct sequence to be integrated is flanked by a pair of homology arms responsible for targeting the construct to the target locus. A 5’ homology arm that hybridizes to a 5’ genomic target sequence and a 3’ homology arm that hybridizes to a 3 ’ genomic target sequence can be introduced into the construct. The homology arms are referred to herein as 5’ and 3’ (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms in the construct. The 5’ and 3’ homology arms hybridize to regions within the target locus where the construct is integrated, which are referred to herein as the “5 ’ target sequence” and “3 ’ target sequence,” respectively. In certain embodiments, the 5’ and 3’ target sequences flank the specific site for cleavage. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher targeting rate. A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.

[0099] In some embodiments, the one or more exogenous copies of the gene are integrated into the genome using a site-specific recombinase. A target locus for integration may include one or more transcriptionally active sites. Examples of transcriptionally active sites include DNasel hypersensitive sites (DHSs). A construct comprising the one or more exogenous copies of the gene can be site-specifically integrated into the genome by introducing a first recombination site into the construct and expressing a site-specific recombinase in the cell. The target site of the cell comprises a second recombination site, wherein recombination between the first and second recombination sites mediated by the site-specific recombinase results in integration of the construct at the target locus. The target locus may comprise either an endogenous recombination site or an engineered recombination site recognized by the site-specific recombinase. Various recombinases may be used for site-specific integration of vector constructs, including, but not limited to phi C31 phage recombinase, TP901-1 phage recombinase, and R4 phage recombinase.

[0100] In some embodiments, the one or more exogenous copies of the gene are not integrated into the genome of the engineered cell or cell line, and instead are maintained in the cell extrachromosomally. Examples of extrachromosomal constructs include those that persist as stable/persistent plasmids or episomal plasmids. In some embodiments, an extrachromosomal construct comprises Epstein-Barr virus (EBV) sequences, including the EBV origin of replication, oriP, and the EBV gene, EBNA1, to provide stable extrachromosomal maintenance and replication of the construct. For a description of methods of using EBV sequences to stably maintain vectors extrachromosomally. In some embodiments, the extrachromosomal constructs may be introduced into the engineered cell or cell line in manner similar to the transfection methods for production of rAAVs. In some embodiments, the extrachromosomal constructs may be introduced into the engineered cell or cell line via one of the plasmids used in rAAV production, e.g., the packaging plasmid or adenoviral helper plasmid.

[0101] It is to be understood that the present disclosure is not limited to the aforementioned methods for delivering additional copies of the gene and that any method than can deliver additional copies of the gene may be used whether it delivers them in a manner that integrates them into the genome or not.

Effect of Modulation on Expression and/or Activity of One or more endogenous genes and/or Gene Products

[0102] In certain embodiments, methods described in the present disclosure can be utilized to generate an engineered cell or cell line that produces higher titers of rAAV than a control parental cell line.

[0103] In certain embodiments, methods described in the present disclosure can be utilized to generate an engineered cell or cell line with an improved phenotype for rAAV production, e.g., a cell or cell line that is less prone to aggregation than a control parental cell line.

[0104] In certain embodiments, methods described in the present disclosure can result in a significant reduction or increase in expression and/or activity of one or more target genes (e.g., a reduction or increase of at least 5%, at least 10%, at least 20%). In certain embodiments, expression and/or activity of a target gene is reduced or increased by from about 20% to about 100% or from 40% to about 100% (for example, from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 40% to about 55%, from about 40% to about 50%, from about 40% to about 45%, from about 45% to about 100%, from about 50% to about 100%, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0105] In certain embodiments, methods described in the present disclosure can result in a significant reduction or increase in expression and/or activity of a gene product (e.g., a protein or non-coding RNA) expressed by one or more target genes (e.g., a reduction or increase of at least 5%, at least 10%, at least 20%). In certain embodiments, expression and/or activity is reduced or increased by from about 20% to about 100% or from 40% to about 100% (for example, from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 40% to about 55%, from about 40% to about 50%, from about 40% to about 45%, from about 45% to about 100%, from about 50% to about 100%, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0106] In certain embodiments, methods described in the present disclosure can result in a significant reduction or increase in the activity of a gene product (e.g., a protein or non-coding RNA) expressed by one or more target genes e.g., a reduction or increase of at least 5%, at least 10%, at least 20%). In certain embodiments, activity is reduced or increased by from about 20% to about 100% or from 40% to about 100% (for example, from about 40% to about 95%, from about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, from about 40% to about 60%, from about 40% to about 55%, from about 40% to about 50%, from about 40% to about 45%, from about 45% to about 100%, from about 50% to about 100%, from about 55% to about 100%, from about 60% to about 100%, from about 65% to about 100%, from about 70% to about 100%, from about 75% to about 100%, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, from about 95% to about 100%; or about 40%. about 50%. about 60%, about 70%, about 80%, about 90%, about 100%).

[0107] In certain embodiments, the modulation is maintained for at least about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or at least about 10 days.

[0108] In certain embodiments, the modulation is intended to be maintained indefinitely or permanently, e.g. through the use of a gene disruption or a partial or complete gene deletion or through stable integration or expression of certain exogenous reagents of the present disclosure.

[0109] In certain embodiments, modulation is maintained for at least one, at least two, at least three, at least four, at least five, at least ten, at least 20, at least 30, at least 40 or more passages of the engineered cell or cell line in culture.

Effect of Modulation on rAAV Production

[0110] Modulation of one or more endogenous genes and/or gene products in an engineered cell or cell line may result in an increase in the titer of rAAV. In some embodiments, modulation results in an increase in the titer of rAAV produced from the engineered cell or cell line is increased at least 1.5 fold, 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 15 fold or more. In some embodiments, modulation results in an increase in the titer of rAAV produced from the engineered cell or cell line is increased up to 20 fold, 15 fold, 10 fold, 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold or 2 fold. Any increase in the rAAV titer resulting from modulation of one or more endogenous genes and/or gene products can be compared with the rAAV titer produced from a control parental cell line.

[0111] In some embodiments, modulation of one or more endogenous genes and/or gene products in an engineered cell or cell line may increase the rAAV titer production for at least 2 days, at least 5 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days or more.

Methods of Producing rAAV

[0112] In some embodiments, rAAV vectors are produced by transfection of an engineered cell or cell line of the present disclosure. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid. In some embodiments, the engineered cell or cell line is transfected with at least an adenoviral helper plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a proviral plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid and an adenoviral helper plasmid. In some embodiments, the engineered cell or cell line is transfected with at least a packaging plasmid and a proviral plasmid. In some embodiments, the engineered cell or cell line is transfected with at least an adenoviral helper plasmid and a proviral plasmid. In some embodiments, rAAV vectors are produced by transfection of an engineered cell or cell line of the present disclosure with a packaging plasmid, an adenoviral helper plasmid, and a proviral plasmid. It is to be understood that the present disclosure also encompasses methods where one or more of these plasmids are combined into a single plasmid, e.g. , where the packaging and adenoviral helper plasmids are combined into a single plasmid and a dual transfection is used instead of a traditional triple transfection.

[0113] In some embodiments, rAAV vectors are produced by infection of an engineered cell or cell line of the present disclosure. In some embodiments, the engineered cell or cell line are infected with a helper virus e.g. , an adenovirus or herpes simplex virus), which allows the rAAV to replicate. In some embodiments, the engineered cell or cell line are infected with rAAV and transfected with a packaging plasmid and an adenoviral helper plasmid.

[0114] In some embodiments, rAAV vectors are produced using an engineered cell or cell line of the present disclosure that has been engineered to reduce aggregation. As described in the Examples, in some embodiments an engineered cell or cell line of the present disclosure, e.g. , an engineered HEK293 cell or cell line can be engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells (e.g. , by way of knockout of the one or more genes). In some embodiments, expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells. In some embodiments, the engineered cell or cell line cell, e.g. , engineered HEK293 cell or cell line is used in a method of production at a viable cell density (VCD) that exceeds 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL. In some embodiments, the engineered HEK293 cell or cell line remains in a non-aggregated form at a viable cell density (VCD) that exceeds 2.5xI0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL and the present disclosure provides a culture of these cells at a viable cell density (VCD) that exceeds 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL.

Methods of Harvesting rAAV

[0115] In some embodiments, rAAV may be obtained from an engineered cell or cell line by lysing the cells. Lysis can be accomplished by methods that chemically or enzymatically treat the cells in order to release rAAV. These methods include the use of nucleases such as benzonase or DNAse, proteases such as trypsin, or detergents or surfactants. Physical disruption, such as homogenization or grinding, or the application of pressure via a microfluidizer pressure cell, or freeze-thaw cycles may also be used. In certain embodiments, lysates from the cells can be used to harvest the rAAVs.

[0116] In come embodiments, cell culture supernatant may be collected from an engineered cell or cell line without the need for cell lysis. In certain embodiments of the present disclosure, the cells secrete rAAV that can be collected from the cell culture supernatant without the need for cell lysis.

[0117] After harvesting the rAAV, it may be necessary to purify the sample containing rAAV, to remove, for example, the cellular debris resulting from cell lysis. Methods of minimal purification of rAAV are known in the art. Two exemplary purification methods are Cesium chloride (CsCl)- and iodixanol-based density gradient purification. Minimal purification can also be accomplished using affinity chromatography. Purification may involve other downstream steps including steps designed to separate empty from full rAAVs, e.g. , using ion-exchange-based purification, optionally in combination with other purification methods. Following purification, rAAV may be filtered and stored.

Methods of Quantifying r AV

[0118] rAAVs can be quantified using a number of methods including quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), dot-blot hybridization, and by optical density of highly purified preparations. Viral genome amplification can also be measured using qPCR techniques similar to those described above.

[0119] rAAV s can also be quantified by any known functional assay. In some embodiments, a reporter gene, e.g., NanoLuc luciferase (Nluc), eGFP and/or mCherry is included in the rAAV payload for optical detection and quantification of rAAV s. For example, for functional assay titering of an rAAV expressing Nluc, a CHO-Lec2 cell-based assay can be used. CHO-Lec2 cells are plated in a multiwell plate and grown overnight. The following day, cell media is replaced to slow down cell growth, and a volume of crude AAV lysate is added. After transducing overnight, CHO-Lec2 cells are washed to remove contaminating Nluc transgene and cultured for an additional 48 hours. The amount of Nluc transgene expressed is then measured by optical detection.

[0120] In some embodiments, rAAVs can be quantified using an engineered cell or cell line of the present disclosure that has been engineered to reduce aggregation. As described in the Examples, in some embodiments an engineered cell or cell line of the present disclosure, e.g. , an engineered HEK293 cell or cell line can be engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells (e.g. , by way of knockout of the one or more genes). In some embodiments, expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells. In some embodiments, the engineered cell or cell line, e.g., engineered HEK293 cell or cell line is used in a method of quantifying rAAVs, e.g., where titer is measured using ddPCR and the quantification is repeated at more than one viable cell density (VCD). In some embodiments cell mass is obtained, e.g. , via cell counting. In some embodiments, these methods are performed across a series of VCD values, e.g., including values that exceed 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL. The ability to enumerate cell mass reliably without aggregation above 2.5x10⁶ vc/mL (when wild-type HEK293 cells tend to aggregate) is advantageous in that it enables more accurate determination of optimal VCD for production purposes. In some embodiments the titer at each value is used to identify an optimal VCD value or range for production or rAAV using the engineered cell or cell line, e.g., HEK293 cell or cell line. In some embodiments, the optimal VCD value exceeds 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL.

EXEMPLIFICATION

Example 1: rAAV sgRNA library design for pooled CRISPR screening method

[0121] The present example demonstrates a method for the design of a rAAV sgRNA library targeting genes in the human genome. A graphical overview of the method is depicted in Figure 1. sgRNAs for a CRISPRko (“CRISPR-knockout”) or CRISPRa (“CRISPR-activation”) screen were designed and subcloned into a proviral plasmid with the sgRNA (and an mCherry reporter transgene and WPRE sequence) under the control of a U6 promoter. Overall, 2 distinct libraries for each screen were produced, library Al and Bl for CRISPRko and library Cl and DI for CRISPRa, where each library encoded 4 unique sgRNAs for each of the about 19,000 genes in the human genome (for a total of 8 sgRNAs per gene across the two libraries). Each library also contained over 500 non-targeting control sgRNAs. These sgRNA library plasmids were combined with packaging and adenoviral helper plasmids for triple transfection into HEK293 P1B2 suspension cells. This triple transfection yields infective rAAV sgRNA libraries with rAAVs that each contain a sgRNA transgene that is a reflection of the sgRNA library used to produce them. rAAVs produced from these libraries were titered based on the mCherry reporter gene using fluorescence- activated cell sorting (FACS). Whole genome coverage and bias were determined by NGS of the rAAVs.

Example 2; Pooled CRISPR screening method [0122] The present example demonstrates a pooled CRISPR screening method that was used to identify genes in a HEK293 P1B2 suspension cell that affect rAAV titer. A graphical overview of the method is depicted in Figure 2. HEK293 P1B2 suspension cells stably expressing either Cas9 or dCas9-VPR were grown to a density of 8e5 cells/mL and transduced with one of the rAAV sgRNA libraries described in Example 1, at an MOI of 0.3 such that each sgRNA was packaged within 500-1000 rAAVs (functional titer). Cells in the CRISPRa screen were grown for 3 days, while cells in the CRISPRko screen were grown for 12 days. If cells became too confluent, cells were diluted to lower densities without discarding cell numbers.

[0123] After this growth period, cells were collected in 50 mL conical tubes, spun down at 300xg for 2 min, and the old growth media was discarded. Each cell pellet was gently washed 3x with suspension culturing media before recombining the cells and diluting to a density of 3e6 cells/mL (CRISPRa) or 6.5e6 cells/mL (CRISPRko).

[0124] A double transfection on rAAV-transduced cells was carried out by mixing packaging and adenoviral helper plasmids at a 0.5:2 molar ratio and combining them with polyethylenimine (PEI) in OptiMEM. After complexing for 5 min at room temperature, the transfection mix was added to rAAV-transduced cells such that the final concentration of rAAV transfection mix DNA and PEI was 6.7 ug/mL and 6 uL/mL, respectively. Cells were cultured for an additional 4 days with shaking at 110 rpm at 37 C and 10% CO2.

[0125] rAAV contained within cells (“pellet”) and the cell media (“supernatant”) were processed separately. To collect each fraction, cells were combined and pelleted at lOOOxg at 4 C for 10 min. After collection and reservation of the supernatant at 4 C, pellets were re-suspended in PBS, pH 7.4, 200 mM NaCl, and 0.001% pluronic F-68 and lysed through 3x rounds of freeze-thaw using a dry ice/ethanol bath. Lysate was clarified by spinning at 3220xg at 4 C for 10 min, and then treated with 50 U/mL benzonase + 2 mM MgC12 at 37 C for 45 min. Lysate was clarified again spinning at 2415xg at 4 C for 10 min.

[0126] rAAV from pellet and supernatant fractions was enriched with an affinity resin at a volume ratio of 17: 1 or 200: 1, respectively. rAAV bound to resin were incubated with rotation at room temperature for 90 min, followed by removal of the flow through and elution of rAAV from the resin using 20 mM sodium citrate, 400 mM NaCl, pH 2.5. rAAVs were neutralized with basic buffer, and buffer exchanged to PBS, pH 7.4, and 0.001% pluronic F-68 in an Amicon Ultra-15 100 kDa concentrator. Titers were monitored with qPCR using a Taqman probe targeting the WPRE sequence.

[0127] 1.5e9 vg of rAAV was used per reaction. rAAVs were treated with DNase

(Invitrogen, 0.025 U/uL final concentration) in lx DNase buffer (Invitrogen, AM8170G) at 37 C for 1 hour, followed by inactivation of the DNase by heating at 65 C for 10 min. rAAV s were broken apart by treatment with 0.4 units of proteinase K and heating for 1 hour at 50 C, followed by inactivation of the enzyme at 95 C for 20 min. The rAAV sgRNA genomic region was PCR amplified with KAPA HiFi polymerase, and the PCR product was purified and analyzed by gel electrophoresis. Samples were analyzed by NGS using NovaSeq technology.

[0128] RPM+1 ratios were calculated between the input rAAV and the rAAV generated after one round of screening to generate a value present extent of enrichment of each sgRNA after one round of selection. The magnitude of this enrichment and the reproducibility of this enrichment were considered during hit calling using both MAGeCK and t-test analysis pipelines.

[0129] Figures 3a and 3b show RPM+1 of input libraries (x-axis) and libraries produced from one round of the pooled CRISPR screen (y-axis). As shown there were 2 genome- wide libraries each for CRISPRko (Al and Bl) and CRISPRa (Cl and DI).

Example 3: Arrayed transient transfection (aTT) and functional titer measurements for targeted CRISPR mediated gene knockout or activation and screening of gene candidates that affect rAAV productivity

[0130] The present example describes an arrayed screening method that was used to screen some of the genes identified in Example 2 to identify genes that affect rAAV titer in a HEK293T cell. A graphical overview of the method is depicted in Figure 4.

[0131] HEK293T cell lines expressing Cas9 or dCas9-VPR were generated as follows. HEK293T cells were cultured in DMEM + 10% FBS with Penicillin-Streptomycin. CRISPRko (“CRISPR-knockout”) cell lines were generated from blasticidin selection of cells transduced with the Edit-R hEFla-Cas9n lentivirus (Horizon, VCAS10126). CRISPRa (“CRIS PR- activation”) cell lines were generated from blasticidin selection of cells transduced with the Edit-R hEFla-dCas9-VPR lentivirus (Horizon, VCAS 11922). Clonal lines were produced by limiting dilution and subsequent scale up.

[0132] crRNAs were pre-complexed with tracrRNA (crRNA/tracrRNA) in DharmaFECT 1 and OptiMEM solution at room temperature for 15-20 min. crRNA/tracrRNA complexes, each targeting a unique gene target, were then added to individual wells of a 96 well plate (Costar, polystyrene) at a final concentration of 25 nM crRNA/tracrRNA and a final volume of 0.2 uL DharmaFECT 1 per well. HEK293T cells stably expressing either Cas9 or dCas9-VPR were then independently reverse transfected when added to the crRNA/tracrRNA containing wells at 12,000 HEK293T cells per well in a final volume of 100 uL antibiotic-free DMEM + 10% FBS. Transfected HEK293T cells expressing Cas9 result in gene knockout while transfected HEK293T cells expressing dCas9-VPR result in gene activation; thereby creating a screening process that ultimately studies the effect either HEK293T gene knockout and/or gene activation has on rAAV titer. All crRNA, tracrRNA and transfection reagents were purchased from Horizon.

[0133] 3-days post crRNA/tracrRNA transfection, 70-80 uL of media was removed, and the cells were transfected with three types of rAAV plasmids (adenoviral helper, packaging, and proviral). Briefly, the three types of plasmids (adenoviral helper, packaging, and proviral) were mixed at a 20:10: 1 mass ratio and combined with polyethylenimine (PEI) at a mass ratio of 1: 1.75 in OptiMEM. The GOI plasmid included a transgene encoding NanoLuc luciferase (Nluc) that was packaged into the resulting rAAV. After complexing for 10 min at room temperature, antibiotic-free quench media (DMEM + 10% FBS) was added, and 125 uL of the transfection mix was added to the 70-80 uL of the HEK293T cells reverse transfected 3 days earlier, such that the final concentration of rAAV plasmid DNA was 0.5 ug/le6 cells (assuming 64,000 cells at confluency).

[0134] 4-days post rAAV plasmid transfection, rAAV-producing cells and growth media were manually harvested by pipetting and transferring cells to a 96-well PCR plate (Thermo AB1400L, polypropylene). Plates were sealed and flash frozen in liquid nitrogen followed by rapid thawing at 37 C in a thermocycler or water bath, for a total of 3 cycles. Crude rAAV lysates were cleared of cell debris by centrifugation at 2000 rpm for 5 min, and were used to directly transduce CHO-Lec2 cells for functional titer assays.

[0135] For functional assay titering, CHO-Lec2 cells were plated in alpha-MEM + 10% FBS in a 384 well PhenoPlate (PerkinElmer) at a density of 6000 cells per well and grown overnight at 37 C and 5% CO2. The following day, cell media was replaced with 25 uL alpha-MEM + 1% FBS to slow down cell growth, and 5 uL of crude rAAV lysate was added. After transducing overnight, CHO-Lec2 cells were washed 2 cycles with 25 uL alpha-MEM + 1% FBS to remove contaminating Nluc transgene and cultured in 25 uL alpha-MEM + 1% FBS for an additional 48 hours. The amount of Nluc transgene expressed was measured using NanoGio (Promega, Nil 30) and an Envision plate-reader. Raw luminescence values were reported relative to the luminescence values generated by rAAV produced from cells transfected with a non-targeting (NT) crRNA/tracrRNA control (“rAAV functional-titer relative to NT”). rAAVs were titered by this CHO-Lec2 functional assay, and expressed as relative fold change compared to the negative control (NT_n).

Example 4; Functional titer of rAAV produced in HEK293T cells that have undergone a CRISPR knockout screen

[0136] The present example demonstrates that HEK293T cells engineered to knockout specific genes result in an rAAV titer increase relative to the negative control. HEK293T cells were engineered to knockout genes and assess rAAV functional titer via the methods described in Example 3. Figure 5 shows that engineered HEK293T cells with a knockout of CDK5R2, DNTTIP2, S100A6, AFG3L2, EVI2B, RAB5C, CD2, NSD1, UCKL1, PRAMEF19, CSNK1D, CHSY3, DCLRE1C, NAGLU, CHMP7, CYFIP2, PPP3R1, CPSF7, FUS, NUP155, MCL1, CABLES1, MECR, PRKAB1, HAAG, and NFE2L1 genes resulted in higher rAAV functional titer relative to the negative control. CDK5R2 was among the first genes identified, during the pooled CR1PSR screening method described in Example 2, to increase rAAV functional titer after knockout and was subsequently used as a positive control and benchmark to assess the performance of other knockout cell lines. The knockout of DNTTIP2, S100A6, AFG3L2, EVI2B, RAB5C, CD2, NSD1, PRAMEF19, CSNK1D, CHSY3, DCLRE1C, NAGLU, CHMP7, CYFIP2, PPP3R1, CPSF7, FUS, NUP155, MCL1, CABLES1, MECR, PRKAB1, HAAO, and NFE2L1 showed functional titers equal to or greater than the knockout of CDK5R2. The knockout of CSNK1D, NUP155, and DNTTIP2 genes resulted in the highest rAAV functional titer relative to other knockout genes in Figure 5. Negative control and positive control (CDK5R2 p) samples were run as n=10 replicates per plate, while all other samples were run as n=2. In (B), CSNK1D is plotted on the right y-axis for ease of visualization.

Example 5: Functional titer of rAAV produced in HEK293T cells that have undergone a CRISPR activation screen

[0137] The present example demonstrates that HEK293T cells engineered to have specific genes up-regulated results in an rAAV titer increase relative to a negative control. HEK293T cells were engineered to activate genes and assess rAAV functional titer via the methods described in Example 3. Figure 6 shows that engineered HEK293T cells with VPS52, TRAF5, LBR, RNF2, TPM4, NRTN, FGF4, ENPP2, ELF3, DDX58, KMT2E, and PCDHGC3 genes up-regulated resulted in higher rAAV functional-titer relative to the negative control. VPS52 was among the first genes identified, during the pooled CRIPSR screening method described in Example 2, to increase rAAV functional titer after activation and was subsequently used as a positive control and benchmark to assess the performance of other activation cell lines. While the differences were slight, activated VPS52, TRAF5, and LBR genes resulted in the highest rAAV functional titer relative to other activated genes in Figure 6. Negative control and positive control (VPS52_p) samples were run as n=10 replicates per plate, while all other samples were run as n=2.

Example 6: Suspension transient transfection (sTT) and functional titer measurements for targeted CRISPR mediated gene knockout and screening of gene candidates that affect rAAV productivity

[0138] The present example describes a suspension screening method that was used to screen some of the genes identified in Example 2 to identify genes that affect rAAV titer in a HEK293 P1B2 suspension cells.

[0139] An HEK293 P1B2 cell line expressing Cas9 was generated as follows. HEK293 P1B2 cells were cultured in DMEM + 10% FBS with Penicillin-Strep tomycin. CRISPRko (“CRISPR-knockout”) cell lines were generated from blasticidin selection of cells transduced with the Edit-R hEFla-Cas9n lentivirus (Horizon, VCAS10126). The most active clonal lines were cultured in Freestyle F17 + GlutaMax + 0.1% pluronic F-68 + blastidicin to grow as suspension cells, with shaking at 110 rpm at 37 C and 10% CO2.

[0140] crRNAs were pre-complexed with tracrRNA (crRNA/tracrRNA) in DharmaFECT 1 and OptiMEM solution at room temperature for 15-20 min. HEK293 P1B2 cells stably expressing Cas9 were seeded in 6 or 12-well plates (Costar, polystyrene) at a density of 5e5 cells/mL in a volume of 1 mL The HEK293 P1B2 cells were then reverse transfected with the crRNA/tracrRNA complexes (final concentration 25 nM) and DharmaFECT 1 (final volume of 2.6 uL per well) such that the final concentration of suspension cells was 4e5/mL. Transfected HEK293 P1B2 cells expressing Cas9 result in gene knockout; thereby creating a screening process that ultimately studies the effect HEK293 P1B2 gene knockout has on rAAV titer. All crRNA, tracrRNA and transfection reagents were purchased from Dharmacon.

[0141] 3-days post crRNA/tracrRNA transfection, cell densities had grown to roughly le6 cells per mL. The cells were then transfected with three types of rAAV plasmids (adenoviral helper, packaging, and pro viral). Briefly, the three types of plasmids (packaging, proviral, and adenoviral helper) were mixed at a 0.5: 1:2 molar ratio and combined with polyethylenimine (PEI) in OptiMEM. After complexing for 5 min at room temperature, the transfection mix was added to crRNA/tracrRNA-transfected cells such that the final concentration of rAAV transfection mix DNA and PEI was 3 ug/mL and 6 uL/mL, respectively. The GOI plasmid included a transgene encoding NanoLuc luciferase (Nluc) that was packaged into the resulting rAAV.

[0142] 4-days post rAAV plasmid transfection, rAAV-producing cells and growth media were manually harvested by pipetting and transferring to Eppendorf tubes. Crude AAV lysates were cleared of cell debris by centrifugation at 2000 rpm for 5 min, and were used to directly transduce CHO-Lec2 cells for functional titer assays as described in Example 3.

[0143] Figure 7 shows that engineered HEK293 P1B2 cells with a knockout of CSNK1D, NAGLU, DNTTIP2, CABLES1, UCKL1, EVI2B, MECR, DCLRE1C, HAAG, PRAMEF19, NSD1, CHSY3, NFE2L1, and NUP155 genes resulted in higher rAAV functional-titer relative to the negative control. The knockout of NUP155 gene resulted in the highest rAAV functional titer relative to other genes in Figure 7. Negative control samples were run across multiple plates (n=8), while other samples were run as n=2 or n=6 replicates.

Example 7: Targeted gene knockout in HEK293 cells for rAAV production and titer determination

[0144] The present example describes a method that was used to knockout target genes or combinations of target genes in HEK293 cells. For each knockout a stable pool of engineered HEK293 cells was generated. For each knockout pool, Cas9-GFP protein and sgRNAs targeting the candidate gene(s) were transfected into TFS HEK293 (ThermoFisher Scientific, A50232) or HEK293 P1B2 cell lines using Neon transfection system (Invitrogen). Approximately three hours after transfection, GFP-positive cells were sorted out from the transfected cells using fluorescence- activated cell sorting (FACS). Cells were maintained by passaging until populations maintained consistent growth with high viability followed by frozen cell bank generation.

[0145] Three types of plasmids (packaging, proviral and adenoviral helper) were mixed at 2: 1 : 1 molar ratio and transfected to each knockout pool using polyethylenimine (PEI) to produce rAAV. Four days after transfection, cells were collected, and viruses were released by cell lysis. The cell lysates were analyzed by droplet digital Polymerase Chain Reaction (ddPCR) to measure rAAV vector genome titer.

Example 8: Genome titer from various knockout pools of engineered HEK293 cells

[0146] The present example demonstrates that HEK293 P1B2 cells and TFS HEK293 cells engineered to knockout specific genes result in an rAAV titer increase relative to a negative control (HEK293 P1B2 cells or TFS HEK293 cells that were unmodified). HEK293 P1B2 cells and TFS HEK293 cells were engineered to knockout certain genes or combinations of genes and assess rAAV titer using the methods described in Example 7. Figure 8a shows that engineered HEK293 P1B2 cells with BAX/BAK, BAX/STAT1, CASP3/7, CASP3/6/7/AIF1, ICAM1, CDH1, OCLN, VIM, and DSG2 genes knocked out resulted in higher rAAV titer relative to the negative control. Figure 8b shows that engineered TFS HEK293 cells with CASP3/7, CDH2, CDH1, OCLN, and VIM, genes knocked out resulted in higher rAAV titer while cells with YBX1, BAX/BAK, BAX/STAT1, CASP3/6/7/AIF1, ICAM1, and DSG2 genes knocked out resulted in slightly lower or equivalent titer, relative to the negative control. Genome titer data (viral genome/mL) were normalized to each control cell line to obtain the relative fold change. The error bars indicate the standard deviation (n=4).

Example 9: Cell aggregation profile of an occludin (OCLN) knockout pool of engineered HEK293 cells

[0147] The present example demonstrates how elimination of the OCLN gene reduces aggregation of HEK293 cells. To assess and quantify host cell aggregation, both unmodified HEK293 cells (control) and HEK293 cells with the OCLN gene eliminated were treated with Accumax (Invitrogen), a cell dissociation reagent, and viable cell number was counted. Figure 9a depicts the fold change in viable cell number of Accumax treated cells to untreated cells in either OCLN knockout or control cells. The fold change in viable cell number between Accumax treated and untreated was about 1 for OCLN knockout cells and about 1.75 in the control. Figure 9b shows a microscopic image of control or OCLN knockout cells treated or untreated with Accumax. The red arrow points to an aggregation of control cells that have not been treated with Accumax. Cells that either have OCLN knocked out or have been treated with cell disassociation agent (Accumax) do not show visible aggregation. Images are representative of each cell population. Figure 9c is a depiction of the role OCLN plays in cell-to-cell adhesion, and other tight junction molecules that could be targeted similarly to OCLN to mitigate cell aggregation and thereby allow for accurate cell count and process robustness for rAAV manufacturing. Depicted molecular targets to reduce host cell aggregation are indicated by the box outline: Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) (Brunner et al., Adv. Drug Deliv. Rev. 171:266- 288, 2021).

[0148] Being able to generate a non-aggregative HEK293 cell line has several advantages for rAAV production using transient transfection since it allows for accurate cell counting which is useful for rAAV production process robustness and manufacturability, particularly GMP manufacturing. For example, Figure 10a shows how titer varied with viable cell density (VCD) for a wild-type HEK293 cell line which is prone to aggregation at higher VCD values. Cell mass was enumerated via optical cell counting using a Vi-CELL™ XR cell counter. Cell lysates were analyzed by droplet digital Polymerase Chain Reaction (ddPCR) to measure rAAV vector genome titer. The Vi-CELL XR ™ cell counter is heavily influenced by aggregation and, as shown in Figure 10a, there is no clear trend between VCD and titer. Figure 10b shows a graphic of titer vs. LDH (Lactate dehydrogenase) concentration for wild-type HEK293 cells. LDH was measured using a CEDEX BioHT® device and is proportional to TCD (or total cell mass) present in a sample. As shown in Figure 10b, this approach reduces noise due to aggregation and produces a better trend but this approach is difficult to use in GMP manufacturing. The ability to enumerate cell mass reliably using a cell counter without aggregation would be advantageous in that it would enable more accurate determination of optimal VCD for production purposes, particularly in GMP manufacturing. The engineered HEK293 cells also open up the possibility of using higher VCD values during production without aggregation than could be achieved using wild-type HEK293 cells. This could in turn lead to improved rAAV production, including an improvement in titer.

Example 10: Engineered cell line has reduced cell aggregation profile

[0149] The present example demonstrates how elimination of the OCLN gene reduces aggregation of engineered HEK293 P1B2 cells (Figure lib). When the OCLN gene was eliminated from the HEK293 P1B2 engineered cell line it still achieved an rAAV genome titer comparable to a commercial cell line (TFS HEK293 cells) (Figure Ila). Genome titer data (viral genome/mL) were normalized to that of a commercial cell line to obtain the relative fold change. The error bars indicate the standard deviation (n=4).

Example 11: Genome titer of rAAV achieved in cells that have undergone a CRISPR knockout screen

[0150] The present example demonstrates that cells engineered to knockout specific genes achieve an increase and/or comparable rAAV genome titer relative to a control. HEK293 cells were engineered to eliminate (e.g., knockout) genes and assess rAAV genome titer via the methods described below. Engineered HEK293 P1B2-OCLN cells were used for sTT evaluation to identify individual candidate gene knockouts that achieved similar or better rAAV genome titer relative to a commercial cell line (TFS HEK293 cells). A schematic view of an engineered cell line evaluation workflow is depicted in Figure 11b.

[0151] Knockout pool sene ration

[0152] Cas9-GFP protein (Cat. #ECAS9GFPPR, Millipore Sigma) and a mixture of three single guide RNAs (CRISPR Gene Knockout Kit, Synthego) targeting each candidate gene were transfected into commercial TFS HEK293 cells or engineered HEK293 P1B2- OCLN cells using Neon transfection system (Invitrogen). Approximately three hours after transfection, GFP-positive cells were sorted out from the transfected cells using fluorescence-activated cell sorting (Sony SH800 sorter). Cells were maintained by passaging until populations maintained consistent growth with high viability, followed by frozen cell bank generation.

[0153] Transfection for rAAV production and titer determination

[0154] Three types of plasmids (Rep/Cap, GOI, and helper) were mixed at a 2:1: 1 molar ratio and transfected to each knockout pool using polyethyleneimine (PEI). Four days after transfection, cells were collected, and viruses were released by lauryl dimethyl amine oxide (LDAO), MgCh. and Benzonase-based cell lysis. The cell lysates were analyzed by droplet digital Polymerase Chain Reaction (ddPCR) to measure rAAV genome titer.

[0155] Results

[0156] rAAV genome titer from various knockout pools were measured. rAAV genome titer of a gene of interest produced by knockout pools derived from a commercial cell line (TFS HEK293) (Figure 12a) or the engineered HEK293 P1B2-OCLN cells (Figure 12b) was assessed. rAAV genome titer data (viral genome/mL) were normalized to that of the commercial cell line to obtain the relative fold change. The error bars indicate the standard deviation (n=4).

[0157] Figure 12a shows results for the commercial cell line with a knockout pool of AFG3L2, CABLES 1, CD2, CDK5R2, CHMP7, CHSY3, CPSF7, CSNK1D, CYFIP2, DCLRE1C, DNTTIP2, EVI2B, FUS, HAAG, MCL1, NAGLU, NFE2L1, NSD1, NUP155, PPP3R1, PRAMEF19, PPKAB 1, RAB5C, S 100A6, or UCKL1 genes. Commercial cells with a knockout of gene RAB5C achieved higher rAAV genome titer relative to the control.

[0158] Figure 12b shows results for the engineered HEK293 P1B2-OCLN cells with a knockout pool of CABLES1, CD2, CDK5R2, CHMP7, CHSY3, CPSF7, CYFIP2, DCLRE1C, EVI2B, FUS, HAAO, NAGLU, NFE2L1, NSD1, PPP3R1, PRAMEF19, PPKAB1, RAB5C, S100A6, and UCKL1 genes. HEK293 P1B2-OCLN knockout pool with a knockout of EVI2B, FUS, or NFE2L1 achieved higher rAAV genome titer relative to the control.

[0159] Additional gene candidate results

[0160] Additional gene candidates useful in achieving rAAV production in the commercial cell line and engineered HEK293 P1B2-OCLN cells, were tested.

[0161] Figures 13a-13b show results for the commercial cell line with a knockout pool of BAX-BAK, CASP 3/7, CASP 3/6/7/ A1F1, CDH1, CDH2, DSG2, ICAM1, OCLN, STAT1-BAX, TIPI, VIM, YBX1, CGN, CLDN1, FUR, KIAA0319L, MYD88, TICAM1, TLR2, TLR3, and TMEM173 genes. Commercial cells with a knockout of gene CASP 3/7, TJP1, CGN, or TICAM1 achieved higher rAAV genome titer relative to the control.

[0162] Figures 13c-13d shows results for the engineered HEK293 P1B2-OCLN cells with a knockout pool of CASP 3/6/7/AIF1, CDH1, CDH2, DSG2, TJP1, VIM, CGN, CLDN1, FUR, KIAA0319L, MYD88, TICAM1, TLR2, TLR3, and TMEM173 genes. HEK293 P1B2-OCLN cells with a knockout of gene CASP 3/6/7/AIF1 or TJP1 achieved higher rAAV genome titer relative to the control. HEK293 P1B2-OCLN cells with a knockout of gene CGN or MYD88 achieved comparable rAAV genome titer relative to the control.

NUMBERED EMBODIMENTS

[0163] Embodiment 1. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells in which (a) expression and/or activity of one or more genes and/or gene products in Tables 2-9 is reduced compared to control parental cells and/or (b) expression and/or activity of one or more genes and/or gene products in Table 10 or 11 is increased compared to control parental cells.

[0164] Embodiment 2. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 2 is reduced compared to control parental cells.

[0165] Embodiment 3. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 3 is reduced compared to control parental cells.

[0166] Embodiment 4. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 4 is reduced compared to control parental cells.

[0167] Embodiment 5. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 5 is reduced compared to control parental cells.

[0168] Embodiment 6. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 6 is reduced compared to control parental cells.

[0169] Embodiment 7. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 7 is reduced compared to control parental cells.

[0170] Embodiment 8. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 8 is reduced compared to control parental cells.

[0171] Embodiment 9. The engineered cell or cell line of embodiment 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 9 is reduced compared to control parental cells. [0172] Embodiment 10. The engineered cell or cell line of any one of embodiments 1-7 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 10 or 11 is increased compared to control parental cells.

[0173] Embodiment 11. The engineered cell or cell line of embodiment 8 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 10 is increased compared to control parental cells.

[0174] Embodiment 12. The engineered cell or cell line of embodiment 8 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 11 is increased compared to control parental cells.

[0175] Embodiment 13. The engineered cell or cell line of any one of embodiments 1-9, wherein expression and/or activity is reduced using a nuclease, a transcriptional repressor, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO), a gene disruption, or a partial or complete gene deletion.

[0176] Embodiment 14. The engineered cell or cell line of embodiment 13, wherein the nuclease is selected from the group consisting of a Zinc Finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein.

[0177] Embodiment 15. The engineered cell or cell line of any one of embodiments 1-9, wherein expression and/or activity is reduced using a CRISPR -based system.

[0178] Embodiment 16. The engineered cell or cell line of any one of embodiments 1-9, wherein expression and/or activity of one or more genes and/or gene products in Tables 2-9 is eliminated compared to control parental cells.

[0179] Embodiment 17. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 2 is eliminated compared to control parental cells. [0180] Embodiment 18. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 3 is eliminated compared to control parental cells.

[0181] Embodiment 19. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 4 is eliminated compared to control parental cells.

[0182] Embodiment 20. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 5 is eliminated compared to control parental cells.

[0183] Embodiment 21. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 6 is eliminated compared to control parental cells.

[0184] Embodiment 22. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 7 is eliminated compared to control parental cells.

[0185] Embodiment 23. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 8 is eliminated compared to control parental cells.

[0186] Embodiment 24. The engineered cell or cell line of embodiment 16, wherein expression and/or activity of one or more genes and/or gene products in Table 9 is eliminated compared to control parental cells.

[0187] Embodiment 25. The engineered cell or cell line of any one of embodiments 1-9, wherein expression and/or activity is reduced using a Zinc Finger (ZF), a transcription activator-like effector (TALE), or an inactivated clustered regularly interspaced short palindromic repeats (CRISPR) associated protein that is fused or non-covalently associated with a transcriptional repression domain. [0188] Embodiment 26. The engineered cell or cell line of any one of embodiments 1 and 10-12, wherein expression and/or activity is increased using a Zinc Finger Protein (ZFP), a transcription activator-like effector protein (TALE), or an inactivated clustered regularly interspaced short palindromic repeats (CRISPR) associated protein that is fused or non-covalently associated with a transcriptional activation domain.

[0189] Embodiment 27. The engineered cell or cell line of any one of embodiments 1 and 10-12, wherein expression and/or activity is increased by introducing additional copies of the one or more genes into the cell or cell line.

[0190] Embodiment 28. The engineered cell or cell line of embodiment 27, wherein the additional copies of the one or more genes are integrated into the genome of the cell or cell line.

[0191] Embodiment 29. The engineered cell or cell line of embodiment 27, wherein the additional copies of the one or more genes are not integrated into the genome of the cell or cell line.

[0192] Embodiment 30. The engineered cell or cell line of any one of embodiments 1-29, wherein the cell or cell line is a human cell or cell line.

[0193] Embodiment 31. The engineered cell or cell line of embodiment 30, wherein the human cell or cell line is a HeLa, COS, HEK293, A549, BHK, or Vero cell or cell line, e.g., an HEK293T or HEK293 P1B2 cell or cell line.

[0194] Embodiment 32. The engineered cell or cell line of any one of embodiments 1-31, wherein the titer of rAAV is increased by at least 1.5 fold, e.g., by at least 2 fold, at least 5 fold, at least 10 fold or at least 15 fold compared to the titer of rAAV produced from control parental cells.

[0195] Embodiment 33. The engineered cell or cell line of embodiment 32, wherein the titer of rAAV is increased by up to 20 fold, e.g., up to 15 fold, up to 10 fold, or up to 2 fold compared to the titer of rAAV produced from control parental cells. [0196] Embodiment 34. The engineered cell or cell line of embodiment 32, wherein the cell or cell line is less prone to aggregation than the control parental cells.

[0197] Embodiment 35. A lysate of an engineered cell or cell line of any one of embodiments 1-34.

[0198] Embodiment 36. A cell culture supernatant from an engineered cell or cell line of any one of embodiments 1-34.

[0199] Embodiment 37. A method of generating an engineered cell or cell line of any one of embodiments 1-34, the method comprising modulating expression and/or activity of one or more genes and/or gene products in Tables 2-11 in a parental cell line.

[0200] Embodiment 38. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Tables 2-9 compared to control parental cells.

[0201] Embodiment 39. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 2 compared to control parental cells.

[0202] Embodiment 40. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 3 compared to control parental cells.

[0203] Embodiment 41. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 4 compared to control parental cells.

[0204] Embodiment 42. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 5 compared to control parental cells.

[0205] Embodiment 43. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 6 compared to control parental cells. [0206] Embodiment 44. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 7 compared to control parental cells.

[0207] Embodiment 45. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 8 compared to control parental cells.

[0208] Embodiment 46. The method of embodiment 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 9 compared to control parental cells.

[0209] Embodiment 47. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Tables 2-9 compared to control parental cells.

[0210] Embodiment 48. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 2 compared to control parental cells.

[0211] Embodiment 49. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 3 compared to control parental cells.

[0212] Embodiment 50. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 4 compared to control parental cells.

[0213] Embodiment 51. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 5 compared to control parental cells.

[0214] Embodiment 52. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 6 compared to control parental cells. [0215] Embodiment 53. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 7 compared to control parental cells.

[0216] Embodiment 54. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 8 compared to control parental cells.

[0217] Embodiment 55. The method of embodiment 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 9 compared to control parental cells.

[0218] Embodiment 56. The method of embodiment 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 10 or 11 compared to control parental cells.

[0219] Embodiment 57. The method of embodiment 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 10 compared to control parental cells.

[0220] Embodiment 58. The method of embodiment 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 11 compared to control parental cells.

[0221] Embodiment 59. The method of any one of embodiments 37-58, wherein the cell or cell line is a human cell or cell line.

[0222] Embodiment 60. The method of embodiment 59, wherein the human cell or cell line is a HeLa, COS, HEK293, A549, BHK, or Vero cell or cell line, e.g., an HEK293T or HEK293 P1B2 cell or cell line.

[0223] Embodiment 61. A method of producing rAAV, the method comprising transfecting an engineered cell or cell line of any one of embodiments 1-34 with one or more plasmids. [0224] Embodiment 62. The method of embodiment 61, wherein the one or more plasmids comprise a packaging plasmid, an adenoviral helper plasmid, and/or a proviral plasmid.

[0225] Embodiment 63. The method of embodiment 61, wherein the one or more plasmids comprise a packaging plasmid and a proviral plasmid.

[0226] Embodiment 64. The method of embodiment 61, wherein the one or more plasmids comprise an adenoviral helper plasmid and a proviral plasmid.

[0227] Embodiment 65. The method of embodiment 61, wherein the one or more plasmids comprise a packaging plasmid, an adenoviral helper plasmid and a proviral plasmid.

[0228] Embodiment 66. A method of producing rAAV, the method comprising infecting an engineered cell or cell line of any one of embodiments 1-34 with one or more helper viruses.

[0229] Embodiment 67. The method of any one of embodiments 61-66, wherein the rAAV is harvested from the cell or cell line, e.g., from a lysate or cell culture supernatant of the cell or cell line.

[0230] Embodiment 68. The method of any one of embodiments 61-67, wherein production of rAAV is enhanced as compared to control parental cells.

[0231] Embodiment 69. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which have been engineered to (a) reduce or eliminate expression and/or activity of a gene product expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increase expression and/or activity of a gene product expressed from one or more genes in Tables 10 or 11 as compared to control parental cells.

[0232] Embodiment 70. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which exhibit (a) reduced or eliminated expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increased expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 10 or 11 as compared to control parental cells.

[0233] Embodiment 71. A method of producing rAAV, the method comprising transducing or infecting an engineered cell or cell line that that has been engineered to reduce aggregation with one or more plasmids or helper viruses.

[0234] Embodiment 72. The method of embodiment 71, wherein the method is repeated at one or more viable cell density (VCD) of the engineered cell or cell line and an rAAV titer is measured at each VCD.

[0235] Embodiment 73. The method of embodiment 72, wherein the VCD is calculated based on the cell mass.

[0236] Embodiment 74. The method of embodiment 73, wherein the cell mass is measured using a cell counter.

[0237] Embodiment 75. The method of any one of embodiments 71-74, wherein the engineered cell or cell line has been engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells.

[0238] Embodiment 76. The method of embodiment 75, wherein the expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells.

[0239] Embodiment 77. The method of any one of embodiments 71-76, wherein the engineered cell or cell line is an engineered HEK293 cell or cell line.

[0240] Embodiment 78. The method of embodiment 77, wherein the method is repeated across a series of VCD values including values that exceed 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL. [0241] Embodiment 79. A cell culture comprising an engineered cell or cell line that that has been engineered to reduce aggregation, wherein the engineered cell or cell line has been transduced of infected with one or more plasmids or helper viruses.

[0242] Embodiment 80. The cell culture of embodiment 79, wherein the engineered cell or cell line has been engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells.

[0243] Embodiment 81. The cell culture of embodiment 80, wherein the expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells.

[0244] Embodiment 82. The cell culture of embodiment 80 or 81, wherein the engineered cell or cell line is an engineered HEK293 cell or cell line.

[0245] Embodiment 83. The cell culture of embodiment 82, wherein the viable cell density (VCD) of the engineered HEK293 cell or cell line exceeds 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL.

[0246] Embodiment 84. The cell culture of embodiment 83, wherein the engineered HEK293 cell or cell line is in a non- aggregated form.

EQUIVALENTS

[0247] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS We claim:

1. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells in which (a) expression and/or activity of one or more genes and/or gene products in Tables 2-9 is reduced compared to control parental cells and/or (b) expression and/or activity of one or more genes and/or gene products in Table 10 or 11 is increased compared to control parental cells.

2. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 2 is reduced compared to control parental cells.

3. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 3 is reduced compared to control parental cells.

4. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 4 is reduced compared to control parental cells.

5. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 5 is reduced compared to control parental cells.

6. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 6 is reduced compared to control parental cells.

7. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 7 is reduced compared to control parental cells.

8. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 8 is reduced compared to control parental cells.

9. The engineered cell or cell line of claim 1 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 9 is reduced compared to control parental cells.

10. The engineered cell or cell line of any one of claims 1-9 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 10 or 11 is increased compared to control parental cells.

11. The engineered cell or cell line of claim 10 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 10 is increased compared to control parental cells.

12. The engineered cell or cell line of claim 10 comprising a cell or cells in which expression and/or activity of one or more genes and/or gene products in Table 11 is increased compared to control parental cells.

13. The engineered cell or cell line of any one of claims 1-9, wherein expression and/or activity is reduced using a nuclease, a transcriptional repressor, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO), a gene disruption, or a partial or complete gene deletion.

14. The engineered cell or cell line of claim 13, wherein the nuclease is selected from the group consisting of a Zinc Finger nuclease (ZFN), a meganuclease, a transcription activatorlike effector nuclease (TALEN), or a clustered regularly interspaced short palindromic repeats (CRISPR) associated protein.

15. The engineered cell or cell line of any one of claims 1-9, wherein expression and/or activity is reduced using a CRISPR-based system.

16. The engineered cell or cell line of any one of claims 1-9, wherein expression and/or activity of one or more genes and/or gene products in Tables 2-9 is eliminated compared to control parental cells.

17. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 2 is eliminated compared to control parental cells.

18. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 3 is eliminated compared to control parental cells.

19. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 4 is eliminated compared to control parental cells.

20. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 5 is eliminated compared to control parental cells.

21. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 6 is eliminated compared to control parental cells.

22. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 7 is eliminated compared to control parental cells.

23. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 8 is eliminated compared to control parental cells.

24. The engineered cell or cell line of claim 16, wherein expression and/or activity of one or more genes and/or gene products in Table 9 is eliminated compared to control parental cells.

25. The engineered cell or cell line of any one of claims 1-9, wherein expression and/or activity is reduced using a Zinc Finger (ZF), a transcription activator-like effector (TALE), or an inactivated clustered regularly interspaced short palindromic repeats (CRISPR) associated protein that is fused or non-covalently associated with a transcriptional repression domain.

26. The engineered cell or cell line of any one of claims 1 and 10-12, wherein expression and/or activity is increased using a Zinc Finger Protein (ZFP), a transcription activator- like effector protein (TALE), or an inactivated clustered regularly interspaced short palindromic repeats (CRISPR) associated protein that is fused or non-covalently associated with a transcriptional activation domain.

27. The engineered cell or cell line of any one of claims 1 and 10-12, wherein expression and/or activity is increased by introducing additional copies of the one or more genes into the cell or cell line.

28. The engineered cell or cell line of claim 27, wherein the additional copies of the one or more genes are integrated into the genome of the cell or cell line.

29. The engineered cell or cell line of claim 27, wherein the additional copies of the one or more genes are not integrated into the genome of the cell or cell line.

30. The engineered cell or cell line of any one of claims 1-29, wherein the cell or cell line is a human cell or cell line.

31. The engineered cell or cell line of claim 30, wherein the human cell or cell line is a HeLa, COS, HEK293, A549, BHK, or Vero cell or cell line, e.g. , an HEK293T or HEK293 P1B2 cell or cell line.

32. The engineered cell or cell line of any one of claims 1-31, wherein the titer of rAAV is increased by at least 1.5 fold, e.g., by at least 2 fold, at least 5 fold, at least 10 fold or at least 15 fold compared to the titer of rAAV produced from control parental cells.

33. The engineered cell or cell line of claim 32, wherein the titer of rAAV is increased by up to 20 fold, e.g., up to 15 fold, up to 10 fold, or up to 2 fold compared to the titer of rAAV produced from control parental cells.

34. The engineered cell or cell line of claim 32, wherein the cell or cell line is less prone to aggregation than the control parental cells.

35. A lysate of an engineered cell or cell line of any one of claims 1-34.

36. A cell culture supernatant from an engineered cell or cell line of any one of claims 1- 34.

37. A method of generating an engineered cell or cell line of any one of claims 1-34, the method comprising modulating expression and/or activity of one or more genes and/or gene products in Tables 2-11 in a parental cell line.

38. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Tables 2-9 compared to control parental cells.

39. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 2 compared to control parental cells.

40. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 3 compared to control parental cells.

41. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 4 compared to control parental cells.

42. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 5 compared to control parental cells.

43. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 6 compared to control parental cells.

44. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 7 compared to control parental cells.

45. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 8 compared to control parental cells.

46. The method of claim 37, wherein modulating expression and/or activity comprises reducing expression and/or activity of one or more genes and/or gene products in Table 9 compared to control parental cells.

47. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Tables 2-9 compared to control parental cells.

48. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 2 compared to control parental cells.

49. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 3 compared to control parental cells.

50. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 4 compared to control parental cells.

51. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 5 compared to control parental cells.

52. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 6 compared to control parental cells.

53. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 7 compared to control parental cells.

54. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 8 compared to control parental cells.

55. The method of claim 37, wherein modulating expression and/or activity comprises eliminating expression and/or activity of one or more genes and/or gene products in Table 9 compared to control parental cells.

56. The method of claim 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 10 or 11 compared to control parental cells.

57. The method of claim 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 10 compared to control parental cells.

58. The method of claim 37, wherein modulating expression and/or activity comprises increasing expression and/or activity of one or more genes and/or gene products in Table 11 compared to control parental cells.

59. The method of any one of claims 37-58, wherein the cell or cell line is a human cell or cell line.

60. The method of claim 59, wherein the human cell or cell line is a HeLa, COS, HEK293, A549, BHK, or Vero cell or cell line, e.g., an HEK293T or HEK293 P1B2 cell or cell line.

61. A method of producing rAAV, the method comprising transfecting an engineered cell or cell line of any one of claims 1-34 with one or more plasmids.

62. The method of claim 61, wherein the one or more plasmids comprise a packaging plasmid, an adenoviral helper plasmid, and/or a proviral plasmid.

63. The method of claim 61, wherein the one or more plasmids comprise a packaging plasmid and a proviral plasmid.

64. The method of claim 61, wherein the one or more plasmids comprise an adenoviral helper plasmid and a proviral plasmid.

65. The method of claim 61, wherein the one or more plasmids comprise a packaging plasmid, an adenoviral helper plasmid and a proviral plasmid.

66. A method of producing rAAV, the method comprising infecting an engineered cell or cell line of any one of claims 1-34 with one or more helper viruses.

67. The method of any one of claims 61-66, wherein the rAAV is harvested from the cell or cell line, e.g., from a lysate or cell culture supernatant of the cell or cell line.

68. The method of any one of claims 61-67, wherein production of rAAV is enhanced as compared to control parental cells.

69. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which have been engineered to (a) reduce or eliminate expression and/or activity of a gene product expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increase expression and/or activity of a gene product expressed from one or more genes in Table 10 or 11 as compared to control parental cells.

70. An engineered cell or cell line for producing recombinant adeno-associated virus (rAAV) comprising a cell or cells which exhibit (a) reduced or eliminated expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Tables 2-9 as compared to control parental cells and/or (b) increased expression and/or activity of a protein or non-coding RNA expressed from one or more genes in Table 10 or 11 as compared to control parental cells.

71. A method of producing rAAV, the method comprising transducing or infecting an engineered cell or cell line that that has been engineered to reduce aggregation with one or more plasmids or helper viruses.

72. The method of claim 71, wherein the method is repeated at one or more viable cell density (VCD) of the engineered cell or cell line and an rAAV titer is measured at each

VCD.

73. The method of claim 72, wherein the VCD is calculated based on the cell mass.

74. The method of claim 73, wherein the cell mass is measured using a cell counter.

75. The method of any one of claims 71-74, wherein the engineered cell or cell line has been engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells.

76. The method of claim 75, wherein the expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells.

77. The method of any one of claims 71-76, wherein the engineered cell or cell line is an engineered HEK293 cell or cell line.

78. The method of claim 77, wherein the method is repeated across a series of VCD values including values that exceed 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL.

79. A cell culture comprising an engineered cell or cell line that that has been engineered to reduce aggregation, wherein the engineered cell or cell line has been transduced of infected with one or more plasmids or helper viruses.

80. The cell culture of claim 79, wherein the engineered cell or cell line has been engineered by having expression and/or activity of one or more genes and/or gene products selected from Claudins (CLDNs), Zonula occludens (TJPs), and Occludin (OCLN) reduced compared to control parental cells.

81. The cell culture of claim 80, wherein the expression and/or activity of Occludin (OCLN) is reduced compared to control parental cells.

82. The cell culture of claim 80 or 81, wherein the engineered cell or cell line is an engineered HEK293 cell or cell line.

83. The cell culture of claim 82, wherein the viable cell density (VCD) of the engineered HEK293 cell or cell line exceeds 2.5xl0⁶ vc/mL, 3xl0⁶ vc/mL, 3.5xl0⁶ vc/mL, 4xl0⁶ vc/mL, 4.5xl0⁶ vc/mL, 5xl0⁶ vc/mL, 5.5xl0⁶ vc/mL, or 6xl0⁶ vc/mL.

84. The cell culture of claim 83, wherein the engineered HEK293 cell or cell line is in a non-aggregated form.