Patent Publication Number: US-2022228129-A1

Title: Cells for enhanced production of adeno-associated virus

Description:
CROSS-REFERENCE 
     This application claims the benefit of U.S. Provisional Patent Application No. 62/839,375, filed Apr. 26, 2019, which application is incorporated herein by reference in its entirety. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Grant No. EB021572 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     INTRODUCTION 
     Gene therapy is a therapeutic modality that involves the delivery of DNA to a cell to treat genetic disease. Common delivery technologies include viral vectors, lipid delivery, and naked-DNA delivery, and while the latter two technologies boast low immune profiles, repeat administration ability, and lack of transgene size limit, the technologies are highly inefficient in vivo. Viral vectors are far more efficient and include a number of properties that make them advantageous. A currently used viral vector for in vivo delivery is Adeno-Associated Virus (AAV). AAV is exhibits low immunogenicity and low random integration rate, making it one of the safest DNA delivery methods. Current AAV-mediated gene therapy is challenged by limitations in manufacturing sufficient quantities to satisfy demand. 
     There is a need in the field for cell lines that produce AAV in higher quantities than currently available using, e.g., human embryonic kidney (HEK) cells. 
     SUMMARY 
     The present disclosure provides an in vitro mammalian cell that is genetically modified to provide for enhanced production of adeno-associated virus (AAV) virions. The mammalian cells can be used to produce AAV virions, e.g., recombinant AAV virions that include a heterologous nucleic acid encoding a gene product; the present disclosure thus provides methods for producing an AAV virion, which may be a recombinant AAV virion. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  presents schematic depictions of an iterative AAV manufacturing enhancement process through genetic manipulation via either over-expression mediated by dCas9-VP64 (left panel) or genetic knock-out via Cas9 (right panel). 
         FIG. 2A-2D  depict: A) the top hits from the GeCKO selection ranked in descending order; B) the top hits from the SAM selection ranked in descending order; C) fold increase of hits increased by over 1500-fold and the corresponding gene; D) quantification of vg/mL and fold increase over AAV2 production in wildtype HEK293Ts of cells constitutively expressing the guide RNA linked to increased AAV manufacturing capability. 
         FIG. 3A-3B  depict: A) Western blot confirming elevated expression of ITPRIP protein in the cell line associated with increased titer due to ITPRIP expression; B) qRT-PCR confirming overexpression of SKA2 in the cell line associated with increased titer due to SKA2 expression. 
         FIG. 4A-4D  depict: A) Box plot showing viral titers in viral genomes (VG) per 15 mL plate of AAV2 as quantified by qPCR; B) Box plot showing infectious units (IU) per 15 mL plate as quantified by GFP expressing cells relative to total cells; C) Average viral titers per 15 mL plate as well as the fold increase relative to the WT 293T condition; D) Average infectious titers per 15 mL plate as well as the fold increase relative to the WT 293T condition. 
         FIG. 5A-5D  depict: A) Box plot showing viral titers in viral genomes (VG) per 15 mL plate of AAV6 as quantified by qPCR; B) Box plot showing infectious units (IU) per 15 mL plate of AAV6 as quantified by GFP expressing cells relative to total cells; C) Average viral titers per 15 mL plate as well as the fold increase relative to the WT 293T condition; D) Average infectious titers per 15 mL plate as well as the fold increase relative to the WT 293T condition. 
         FIG. 6  provides an amino acid sequence of an inositol 1,4,5-trisphosphate receptor-interacting protein (ITPRIP) isoform Xl. The sequence is set forth in SEQ ID NO: 3. 
         FIG. 7  provides an amino acid sequence of a CUGBP Elav-like family member 2 (CELF2), isoform 3, polypeptide. The sequence is set forth in SEQ ID NO: 4. 
         FIG. 8  provides an amino acid sequence of a centrosomal protein of 128 kDa (CEP128) polypeptide. The sequence is set forth in SEQ ID NO: 5. 
         FIG. 9  provides an amino acid sequence of a protocadherin alpha-2 (PCDHA2), isoform 1, polypeptide. The sequence is set forth in SEQ ID NO: 6. 
         FIG. 10  provides an amino acid sequence of an acetylcholinesterase (Cartwright blood group) 
       (ACHE), isoform E4-E6, polypeptide. The sequence is set forth in SEQ ID NO: 7. 
         FIG. 11  provides an amino acid sequence of an endoplasmic reticulum lectin 1, isoform 1, polypeptide. The sequence is set forth in SEQ ID NO: 8. 
         FIG. 12  provides an amino acid sequence of a kinesin-like protein (KIF23), isoform 1, polypeptide. The sequence is set forth in SEQ ID NO: 9. 
         FIG. 13  provides an amino acid sequence of an interferon-induced protein with tetratricopeptide repeats 5 (IFITS) polypeptide. The sequence is set forth in SEQ ID NO: 10. 
         FIG. 14  provides an amino acid sequence of a caspase recruitment domain-containing protein 8 (CARD8) polypeptide. The sequence is set forth in SEQ ID NO: 11. 
         FIG. 15  provides an amino acid sequence of a chromosome 5 open reading frame 52 (C5orf52) polypeptide. The sequence is set forth in SEQ ID NO: 12. 
         FIG. 16  depicts increase AAV2 (upper panel) and AAV6 (lower panel) in the number of viral genomes with each round, normalized to the wild-type (WT) 293Ts value for that round, from SKA2- and ITPRIP-expressing cells. 
         FIG. 17  depicts increase AAV2 (upper panel) and AAV6 (lower panel) in the number of viral genomes with each round, normalized to the WT 293Ts value for that round. 
         FIG. 18  depicts Western blot analysis of: i) SKA2 production in a cell line expressing SKA2 and in a ‘dual’ cell line expressing both SKA2 and ITPRIP; and ii) ITPRIP production in an ITPRIP expressing cell line and in a ‘dual’ cell line expressing both SKA2 and ITPRIP. 
         FIG. 19  presents Table 1: Compiled AAV2 viral titers from WT 293Ts as well as 293Ts induced to express: i) SKA2; ii) ITPRIP; or iii) both SKA2 and ITPRIP simultaneously (“dual”) 
         FIG. 20  presents Table 2: Compiled AAV6 viral titers from WT 293Ts as well as 293 Ts induced to express: i) SKA2; ii) ITPRIP; or iii) both SKA2 and ITPRIP simultaneously (“dual”). 
       DEFINITIONS 
       “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330:375. The term “AAV” also includes chimeric AAV. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. 
       An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. 
       An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle. 
       “Packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. 
       AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.” 
       A “helper virus” for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC. 
       “Helper virus function(s)” refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. 
       An “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005)  Mol. Ther.  11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999)  Gene Ther.  6:973. 
       A “replication-competent” virus (e.g. a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e. in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In general, rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences in order to minimize the possibility that replication competent AAV are generated by recombination between AAV packaging genes and an incoming rAAV vector. In many embodiments, rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10 2  rAAV particles, less than about 1 rcAAV per 10 4  rAAV particles, less than about 1 rcAAV per 10 8  rAAV particles, less than about 1 rcAAV per 10 12  rAAV particles, or no rcAAV). 
       The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. 
       A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. 
       Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace &amp; Co., San Diego, California, USA. Alignment programs that permit gaps in the sequence can be used. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See  Meth. Mol. Biol.  70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See  J. Mol. Biol.  48: 443-453 (1970). 
       Of interest is the BestFit program using the local homology algorithm of Smith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. Preferably, the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA. 
       Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in 
       Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: 
       Mismatch Penalty: 1.00; 
       Gap Penalty: 1.00; 
       Gap Size Penalty: 0.33; and 
       Joining Penalty: 30.0. 
       A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. 
       The term “guide RNA”, as used herein, refers to an RNA that comprises: i) an “activator” nucleotide sequence that binds to a CRISPR/Cas effector polypeptide (e.g., a class 2 CRISPR/Cas effector polypeptide such as a type II, type V, or type VI CRISPR/Cas effector polypeptide) and activates the CRISPR/Cas effector polypeptide; and ii) a “targeter” nucleotide sequence that comprises a nucleotide sequence that hybridizes with a target nucleic acid. The “activator” nucleotide sequence and the “targeter” nucleotide sequence can be on separate RNA molecules (e.g., a “dual-guide RNA”); or can be on the same RNA molecule (a “single-guide RNA”). 
       A “small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”). An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is “targeted” to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19-25 nucleotides in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. 
       As used herein, the term “microRNA” refers to any type of interfering RNAs, including but not limited to, endogenous microRNAs and artificial microRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAs naturally encoded in the genome which are capable of modulating the productive utilization of mRNA. An artificial microRNA can be any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the activity of an mRNA. A microRNA sequence can be an RNA molecule composed of any one or more of these sequences. MicroRNA (or “miRNA”) sequences have been described in publications such as Lim, et al., 2003, Genes &amp; Development, 17, 991-1008, Lim et al., 2003, Science, 299, 1540, Lee and Ambrose, 2001, Science, 294, 862, Lau et al., 2001, Science 294, 858-861, Lagos-Quintana et al., 2002, Current Biology, 12, 735-739, Lagos-Quintana et al., 2001, Science, 294, 853-857, and Lagos-Quintana et al., 2003, RNA, 9, 175-179. Examples of microRNAs include any RNA that is a fragment of a larger RNA or is a miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNA. See, e.g., US Patent Applications 20050272923, 20050266552, 20050142581, and 20050075492. A “microRNA precursor” (or “pre-miRNA”) refers to a nucleic acid having a stem-loop structure with a microRNA sequence incorporated therein. A “mature microRNA” (or “mature miRNA”) includes a microRNA that has been cleaved from a microRNA precursor (a “pre-miRNA”), or that has been synthesized (e.g., synthesized in a laboratory by cell-free synthesis), and has a length of from about 19 nucleotides to about 27 nucleotides, e.g., a mature microRNA can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt. A mature microRNA can bind to a target mRNA and inhibit translation of the target mRNA. 
       “Recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. 
       A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter. 
       “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained. 
       An “expression vector” is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art. 
       “Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV. 
       The terms “genetic alteration” and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term. 
       A cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. 
       The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a gene product to a mammalian subject (which may be referred to as “transgenes” to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function. 
       An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this disclosure are increasingly more isolated. An isolated nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure. 
       Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. 
       Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. 
       Unless defined otherwise, 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 invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. 
       It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a mammalian cell” includes a plurality of such cells and reference to “the AAV virion” includes reference to one or more AAV virions and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. 
       It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. 
       The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. 
       DETAILED DESCRIPTION 
       The present disclosure provides an in vitro mammalian cell that is genetically modified to provide for enhanced production of adeno-associated virus (AAV) virions. The mammalian cells can be used to produce AAV virions, e.g., recombinant AAV virions that include a heterologous nucleic acid encoding a gene product; the present disclosure thus provides methods for producing an AAV virion, which may be a recombinant AAV virion. The present disclosure also provides methods for identifying genes that, when overexpressed or knocked out in a mammalian cell, provide for enhanced production of AAV by the mammalian cell. 
       Genetically Modified Mammalian Cells 
       The present disclosure provides an in vitro mammalian cell that is genetically modified to provide for enhanced production of AAV virions by the cell. In some cases, the genetic modification provides for increased synthesis of a gene product by the cell, compared to the amount of the gene product synthesized by a control cell that does not comprise the genetic modification. In some cases, the genetic modification provides for decreased synthesis of a gene product by the cell, compared to the amount of the gene product synthesized by a control cell that does not comprise the genetic modification. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises two or more genetic modifications that together result in enhanced production of AAV virions by the cell. For example, in some cases, a genetically modified mammalian cell of the present disclosure comprises: i) a first genetic modification that results in increased production of a first gene product compared to the level of production of the first gene product in a control mammalian cell that does not comprise the first genetic modification; and ii) a second genetic modification that results in increased production of a second gene product compared to the level of production of the second gene product in a control mammalian cell that does not comprise the second genetic modification. As another example, in some cases, a genetically modified mammalian cell of the present disclosure comprises: i) a first genetic modification that results in decreased production (e.g., results in a knock out) of a first gene product compared to the level of production of the first gene product in a control mammalian cell that does not comprise the first genetic modification; and ii) a second genetic modification that results in decreased production (e.g., results in a knock out) of a second gene product compared to the level of production of the second gene product in a control mammalian cell that does not comprise the second genetic modification. As another example, in some cases, a genetically modified mammalian cell of the present disclosure comprises: i) a first genetic modification that results in decreased production (e.g., results in a knock out) of a first gene product compared to the level of production of the first gene product in a control mammalian cell that does not comprise the first genetic modification; and ii) a second genetic modification that results in increased production of a second gene product compared to the level of production of the second gene product in a control mammalian cell that does not comprise the second genetic modification. 
       A genetically modified mammalian cell of the present disclosure provides for enhanced production of AAV virions by the cell. For example, when a genetically modified mammalian cell of the present disclosure comprises an AAV genome (or a recombinant AAV genome, as described below), and is provided with helper functions, the genetically modified mammalian cell produces AAV virions (which may be recombinant AAV virions) at a level that is at least 25%, at least 50%, at least 75%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, from about 5-fold to about 10-fold, or more than 10-fold, higher than the amount of AAV virions produced by a control cell not comprising the genetic modification(s). 
       Overexpressed Gene Products 
       Genetic modifications that result in increased production of a gene product include, e.g., insertion into the genome of a mammalian cell of an exogenous nucleic acid comprising a nucleotide sequence encoding the gene product. 
       Non-limiting examples of gene products that, when overexpressed in a mammalian cell, provide for increased production of AAV virions by the cell, compared to the level of production of AAV virions by a control cell not genetically modified to overexpress the gene product, include, but are not limited to, a spindle and kinetochore associated complex subunit 2 (SKA2) polypeptide; an Inositol Tri-Phosphate Receptor Interacting Protein (ITPRIP2; also referred to herein as “ITPRIP”) polypeptide; a CUGBP, Elav-like family member 2 (CELF2) polypeptide; a centrosomal protein 128 (CEP128) polypeptide; a protocadherin alpha 2 (PCDHA2) polypeptide; and an acetylcholinesterase (Cartwright blood group) (ACHE) polypeptide 
       For example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of a SKA2 polypeptide, compared to the level of the SKA2 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a SKA2 polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the SKA2 polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of SKA2 polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the SKA2 polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the SKA2 polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the SKA2 polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter Amino acid sequences of SKA2 polypeptides are known in the art. See, e.g., GenBank Accession No. NP_001094065. In some cases, the SKA2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following SKA2 amino acid sequence: MASEVGHNLE SPETPGGGGW TRVEFPPPAP KGAATVWCLN RLGSRKLSLI WITFNTGWNM KSRLIILIQQ VSCHH (SEQ ID NO: 1). 
       As another example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of an ITPRIP2 polypeptide, compared to the level of the ITPRIP2 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a ITPRIP2 polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the ITPRIP2 polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of ITPRIP2 polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the ITPRIP2 polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the ITPRIP2 polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the ITPRIP2 polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter. Amino acid sequences of ITPRIP2 polypeptides are known in the art. In some cases, the ITPRIP2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ITPRIP2 amino acid sequence depicted in  FIG. 6 . 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that results in increased production of a SKA2 polypeptide, compared to the level of the SKA2 polypeptide produced by a control cell not comprising the genetic modification, where the genetically modified mammalian cell does not comprise any other genetic modifications that provide for increased production of an AAV virion by the cell. In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that results in increased production of a ITPRIP2 polypeptide, compared to the level of the ITPRIP2 polypeptide produced by a control cell not comprising the genetic modification, where the genetically modified mammalian cell does not comprise any other genetic modifications that provide for increased production of an AAV virion by the cell. In some cases, a mammalian cell of the present disclosure comprises: i) a first genetic modification that results in increased production of a SKA2 polypeptide, compared to the level of the SKA2 polypeptide produced by a control cell not comprising the first genetic modification; and ii) a second genetic modification that results in increased production of a ITPRIP2 polypeptide, compared to the level of the ITPRIP2 polypeptide produced by a control cell not comprising the second genetic modification. 
       As another example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of a CELF2 polypeptide, compared to the level of the CELF2 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a CELF2 polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the CELF2 polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of CELF2 polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the CELF2 polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the CELF2 polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the CELF2 polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter. Amino acid sequences of CELF2 polypeptides are known in the art. In some cases, the CELF2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the CELF2 amino acid sequence depicted in  FIG. 7 . 
       As another example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of a CEP128 polypeptide, compared to the level of the CEP128 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a CEP128 polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the CEP128 polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of CEP128 polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the CEP128 polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the CEP128 polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the CEP128 polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter. Amino acid sequences of CEP128 polypeptides are known in the art. In some cases, the CEP128 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the CEP128 amino acid sequence depicted in  FIG. 8 . 
       As another example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of a PCDHA2 polypeptide, compared to the level of the PCDHA2 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a PCDHA2 polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the PCDHA2 polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of PCDHA2 polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the PCDHA2 polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the PCDHA2 polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the PCDHA2 polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter. Amino acid sequences of PCDHA2 polypeptides are known in the art. In some cases, the PCDHA2 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the PCDHA2 amino acid sequence depicted in  FIG. 9 . 
       As another example, in some cases, a mammalian cell of the present disclosure comprises a genetic modification that provides for increased production of a ACHE polypeptide, compared to the level of the ACHE polypeptide produced by a control cell not comprising the genetic modification. In some cases, a nucleic acid comprising a nucleotide sequence encoding a ACHE polypeptide is introduced into a recipient mammalian cell, generating a genetically modified mammalian cell that produces the ACHE polypeptide at a level that is at least 20%, at least 25%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or more than 25-fold, higher than the level of ACHE polypeptide produced by the recipient (unmodified) cell. All or a portion of the nucleic acid comprising the nucleotide sequence encoding the ACHE polypeptide can integrate into the genome of the genetically modified cell. Alternatively, the nucleic acid comprising the nucleotide sequence encoding the ACHE polypeptide can be present in the genetically modified cell episomally (not integrated into the genome of the genetically modified cell. The nucleotide sequence encoding the ACHE polypeptide can be operably linked to a constitutive promoter or a regulatable (e.g., inducible) promoter. Amino acid sequences of ACHE polypeptides are known in the art. In some cases, the ACHE polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ACHE amino acid sequence depicted in  FIG. 10 . 
       As noted above, in some cases, a nucleotide sequence encoding a polypeptide that, when overexpressed, provides for increased production of an AAV virion in a genetically modified mammalian cell of the present disclosure, is operably linked to a transcriptional control element such as a promoter. Suitable promoters include constitutively active promoters (i.e., a promoter that is constitutively in an active/“ON” state). Suitable promoters include inducible promoters (i.e., a promoter whose state, active/“ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). Suitable promoter and enhancer elements are known in the art. 
       Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. 
       Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), and temperature/heat-inducible promoters (e.g., heat shock promoters). 
       Knock-Out of a Gene Product 
       Genetic modifications that result in reduced production (e.g., knock-out) of a gene product include, e.g., deletion of all or a portion of a coding region encoding the gene product; insertion of one or more nucleotides such that the coding region encoding the gene product is out of frame; and the like. 
       Genes that, when disrupted, result in: i) reduced production of a gene product encoded by the gene; and ii) increased production of an AAV virion by a mammalian cell comprising a genetic modification that disrupts the gene, include, but are not limited to, an endoplasmic reticulum lectin 1 (ERLEC1) gene (e.g., Gene ID 27248), a kinesin-like protein (KIF23) gene (e.g., Gene ID 9493), an interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene (e.g., Gene ID 24138), a caspase recruitment domain-containing protein 8 (CARDS) gene (e.g., Gene ID 22900), a gene encoding hsa-mir-4770 (e.g., Gene ID 100616373), and a chromosome 5 open reading frame (C5orf52) gene (e.g., Gene ID 100190949). 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of an ERLEC1 polypeptide, compared to the level of the ERLEC1 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the ERLEC1 polypeptide at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of ERLEC1 polypeptide produced by the recipient (unmodified) cell. In some cases, an ERLEC1 polypeptide is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for the ERLEC1 polypeptide. Amino acid sequences of ERLEC1 polypeptides are known in the art. In some cases, the ERLEC1 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the ERLEC1 amino acid sequence depicted in  FIG. 11 . Guide RNAs that target an ERLEC1 gene can be readily generated by those skilled in the art, given the known genomic structure and genomic sequence of an ERLEC1 gene. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of a KIF23 polypeptide, compared to the level of the KIF23 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the KIF23 polypeptide at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of KIF23 polypeptide produced by the recipient (unmodified) cell. In some cases, a KIF23 polypeptide is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for the KIF23 polypeptide Amino acid sequences of KIF23 polypeptides are known in the art. In some cases, the KIF23 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the KIF23 amino acid sequence depicted in  FIG. 12 . Guide RNAs that target a KIF23 gene can be readily generated by those skilled in the art, given the known genomic structure and genomic sequence of a KIF23 gene. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of an IFIT5 polypeptide, compared to the level of the IFIT5 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the IFIT5 polypeptide at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of IFIT5 polypeptide produced by the recipient (unmodified) cell. In some cases, an IFIT5 polypeptide is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for the IFIT5 polypeptide Amino acid sequences of IFIT5 polypeptides are known in the art. In some cases, the IFIT5 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the IFIT5 amino acid sequence depicted in  FIG. 13 . Guide RNAs that target an IFIT5 gene can be readily generated by those skilled in the art, given the known genomic structure and genomic sequence of an IFIT5 gene. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of a CARD8 polypeptide, compared to the level of the CARD8 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the CARD8 polypeptide at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of CARD8 polypeptide produced by the recipient (unmodified) cell. In some cases, a CARD8 polypeptide is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for the CARD8 polypeptide. Amino acid sequences of CARD8 polypeptides are known in the art. In some cases, the CARD8 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the CARD8 amino acid sequence depicted in  FIG. 14 . Guide RNAs that target a CARD8 gene can be readily generated by those skilled in the art, given the known genomic structure and genomic sequence of a CARD8 gene. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of a C5orf52 polypeptide, compared to the level of the C5orf52 polypeptide produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the C5orf52 polypeptide at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of C5orf52 polypeptide produced by the recipient (unmodified) cell. In some cases, a C5orf52 polypeptide is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for the C5orf52 polypeptide. Amino acid sequences of C5orf52 polypeptides are known in the art. In some cases, the C5orf52 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the C5orf52 amino acid sequence depicted in  FIG. 15 . Guide RNAs that target a C5orf52 gene can be readily generated by those skilled in the art, given the known genomic structure and genomic sequence of a C5orf52 gene. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises a genetic modification that provides for reduced production of hsa-mir4770 microRNA. compared to the level of the hsa-mir4770 microRNA produced by a control cell not comprising the genetic modification. In some cases, a genetic modification is made in a recipient mammalian cell, generating a genetically modified mammalian cell that produces the hsa-mir4770 at a level that is at least 50% less, at least 75% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, or at least 99% less, than the level of hsa-mir4770 produced by the recipient (unmodified) cell. In some cases, a hsa-mir4770 is not produced in a genetically modified mammalian cell of the present disclosure or is produced at undetectable levels. In some cases, a CRISPR/Cas effector polypeptide and one or more guide RNAs are introduced into a recipient mammalian cell, where the CRISPR/Cas effector polypeptide and guide RNA(s) provide for deletion or all or a part of a coding region for hsa-mir4770. In some cases, the hsa-mir4770 comprises a nucleotide acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, nucleotide sequence identity to the following hsa-mir4770 nucleotide sequence: GAGUUAUGGGGUCAUCUAUCCUUCCCUUGGAAAAUGAUCUGAGAUGACACUGUAGCUC (SEQ ID NO: 2). Guide RNAs that target a hsa-mir4770-encoding gene can be readily generated by those skilled in the art, given the sequence of hsa-mir4770. 
       Recipient Cells 
       Cells (“recipient cells”) that can be used to generate a genetically modified mammalian cell of the present disclosure include any mammalian cell that can be cultured in vitro and that can produce an AAV virion. A recipient cell is genetically modified, as described above, to provide for increased production of an AAV virion, compared to the level of production of the AAV virion by a control cell not comprising the genetic modification. Examples of suitable recipient cells include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. 
       AAV virions 
       A genetically modified mammalian cell of the present disclosure can comprise an AAV genome. In some cases, the AAV genome is a wild-type AAV genome; e.g., where the AAV genome comprises a nucleotide sequence of a naturally-occurring AAV. A genetically modified mammalian cell of the present disclosure can comprise a recombinant AAV genome, e.g., an AAV genome in which part of the AAV genome is replaced with a heterologous nucleic acid. 
       Recombinant AAV and Recombinant AAV Virions 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises: a) a nucleic acid comprising a nucleotide sequence encoding an AAV capsid; and b) a heterologous nucleic acid (e.g., a nucleic acid comprising a nucleotide sequence encoding a gene product that is heterologous to AAV, i.e., a gene product that is not encoded by a naturally occurring AAV). In some cases, the heterologous gene product is a nucleic acid. In some cases, the heterologous gene product is a polypeptide. 
       In some cases, a genetically modified mammalian cell of the present disclosure comprises: a) a nucleic acid comprising a nucleotide sequence encoding an AAV capsid, where the AAV capsid is a variant AAV capsid that provides for one or more of: i) increased infectivity of a non-permissive cell compared to the infectivity of the non-permissive cell by an AAV virion comprising a wild-type AAV capsid protein; ii) reduced susceptibility to neutralization by a neutralizing antibody specific; and iii) increased ability to cross a physiological barrier (e.g., blood-brain barrier; inner limiting membrane; and the like); and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product. Such a genetically modified mammalian cell can produce a recombinant AAV virion (rAAV virion). 
       A recombinant AAV (rAAV) (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products (one or more heterologous gene products). In some cases, the gene product is a polypeptide. In some cases, the gene product is an RNA. In some cases, an rAAV (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises a heterologous nucleotide sequence encoding both a heterologous nucleic acid gene product and a heterologous polypeptide gene product. Where the gene product is an RNA, in some cases, the RNA gene product encodes a polypeptide. Where the gene product is an RNA, in some cases, the RNA gene product does not encode a polypeptide. In some cases, an rAAV (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding a single heterologous gene product. In some cases, an rAAV (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding two heterologous gene products. Where the single heterologous nucleic acid encodes two heterologous gene products, in some cases, nucleotide sequences encoding the two heterologous gene products are operably linked to the same promoter. Where the single heterologous nucleic acid encodes two heterologous gene products, in some cases, nucleotide sequences encoding the two heterologous gene products are operably linked to two different promoters. In some cases, an rAAV (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding three heterologous gene products. Where the single heterologous nucleic acid encodes three heterologous gene products, in some cases, nucleotide sequences encoding the three heterologous gene products are operably linked to the same promoter. Where the single heterologous nucleic acid encodes three heterologous gene products, in some cases, nucleotide sequences encoding the three heterologous gene products are operably linked to two or three different promoters. In some cases, an rAAV (or an rAAV virion produced by a genetically modified mammalian cell of the present disclosure) comprises two heterologous nucleic acids, each comprising a nucleotide sequence encoding a heterologous gene product. 
       In some cases, the gene product is a polypeptide-encoding RNA. In some cases, the gene product is an interfering RNA. In some cases, the gene product is an aptamer. In some cases, the gene product is a polypeptide. In some cases, the gene product is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit. In some cases, the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function. In some embodiments, the gene product is a CRISPR/Cas effector polypeptide that provides for modification of a target nucleic acid. In some cases, the gene products are: i) a CRISPR/Cas effector polypeptide that provides for modification of a target nucleic acid; and ii) a guide RNA that comprises a first segment that binds to a target sequence in a target nucleic acid and a second segment that binds to the CRISPR/Cas effector polypeptide. In some cases, the gene products are: i) a CRISPR/Cas effector polypeptide that provides for modification of a target nucleic acid; ii) a first guide RNA that comprises a first segment that binds to a first target sequence in a target nucleic acid and a second segment that binds to the CRISPR/Cas effector polypeptide; and iii) a first guide RNA that comprises a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the CRISPR/Cas effector polypeptide. CRISPR/Cas effector polypeptides include type II CRISPR/Cas effector polypeptides (e.g., Cas9 polypeptides); type V CRISPR/Cas effector polypeptides (e.g., a Cas12a polypeptide; a Cas12b polypeptide; a Cas12c polypeptide; a Cas12d polypeptide; a Cas12e polypeptide; a Cas13a polypeptide); a CasX polypeptide; a CasY polypeptide; and a CasZ polypeptide. 
       A nucleotide sequence encoding a heterologous gene product in an rAAV virion of the present disclosure can be operably linked to a promoter. For example, a nucleotide sequence encoding a heterologous gene product in an rAAV virion can be operably linked to a constitutive promoter, a regulatable promoter, a tissue-specific promoter, or a cell type-specific promoter. 
       Suitable nucleic acid gene products include interfering RNA, antisense RNA, ribozymes, CRISPR/Cas guide RNAs, and aptamers. Where the gene product is an interfering RNA (RNAi), suitable RNAi include RNAi that decrease the level of an angiogenic factor in a cell. For example, an RNAi can be a miRNA, an shRNA, or an siRNA that reduces the level of vascular endothelial growth factor (VEGF) in a cell. 
       Where the gene product is a polypeptide, exemplary polypeptides include, e.g., an interferon (e.g., IFN-γ, IFN-α, IFN-β, IFN-ω; IFN-τ; an insulin (e.g., Novolin, Humulin, Humalog, Lantus, Ultralente, etc.); an erythropoietin (“EPO”; e.g., Procrit®, Eprex®, or Epogen® (epoetin-α); Aranesp® (darbepoietin-α); NeoRecormon®, Epogin® (epoetin-(β); and the like); an antibody (e.g., a monoclonal antibody) (e.g., Rituxan® (rituximab); Remicade® (infliximab); Herceptin® (trastuzumab); Humira™ (adalimumab); Xolair® (omalizumab); Bexxar® (tositumomab); RaptivaTM (efalizumab); Erbitux™ (cetuximab); and the like), including an antigen-binding fragment of a monoclonal antibody; a blood factor (e.g., Activase® (alteplase) tissue plasminogen activator; NovoSeven® (recombinant human factor VIIa); Factor VIIa; Factor VIII (e.g., Kogenate®); Factor IX; β-globin; hemoglobin; and the like); a colony stimulating factor (e.g., Neupogen® (filgrastim; G-CSF); Neulasta (pegfilgrastim); granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte colony stimulating factor, macrophage colony stimulating factor, megakaryocyte colony stimulating factor; and the like); a growth hormone (e.g., a somatotropin, e.g., Genotropin®, Nutropin®, Norditropin®, Saizen®, Serostim®, Humatrope®, etc.; a human growth hormone; and the like); an interleukin (e.g., IL-1; IL-2, including, e.g., Proleukin®; IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9; etc.); a growth factor (e.g., Regranex® (beclapermin; PDGF); Fiblast® (trafermin; bFGF); Stemgen® (ancestim; stem cell factor); keratinocyte growth factor; an acidic fibroblast growth factor, a stem cell factor, a basic fibroblast growth factor, a hepatocyte growth factor; and the like); a soluble receptor (e.g., a TNF-α-binding soluble receptor such as Enbrel® (etanercept); a soluble VEGF receptor; a soluble interleukin receptor; a soluble γ/δ T cell receptor; and the like); an enzyme (e.g., a-glucosidase; Cerazyme® (imiglucarase; β-glucocerebrosidase, Ceredase® (alglucerase;); an enzyme activator (e.g., tissue plasminogen activator); a chemokine (e.g., IP-10; Mig; Groa/IL-8, RANTES; MIP-1α; MIP-1β; MCP-1; PF-4; and the like); an angiogenic agent (e.g., vascular endothelial growth factor (VEGF) ; an anti-angiogenic agent (e.g., a soluble VEGF receptor); a protein vaccine; a neuroactive peptide such as bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, etc.; other proteins such as a thrombolytic agent, an atrial natriuretic peptide, bone morphogenic protein, thrombopoietin, relaxin, glial fibrillary acidic protein, follicle stimulating hormone, a human alpha-1 antitrypsin, a leukemia inhibitory factor, a transforming growth factor, an insulin-like growth factor, a luteinizing hormone, a macrophage activating factor, tumor necrosis factor, a neutrophil chemotactic factor, a nerve growth factor a tissue inhibitor of metalloproteinases; a vasoactive intestinal peptide, angiogenin, angiotropin, fibrin; hirudin; a leukemia inhibitory factor; an IL-1 receptor antagonist (e.g., Kineret® (anakinra)); an ion channel, e.g., cystic fibrosis transmembrane conductance regulator (CFTR); dystrophin; utrophin, a tumor suppressor; lysosomal enzyme acid α-glucosidase (GAA); and the like. Suitable nucleic acids also include those that encode a functional fragment of any of the aforementioned proteins; and nucleic acids that encode functional variants of any of the aforementioned proteins. 
       Where the gene product is a polypeptide, exemplary polypeptides include neuroprotective polypeptides and anti-angiogenic polypeptides. Suitable polypeptides include, but are not limited to, glial derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), nurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-β), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelium derived factor (PEDF), a Wnt polypeptide, soluble Flt-1, angiostatin, endostatin, an anti-VEGF antibody, a soluble VEGFR, and a member of the hedgehog family (sonic hedgehog, indian hedgehog, and desert hedgehog, etc.). 
       Where the rAAV virion comprises a variant capsid protein, in some cases, the variant AAV capsid protein comprises from 1 to about 10 amino acid differences (e g , amino acid substitutions and/or amino acid insertions and/or amino acid deletions) compared to a wild-type AAV capsid protein. The amino acid difference(s) can be located in a solvent accessible site in the capsid, e.g., a solvent-accessible loop. For example, the amino acid substitution(s) can be located in a GH loop in the AAV capsid protein. As one non-limiting example, the variant capsid protein can comprise an amino acid substitution at amino acid 451 of AAV6 capsid, or the corresponding position in another AAV serotype. As another non-limiting example, the variant capsid protein can comprise an amino acid substitution at amino acid 532 of AAV6 capsid, or the corresponding position in another AAV serotype. In some cases, the variant capsid comprises an insertion of a peptide comprising LGETTRP, where the insertion site is between amino acids corresponding to 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. In some cases, the variant capsid comprises an insertion of a peptide comprising LALGETTRPA, where the insertion site is between amino acids corresponding to 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. See, e.g., U.S. Pat. Nos. 9,193,956; 8,663,624; 9,457,103. 
       Method of Producing an AAV Virion 
       The present disclosure provides a method for producing an AAV virion, the method comprising: 
       a) culturing a genetically modified mammalian cell of the present disclosure in vitro in a liquid culture medium, where the genetically modified mammalian cell comprises an AAV genome, such that the genetically modified mammalian cell produces an AAV virion comprising the AAV genome; and b) harvesting the AAV virion from the culture medium. In some cases, the genetically modified mammalian cell comprises: a) a nucleic acid comprising a nucleotide sequence encoding an AAV capsid; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more heterologous gene products. In some cases, the AAV capsid is a variant AAV capsid. In some cases, the one or more heterologous gene products is a nucleic acid. In some cases, the one or more heterologous gene products is a polypeptide. In some cases, the heterologous gene products comprise a nucleic acid gene product and a polypeptide gene product. 
       A genetically modified mammalian cell of the present disclosure can function as “producer” cell for AAV vector replication and packaging. Such a producer cell generally comprises or is modified to comprise several different types of components for AAV (including rAAV) production. As noted above, in some cases, a genetically modified mammalian cell of the present disclosure can comprise an AAV genome or a recombinant adeno-associated viral (rAAV) vector genome (or “rAAV pro-vector”) that can be replicated and packaged into particles by the host packaging cell. The rAAV pro-vector comprises a heterologous nucleic acid (or “transgene”), where the heterologous nucleic acid can be flanked by two AAV inverted terminal repeats (ITRs) which comprise sequences that are recognized during excision, replication and packaging of the AAV vector, as well as during integration of the vector into a host cell genome. 
       A second component is a helper virus that can provide helper functions for AAV replication, or a helper recombinant vector that comprises a nucleotide sequence encoding helper functions. Although adenovirus is commonly employed, other helper viruses can also be used as is known in the art. Alternatively, the requisite helper virus functions can be isolated genetically from a helper virus and the encoding genes can be used to provide helper virus functions in trans. The AAV vector elements and the helper virus (or helper virus functions) can be introduced into the host cell either simultaneously or sequentially in any order. 
       The final components for AAV production to be provided in the producer cell are “AAV packaging genes” such as AAV rep and cap genes that provide replication and encapsidation proteins, respectively. Several different versions of AAV packaging genes can be provided (including rep-cap cassettes and separate rep and/or cap cassettes in which the rep and/or cap genes can be left under the control of the native promoters or operably linked to heterologous promoters. Such AAV packaging genes can be introduced either transiently or stably into the host packaging cell (e.g., a genetically modified mammalian cell of the present disclosure), as is known in the art. 
       In some cases, a genetically modified mammalian cell of the present disclosure is further genetically modified with one or more nucleic acids comprising nucleotide sequences encoding one or more of: AAV rep proteins; and AAV cap protein. In some cases, such nucleic acids encoding helper functions are integrated into the genome of a genetically modified mammalian cell of the present disclosure. 
       To produce an AAV virion (which may be an rAAV virion), a genetically modified mammalian cell of the present disclosure (where the genetically modified mammalian cell comprises an AAV genome; or comprises: a) a nucleic acid comprising a nucleotide sequence encoding an AAV capsid; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more heterologous gene products) is modified with a helper virus or one or more recombinant expression vectors comprising nucleotide sequences encoding helper functions. Thus, in some cases, a method of the present disclosure comprises: A) culturing a genetically modified mammalian cell of the present disclosure in vitro in a liquid culture medium, where the genetically modified mammalian cell comprises: a) an AAV genome; or b) i) a nucleic acid comprising a nucleotide sequence encoding an AAV capsid; and ii) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more heterologous gene products; B) introducing into the genetically modified mammalian cell a nucleic acid comprising a nucleotide sequence encoding helper functions, such that the genetically modified mammalian cell produces an AAV virion or an rAAV virion; and C) harvesting the AAV virion from the culture medium. 
       In some cases, the harvested AAV virions are purified. In some cases, the AAV virions are purified to at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99%, purity. 
       Suitable liquid culture media include any culture medium that provides for growth and/or viability of a mammalian cell in in vitro culture. 
       Methods of Identifying Genes that, When Overexpressed, Provide for Increased Production of AAV Virions 
       The present disclosure provides a method of identifying a gene product that, when overexpressed, enhances production of a virus by a mammalian cell. The method comprises: A) introducing into a plurality of mammalian cells: a) a nucleic acid comprising a fusion polypeptide comprising: i) CRISPR-Cas effector polypeptide that, when complexed with a CRISPR-Cas guide RNA, binds, but does not cleave, a target nucleic acid in the mammalian cell; and ii) a transcriptional activator polypeptide; b) a library of CRISPR-Cas guide RNAs, or a nucleic acid comprising a nucleotide sequence encoding the library of CRISPR-Cas guide RNAs, thereby producing a plurality of CRISPR-Cas effector polypeptide/guide RNA-modified cells; B) introducing into the CRISPR-Cas effector polypeptide/guide RNA-modified cells a viral genome and a nucleic acid comprising a nucleotide sequence encoding helper functions for packaging the viral genome into a viral virion, thereby producing a plurality of packaging cells; and C) determining the number of virions produced by the packaging cell. An increase in virion production by an individual packaging cell in the plurality of packaging cells, compared to the level of virion production by a control mammalian cell not modified with the guide RNA introduced into the individual packaging cell, indicates that the gene product encoded by the target of the guide RNA, when overexpressed, enhances production of the virus by the cell. In some cases, the transcriptional activator polypeptide is a VP64 polypeptide. 
       Suitable the CRISPR-Cas effector polypeptides are known to those skilled in the art and include, e.g., a Cas9 polypeptide. 
       In some cases, the guide RNAs present in the library of guide RNAs comprise a protein-binding RNA aptamer. In some cases, the aptamer is capable of binding an adaptor protein. In some cases, the adaptor protein comprises a bacteriophage coat protein. For example, a suitable bacteriophage coat protein comprises an MS2 polypeptide. As an example, the adaptor protein can be an MS2-P65-HSF1 fusion polypeptide. In some cases, the mammalian cell line is genetically modified with a nucleic acid comprising a nucleotide sequence encoding the adaptor protein. 
       A method of the present disclosure can further comprise sequencing the nucleic acid encoding the identified gene product. 
       Methods of Identifying Genes that, When Knocked Out, Provide for Increased Production of AAV Virions 
       The present disclosure provides a method of identifying a gene product that when knocked out, enhances production of a virus by a mammalian cell. The method comprises: A) introducing into a plurality of mammalian cells: a) a CRISPR-Cas effector polypeptide or a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptide; b) a library of CRISPR-Cas guide RNAs, or a nucleic acid comprising a nucleotide sequence encoding the library of CRISPR-Cas guide RNAs, thereby producing a plurality of CRISPR-Cas effector polypeptide/guide RNA-modified cells; B) introducing into the CRISPR-Cas effector polypeptide/guide RNA-modified cells a viral genome and a nucleic acid comprising a nucleotide sequence encoding helper functions for packaging the viral genome into a viral virion, thereby producing a plurality of packaging cells; and C) determining the number of virions produced by the packaging cell. A decrease in virion production by an individual packaging cell in the plurality of packaging cells, compared to the level of virion production by a control mammalian cell not modified with the guide RNA introduced into the individual packaging cell, indicates that the gene product encoded by the target of the guide RNA, when knocked out, enhances production of the virus by the cell. A method of the present disclosure can further comprise sequencing the nucleic acid encoding the identified gene product. 
       Examples of Non-Limiting Aspects of the Disclosure 
       Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-42 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below: 
       Aspect 1. An in vitro mammalian cell comprising one or more genetic modifications that provide for increased adeno-associated virus (AAV) virion production by the cell. 
       Aspect 2. The mammalian cell of aspect 1, wherein the genetic modification results in decreased production in the cell of a gene product encoded by a coding region. 
       Aspect 3. The mammalian cell of aspect 2, wherein the genetic modification comprises a deletion of all or a portion of the coding region. 
       Aspect 4. The mammalian cell of aspect 2, wherein the genetic modification comprises insertion of one or more nucleotides such that the coding region is out of frame. 
       Aspect 5. The mammalian cell of aspect 1, wherein the genetic modification results in increased production of a polypeptide compared to a control mammalian cell not comprising the one or more genetic modifications. 
       Aspect 6. The mammalian cell of aspect 1, comprising two or more genetic modifications, wherein the two or more genetic modifications provide for: 
       a) decreased production in the cell of one or more gene products encoded by the at least one coding region; and/or 
       b) increased production of one or more polypeptides compared to a control mammalian cell not comprising the one or more genetic modifications. 
       Aspect 7. The mammalian cell of any one of aspects 1, 5, or 6, wherein the one or more genetic modifications provides for increased production of one or more polypeptides selected from: a Spindle and Kinetochore Associated Complex subunit 2 (SKA2) polypeptide; an Inositol Tri-Phosphate Receptor Interacting Protein (ITPRIP2) polypeptide; a CUGBP, Elav-like family member 2 (CELF2) polypeptide; a centrosomal protein 128 (CEP128) polypeptide, a protocadherin alpha 2 (PCDHA2) polypeptide; and an acetylcholinesterase (Cartwright blood group) (ACHE) polypeptide. 
       Aspect 8. The mammalian cell of any one of aspects 1-4 or 6, wherein the one or more genetic modifications provides for reduced production of one or more gene products selected from: an endoplasmic reticulum lectin 1 polypeptide; a kinesin-like protein KIF23 polypeptide; an interferon-induced protein with tetratricopeptide repeats 5 polypeptide; a caspase recruitment domain-containing protein 8 polypeptide; and a mir-4770 nucleic acid. 
       Aspect 9. The mammalian cell of aspect 1, wherein the one or more genetic modifications provides for increased production of: 
       a) a Spindle and Kinetochore Associated Complex subunit 2 (SKA2) polypeptide; or 
       b) an Inositol Tri-Phosphate Receptor Interacting Protein (ITPRIP2) polypeptide; or 
       c) both a SKA2 polypeptide and an ITPRIP2 polypeptide, 
       compared to a control mammalian cell not comprising the one or more genetic modifications. 
       Aspect 10. The mammalian cell of aspect 9, wherein the genetic modification comprises integration into the genome of the cell of a nucleic acid comprising a nucleotide sequence encoding the SKA2 polypeptide and/or the ITPRIP2 polypeptide. 
       Aspect 11. The mammalian cell of aspect 10, wherein the nucleotide sequence is operably linked to a heterologous transcriptional control element. 
       Aspect 12. The mammalian cell of aspect 11, wherein the heterologous transcriptional control element is a regulatable promoter. 
       Aspect 13. The mammalian cell of aspect 12, wherein the regulatable promoter is an inducible promoter. 
       Aspect 14. The mammalian cell of aspect 12, wherein the regulatable promoter is a repressible promoter. 
       Aspect 15. The mammalian cell of any one of aspects 9-14, wherein the SKA2 polypeptide and/or the ITPRIP2 polypeptide are produced at a level that is at least 25% higher than the level of the SKA2 polypeptide and/or the ITPRIP2 polypeptide in a control cell. 
       Aspect 16. The mammalian cell of any one of aspects 9-14, wherein the SKA2 polypeptide and/or the ITPRIP2 polypeptide are produced at a level that is at least 2-fold higher than the level of the SKA2 polypeptide and/or the ITPRIP2 polypeptide in a control cell. 
       Aspect 17. The mammalian cell of any one of aspects 9-14, wherein the SKA2 polypeptide and/or the ITPRIP2 polypeptide are produced at a level that is at least 10-fold higher than the level of the SKA2 polypeptide and/or the ITPRIP2 polypeptide in a control cell. 
       Aspect 18. The mammalian cell of any one of aspects 9-14, wherein the SKA2 polypeptide and/or the ITPRIP2 polypeptide are produced at a level that is at least 50-fold higher than the level of the SKA2 polypeptide and/or the ITPRIP2 polypeptide in a control cell. 
       Aspect 19. The mammalian cell of any one of aspects 9-14, wherein the SKA2 polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to: 
       
         
           
             
                 
                 
              
                 
                     
                   (SEQ ID NO: 1) 
                 
                 
                     
                   MASEVGHNLE SPETPGGGGW TRVEFPPPAP KGAATVWCLN 
                 
                 
                     
                     
                 
                 
                     
                   RLGSRKLSLI WITFNTGWNM KSRLIILIQQ VSCHH. 
                 
              
             
           
         
       
       Aspect 20. The mammalian cell of any one of aspects 9-14, wherein the ITPRIP2 polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence depicted in  FIG. 6 . 
       Aspect 21. The mammalian cell of any one of aspects 1-20, comprising an adeno-associated virus (AAV) genome. 
       Aspect 22. The mammalian cell of aspect 21, wherein the AAV genome is a recombinant AAV genome comprising a heterologous nucleic acid encoding one or more heterologous gene products. 
       Aspect 23. The mammalian cell of aspect 22, wherein the one or more heterologous gene products comprises a polynucleotide. 
       Aspect 24. The mammalian cell of aspect 22, wherein the one or more heterologous gene products comprises a polypeptide. 
       Aspect 25. The mammalian cell of any one of aspects 21-24, wherein the cell, when provided with helper functions, packages the AAV genome into an AAV virion, and produces the AAV virion at a level that is higher than the level of AAV virion produced by a control cell. 
       Aspect 26. The mammalian cell of aspect 25, wherein the AAV virion is produced at a level that is at least 1.5 times higher than the level of AAV virion produced by a control cell. 
       Aspect 27. The mammalian cell of aspect 25, wherein the AAV virion is produced at a level that is at least 2 times higher than the level of AAV virion produced by a control cell. 
       Aspect 28. A method of producing an AAV virion, the method comprising: 
       a) culturing the mammalian cell of any one of aspects 21-27 in vitro in a liquid culture medium such that the mammalian cell produces the AAV virion; and 
       b) harvesting the AAV virion from the culture medium. 
       Aspect 29. The method of aspect 28, comprising purifying the AAV virion from the culture medium. 
       Aspect 30. The method of aspect 28 or aspect 29, wherein the AAV virion is a recombinant 
       AAV virion comprising a heterologous nucleic acid encoding one or more heterologous gene products. 
       Aspect 31. The method of aspect 30, wherein the one or more heterologous gene products comprises a polynucleotide. 
       Aspect 32. The method of aspect 28, wherein the one or more heterologous gene products comprises a polypeptide. 
       Aspect 33. A method of identifying a gene product that, when overexpressed, enhances production of a virus by a mammalian cell, the method comprising: 
       A) introducing into a plurality of mammalian cells: 
       a) a nucleic acid comprising a fusion polypeptide comprising: i) CRISPR-Cas effector polypeptide that, when complexed with a CRISPR-Cas guide RNA, binds, but does not cleave, a target nucleic acid in the mammalian cell; and ii) a transcriptional activator polypeptide; 
       b) a library of CRISPR-Cas guide RNAs, or a nucleic acid comprising a nucleotide sequence encoding the library of CRISPR-Cas guide RNAs, 
       thereby producing a plurality of CRISPR-Cas effector polypeptide/guide RNA-modified cells; 
       B) introducing into the CRISPR-Cas effector polypeptide/guide RNA-modified cells a viral genome and a nucleic acid comprising a nucleotide sequence encoding helper functions for packaging the viral genome into a viral virion, thereby producing a plurality of packaging cells; and 
       C) determining the number of virions produced by the packaging cell, 
       wherein an increase in virion production by an individual packaging cell in the plurality of packaging cells, compared to the level of virion production by a control mammalian cell not modified with the guide RNA introduced into the individual packaging cell, indicates that the gene product encoded by the target of the guide RNA, when overexpressed, enhances production of the virus by the cell. 
       Aspect 34. The method of aspect 33, wherein the guide RNAs present in the library of guide 
       RNAs comprise a protein-binding RNA aptamer. 
       Aspect 35. The method of aspect 34, wherein the aptamer is capable of binding an adaptor protein. 
       Aspect 36. The method of aspect 35, wherein the adaptor protein comprises a bacteriophage coat protein. 
       Aspect 37. The method of aspect 36, wherein the bacteriophage coat protein comprises an MS2 polypeptide. 
       Aspect 38. The method of aspect 37, wherein the adaptor protein is an MS2-P65-HSF1 fusion polypeptide. 
       Aspect 39. The method of any one of aspects 35-38, wherein the mammalian cell line is genetically modified with a nucleic acid comprising a nucleotide sequence encoding the adaptor protein. 
       Aspect 40. The method of any one of aspects 33-39, wherein the transcriptional activator polypeptide is a VP64 polypeptide. 
       Aspect 41. The method of any one of aspects 33-40, comprising sequencing the nucleic acid encoding the identified gene product. 
       Aspect 42. A method of identifying a gene product that, when knocked out, enhances production of a virus by a mammalian cell, the method comprising: 
       A) introducing into a plurality of mammalian cells: 
       a) a CRISPR-Cas effector polypeptide or a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptide; 
       b) a library of CRISPR-Cas guide RNAs, or a nucleic acid comprising a nucleotide sequence encoding the library of CRISPR-Cas guide RNAs, 
       thereby producing a plurality of CRISPR-Cas effector polypeptide/guide RNA-modified cells; 
       B) introducing into the CRISPR-Cas effector polypeptide/guide RNA-modified cells a viral genome and a nucleic acid comprising a nucleotide sequence encoding helper functions for packaging the viral genome into a viral virion, thereby producing a plurality of packaging cells; and 
       C) determining the number of virions produced by the packaging cell, 
       wherein a decrease in virion production by an individual packaging cell in the plurality of packaging cells, compared to the level of virion production by a control mammalian cell not modified with the guide RNA introduced into the individual packaging cell, indicates that the gene product encoded by the target of the guide RNA, when knocked out, enhances production of the virus by the cell. 
       EXAMPLES 
       The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. 
       Example 1 
       Human Embryonic Kidney (HEK) cells are one of the primary methods of AAV production. 
       Cells are transfected with three separate plasmids that contain all of the necessary DNA produce and assemble the proteins and DNA for a viable AAV capsid. Given that HEK cells (a highly transformed cell line) have not ever been a natural target of this parvovirus infection, these cells could be genetically modified to increase their ability to make virus. To do this, a genome wide screen of the Genomic CRISPR Knock-Out (GeCKO) or Synergistic Activation Mediator (SAM) was used to either knock-out or over-express every gene in the human genome using a CRISPR/Cas9 system. The method is depicted schematically in  FIG. 1 .  FIG. 1  presents schematic depictions of an iterative AAV manufacturing enhancement process through genetic manipulation via either over-expression mediated by dCas9-VP64 (left panel) or genetic knock-out via Cas9 (right panel). 
       This method focuses on iteratively altering the genetic profile of a cell and then using that altered cell to make virus to select for genetic modifications that enhance virus production. AAV was packaged, such that the AAV contained a library of guide RNAs (gRNAs) to modify genes either via a Cas9 to knock-out genes or a dCas9-VP64 to over-express genes. The AAV-delivered DNA was present as an episome in the nucleus and encoded the gRNAs used to modify the genes. After infection and genetic modification, the episome was then repackaged into virus in the modified cells, and cells with beneficial genetic modifications produced more virus. This means that virus containing the beneficial gRNA was more populous in the overall viral population. The newly packaged virus was used to infect a new batch of cells containing either Cas9 or dCas9-VP64. By iteratively packaging virus, the gRNA responsible for the increased titer became an increasingly greater percentage of the overall virus collected after each round. Eventually over multiple rounds, this library of gRNAs reached a consensus and provided targets for modification to create cell lines that produced AAV at higher titers than in unmodified HEK cells. 
       Expression constructs were made that contained a U6 promoter followed by a library of guide RNAs as well as an expression cassette for GFP flanked by two ITRs. The expression constructs were then packaged into rAAV2 through the standard triple transfection technique. Two guide RNA libraries were used. In one guide RNA library, each of the guide RNAs binds upstream of a target gene and recruits a transactivator, such that the target gene is activated and the encoded gene product overexpressed. The second guide RNA library included guide RNAs that target and knock out genes. 
       HEK293Ts were made that expressed either: enzymatically active Cas9; or dCas9-VP64 with MS2-P65-HSF1. These cells were first infected at a range of multiplicities of infection (MOI) to find the MOI that ensured that each cell contained at least one guide RNA, as measured by green fluorescent protein (GFP) expression. An MOI of 500 was found to be best in a 24-well dish format. Cells were then given 48 hours to ensure onset of guide activation and change in gene expression. 
       Once gene expression had been given time to take effect, such that the Cas9 or dCas9-VP64 was produced, the cells were then transfected with the rAAV2 libraries and helper plasmid. 
       The episome containing the gRNA responsible for guide expression serves as a third plasmid and can be repackaged in the presence of rAAV2 and the helper plasmid. The episome was repackaged into virus and where some cells had altered genetic profiles that enhanced virus production and thus had a larger viral load. Those viruses were harvested and quantified before re-infecting 293Ts expressing Cas9 or dCas9-VP64 at an MOI of 500. The procedure was done 4 times for the KO library and 5 times for the overexpression library before genomes were recovered and the guides were re-cloned and repackaged in wildtype 293Ts. The process was then repeated 3 more times for each library and deep sequencing was done to quantify the fold increase of guide present as compared to the original amount of guide. 
       The results are shown in  FIG. 2  through  FIG. 5 . 
         FIG. 2A-2D  depict: A) the top hits from the GeCKO selection ranked in descending order; B) the top hits from the SAM selection ranked in descending order; C) fold increase of hits increased by over 1500-fold and the corresponding gene; D) quantification of viral genomes per mL (vg/mL) and fold increase over AAV2 production in wildtype HEK293Ts of cells constitutively expressing the guide RNA linked to increased AAV manufacturing capability. 
         FIG. 3A-3B  depict: A) Western blot confirming elevated expression of ITPRIP protein in the cell line associated with increased titer due to ITPRIP expression; B) quantitative reverse transcription-polymerase chain reaction (qRT-PCR) confirming overexpression of SKA2 in the cell line associated with increased titer due to SKA2 expression. 
         FIG. 4A-4D  depict: A) Box plot showing viral titers in viral genomes (VG) per 15 mL plate of AAV2 as quantified by qPCR; B) Box plot showing infectious units (IU) per 15 mL plate as quantified by GFP expressing cells relative to total cells; C) Average viral titers per 15 mL plate as well as the fold increase relative to the WT 293T condition; D) Average infectious titers per 15 mL plate as well as the fold increase relative to the WT 293T condition. 
         FIG. 5A-5D  depict: A) Box plot showing viral titers in viral genomes (VG) per 15 mL plate of AAV6 as quantified by qPCR; B) Box plot showing infectious units (IU) per 15 mL plate of AAV6 as quantified by GFP expressing cells relative to total cells; C) Average viral titers per 15 mL plate as well as the fold increase relative to the WT 293T condition; D) Average infectious titers per 15 mL plate as well as the fold increase relative to the WT 293T condition. 
       Example 2 
       HEK293T cells were modified to express: i) SKA2; ii) ITPRIP; or iii) both SKA2 and ITPRIP (“dual”). The effect on AAV2 and AAV6 production was assessed. The data are presented in  FIG. 16-20 . 
         FIG. 16  shows AAV2 (upper panel) and AAV6 (lower panel) viral genome fold increase with each round normalized to the wild-type (WT) 293Ts value for that round from SKA2 and ITPRIP expressing cells. Significance is based on Dunnett&#39;s multiple comparisons test where * p≤0.05, ** p≤0.005, *** pA≤1.0005. n≥6. 
         FIG. 17  shows AAV2 (upper panel) and AAV6 (lower panel) viral genome fold increase with each round normalized to the WT 293Ts value for that round. A cell line expressing both genes of interest (“Dual”) exhibited a 3.8-fold increase in packaging AAV2 and 3.5-fold in packaging AAV6. * p≤0.05, *** p≤0.005, **** p≤0.0005 n≥4. 
       As shown in  FIG. 18 , Western blot analysis confirmed upregulation of SKA2 in a cell line expressing SKA2 and in a ‘dual’ cell line expressing both SKA2 and ITPRIP as well as upregulation of ITPRIP in an ITPRIP expressing cell line and in a ‘dual’ cell line expressing both SKA2 and ITPRIP. 
         FIG. 19  presents Table 1: Compiled AAV2 viral titers from WT 293Ts as well as 293Ts induced to express: i) SKA2; ii) ITPRIP; or iii) both SKA2 and ITPRIP simultaneously (“dual”) 
         FIG. 20  presents Table 2: Compiled AAV6 viral titers from WT 293Ts as well as 293Ts induced to express: i) SKA2; ii) ITPRIP; or iii) both SKA2 and ITPRIP simultaneously (“dual”). 
     
    
    
     While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.