Engineered production cell lines and methods for their preparation and use

Abstract

The present application relates to recombinant adeno-associated virus (rAAV) packaging cell lines and / or production cell lines that have been engineered to reduce the expression and / or activity of one or more genes and / or proteins to increase rAAV titers. Methods for generating the engineered cell lines are also described herein.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 833,548, filed on April 12, 2019; U.S. Provisional Patent Application No. 62 / 839,207, filed on April 26, 2019; and U.S. Provisional Patent Application No. 62 / 979,483, filed on February 21, 2020. For all purposes, the disclosures thereof are hereby incorporated by reference in their entireties.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on April 9, 2020, named ULP - 005WO_SL.txt, and is 113 kilobytes in size. Technical Field

[0005] This application generally relates to engineered production cell lines and / or packaging cell lines and methods of generating engineered production cell lines and / or packaging cell lines for increasing the titer of recombinant adeno - associated virus (rAAV). Background Art

[0006] rAAV - based vectors are among the most promising vectors for human gene therapy. rAAV vectors are considered for a wide variety of gene therapy applications. Specifically, rAAV vectors can deliver therapeutic genes to both dividing and non - dividing cells, and these genes can persist for long periods of time without integrating into the genome of the target cells. Although systems for producing rAAV have evolved over the past two decades, several problems remain to be solved. One limitation of rAAV production systems is the low - titer yield of rAAV particles. rAAV - based gene products in pre - clinical stage drug development require large amounts of rAAV vectors for studies in larger species to achieve full toxicology and biodistribution studies that help predict human dosing. In addition, because current rAAV production systems result in low - titer yields, it is challenging to manufacture sufficient levels of rAAV for human trials and commercial applications. Researchers have explored many methods to produce rAAV particles at sufficiently high titers, but there is still a great need to solve this problem. In particular, there is a need for effective cell lines that can produce high - quality rAAV at high - titer yields. Generating high - titer rAAV through the engineered cell lines described herein accelerates the application of this vector system for in vivo gene therapy uses. Summary of the Invention

[0007] The present disclosure addresses the need for obtaining improved rAAV titers for gene therapy applications by providing rAAV packaging cell lines and / or production cell lines comprising cells in which one or more genes and / or proteins have been modified. Also described herein are methods for identifying one or more genes and / or proteins associated with the production of rAAV, and methods for generating engineered rAAV packaging cell lines and / or production cell lines.

[0008] Described herein are compositions and methods for generating rAAV packaging cell lines and / or production cell lines that comprise cells capable of producing higher titers of rAAV compared to control parental cells. More specifically, provided herein are rAAV packaging cell lines and / or production cell lines that comprise cells in which the expression of one or more genes and / or proteins is regulated, resulting in higher rAAV titers compared to control parental cells. In one aspect, the present disclosure provides rAAV packaging cell lines and / or production cell lines that comprise cells in which the expression of one or more genes and / or proteins is reduced compared to control parental cells. For example, the expression of ATP5EP2 (ATP synthase F1 subunit epsilon pseudogene 2), LINC00319 (long intergenic non-protein coding RNA 319), CYP3A7 (cytochrome P450 family 3 subfamily A member 7), ABCA10 (ATP-binding cassette subfamily A member 10), NOG (Noggin), RGMA (repulsive guidance molecule BMP coreceptor A), SPANXN3 (SPANX family member N3), PGA5 (pepsinogen A5), MYRIP (myosin VIIA and Rab interacting protein), KCNN2 (potassium calcium-activated channel subfamily N member 2), and / or NALCN-AS1 (NALCN antisense RNA 1) is reduced compared to control parental cells.

[0009] In some embodiments, the present disclosure provides rAAV packaging cell lines and / or production cell lines that comprise cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parental cells.

[0010] In certain embodiments, the present disclosure provides rAAV packaging cell lines and / or production cell lines that comprise cells engineered to have reduced expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to the corresponding unmodified parental cells. In certain embodiments, the present disclosure provides rAAV packaging cell lines and / or production cell lines that exhibit reduced expression and / or activity of a polypeptide or polyribonucleotide expressed by at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1 as compared to the corresponding parental cell line.

[0011] In one aspect, the present disclosure provides rAAV packaging cell lines and / or production cell lines in which the expression of one or more genes is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO).

[0012] In certain embodiments, the expression of one or more genes is reduced using siRNAs comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1-11. For example, in some embodiments, the expression of ATP5EP2 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 1 in the sense strand and the nucleotide sequence of SEQ ID NO: 32 in the antisense strand. In some embodiments, the expression of LINC00319 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 2 in the sense strand and the nucleotide sequence of SEQ ID NO: 33 in the antisense strand. In some embodiments, the expression of CYP3A7 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 3 in the sense strand and the nucleotide sequence of SEQ ID NO: 34 in the antisense strand. In some embodiments, the expression of NOG is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 4 in the sense strand and the nucleotide sequence of SEQ ID NO: 35 in the antisense strand. In some embodiments, the expression of SPANXN3 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 5 in the sense strand and the nucleotide sequence of SEQ ID NO: 36 in the antisense strand. In some embodiments, the expression of MYRIP is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 6 in the sense strand and the nucleotide sequence of SEQ ID NO: 37 in the antisense strand. In some embodiments, the expression of KCNN2 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 7 in the sense strand and the nucleotide sequence of SEQ ID NO: 38 in the antisense strand. In some embodiments, the expression of NALCN-AS1 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 8 in the sense strand and the nucleotide sequence of SEQ ID NO: 39 in the antisense strand. In some embodiments, the expression of RGMA is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 9 in the sense strand and the nucleotide sequence of SEQ ID NO: 40 in the antisense strand. In some embodiments, the expression of PGA5 is reduced, and the siRNA comprises the sequence of SEQ ID NO: 10 in the sense strand and the sequence of SEQ ID NO: 41 in the antisense strand. In some embodiments, the expression of ABCA10 is reduced, and the siRNA comprises the sequence of SEQ ID NO: 11 in the sense strand and the nucleotide sequence of SEQ ID NO: 42 in the antisense strand.

[0013] In certain embodiments, the nuclease for reducing the expression of one or more genes is selected from the group consisting of zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins.

[0014] In certain embodiments, CRISPR genome editing is used to reduce the expression of one or more genes. In some embodiments, guide RNA pairs are used to target genes to reduce and / or eliminate the expression of the genes. In certain embodiments, guide RNA pairs are used to reduce the expression of one or more genes, wherein each guide RNA: (a) comprises a sequence selected from the nucleotide sequences of SEQ ID NOs: 12-15, and / or (b) targets a target DNA sequence selected from any of the nucleotide sequences of SEQ ID NOs: 16-31. For example, in some embodiments, a gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 12 and a second gRNA molecule containing the sequence of SEQ ID NO: 13. In some embodiments, a gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 14 and a second gRNA molecule containing the sequence of SEQ ID NO: 15. In some embodiments, each gRNA molecule is a 2′O-methyl analogue that contains 3′ phosphorothioate internucleotide linkages in the terminal three nucleotides at either or both of its 5′ and 3′ ends.

[0015] In certain embodiments, a pair of guide RNAs is used to reduce the expression of one gene. In certain other embodiments, multiple pairs of guide RNAs are used to reduce the expression of one or more genes. In certain embodiments, the gene expression of one or more genes and / or the activity of one or more genes and / or proteins in the rAAV packaging cell line and / or production cell line is reduced and / or eliminated as compared to a control parental cell line. In certain embodiments, the gene expression and / or activity in the rAAV packaging and / or production cells is eliminated as compared to control parental cells.

[0016] In some embodiments described herein, the rAAV packaging cell line and / or production cell line is a eukaryotic cell line. In certain embodiments, the rAAV packaging cell line and / or production cell line is a human cell line. In certain embodiments, the rAAV packaging cell line and / or production cell line is an insect cell line. In certain embodiments, the rAAV packaging cell line and / or production cell line is a HeLa cell line. In certain other embodiments, the rAAV packaging cell line and / or production cell line is a human embryonic kidney (HEK) 293 cell line.

[0017] In some embodiments described herein, the rAAV packaging cell lines and / or production cell lines of the present disclosure produce higher rAAV titers than control parental cell lines. In certain embodiments, the titer of rAAV produced by the cells of the rAAV production cell lines of the present disclosure is increased by about 1.5 to about 7-fold compared to the titer of rAAV produced from cell lines containing control parental cells. Also described herein are lysates of engineered cell lines. In certain embodiments, higher titers of rAAV are harvested from the lysates. Also described herein are cell culture supernatants from engineered cell lines. In certain embodiments, higher titers of rAAV are harvested from the cell culture supernatants.

[0018] Also described herein are methods for generating production cell lines, wherein the methods comprise delivering an rAAV vector to cells of a packaging cell line, wherein the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to control parental cells. In certain embodiments, the present disclosure provides methods for generating production cell lines, wherein the methods comprise delivering an rAAV vector to cells of a packaging cell line, wherein the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parental cells.

[0019] Also described herein are methods for generating rAAV by infecting cells of a production cell line produced from a packaging cell line with a helper virus, wherein the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 in the packaging cell line is reduced compared to control parental cells. In certain embodiments, the expression of KCNN2, LINC00319, RGMA, and SPANXN3 in the packaging cell line is reduced compared to control parental cells.

[0020] In one aspect, the present disclosure provides methods for generating rAAV by infecting cells of a production cell line with a helper virus, wherein the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 in the production cell line is reduced compared to control parental cells. In certain embodiments, the present disclosure provides methods for generating rAAV by infecting cells of a production cell line with a helper virus, wherein the expression of KCNN2, LINC00319, RGMA, and SPANXN3 in the production cell line is reduced compared to control parental cells.

[0021] The present disclosure also describes methods for harvesting rAAV from a production cell line, wherein the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to a control parental cell line. Also described are methods for harvesting rAAV from a production cell line, wherein the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to a control parental cell line. In certain embodiments, rAAV production from the production cell lines of the present disclosure is enhanced compared to a control parental cell line.

[0022] The present disclosure also describes methods for identifying one or more genes associated with rAAV production, wherein the methods comprise: i.) adding one or more supplements that increase the rAAV titer in a cell line; ii.) measuring global gene expression across the transcriptome in the supplemented and unsupplemented cell lines; iii.) obtaining a list of genes that are differentially expressed between the supplemented and unsupplemented cell lines; and iv.) identifying one or more genes associated with the production of rAAV. In some embodiments, one or more of the identified genes are responsible for reducing rAAV production.

[0023] The present disclosure also describes methods for generating rAAV packaging cell lines and / or production cell lines to facilitate increased rAAV production. In some embodiments, rAAV production is increased by modulating the expression of one or more genes and / or proteins identified from a list of genes that are differentially expressed between supplemented and unsupplemented rAAV production cell lines. In certain embodiments, the rAAV titer is increased by modulating the expression of one or more genes and / or proteins identified from a list of genes that are differentially expressed between supplemented and unsupplemented rAAV production cell lines. In some embodiments, the modulation of one or more genes and / or proteins increases the rAAV titer by at least 1.5-fold compared to the rAAV titer of an unmodulated cell line. In certain embodiments, modulating the expression comprises reducing the expression of one or more genes. In certain embodiments, modulating the expression comprises reducing the expression of one or more proteins. In certain embodiments, modulating the expression comprises eliminating the expression of one or more genes. In certain embodiments, modulating the expression comprises eliminating the expression of one or more proteins.

[0024] In some embodiments, the rAAV packaging cell line and / or production cell line is a eukaryotic cell line. In certain embodiments, the cell line is a human cell line. In certain embodiments, the cell line is an insect cell line. In certain embodiments, the cell line is a HeLa cell line. In certain embodiments, the cell line is a human embryonic kidney (HEK) 293 cell line.

[0025] The present disclosure also describes recombinant adeno-associated virus (rAAV) packaging cell lines and / or production cell lines that comprise cells engineered to have reduced expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to their respective unmodified parental cells.

[0026] In some embodiments, the expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced indefinitely or permanently.

[0027] In some embodiments, the cell line has been engineered to comprise a gene disruption or a partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0028] In some embodiments, the cell line has been engineered to comprise a gene disruption in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0029] In some embodiments, the cell line has been engineered to comprise gene disruptions in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0030] In some embodiments, the cell line has been engineered to comprise a partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0031] In some embodiments, the cell line has been engineered to contain a partial or complete gene deletion in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0032] Also provided are packaging cell lines and / or production cell lines, wherein the cell lines exhibit a decrease in the expression and / or activity of a polypeptide or polyribonucleotide expressed by at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1 as compared to the corresponding parental cell line.

[0033] Other features and advantages of the present disclosure will be apparent from the following detailed description and claims.

[0034] All publications, references, patents, and / or patent applications cited herein are hereby incorporated by reference in their entirety for all purposes, unless stated to the contrary. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present disclosure can be more fully understood with reference to the following drawings.

[0036] Figure 1 is a schematic diagram of a method for generating the rAAV packaging cells and production cells described herein.

[0037] Figures 2A - 2D shows experimental data generated from the optimization and development of the HPRT1 siRNA knockout experiment. Figures 2A - 2B shows the knockdown percentage ( Figure 2A ) and protein expression ( Figure 2B ) data generated from the HPRT1 siRNA knockdown experiment performed in 24-well plates. Figures 2C - 2D shows the knockdown percentage ( Figure 2C ) and protein expression ( Figure 2D ) data generated from the HPRT1 siRNA knockdown experiment performed in 6-well plates.

[0038] Figures 3A - 3B shows from PGA5 ( Figure 3A ) and SPANXN3 ( Figure 3B) Fold change values of gene expression obtained by bioinformatics analysis of RNA-Seq data, expressed as the fold change in gene expression of cells cultured in unsupplemented cell medium relative to uninfected cells (cells not infected with helper virus), and the fold change in gene expression of cells cultured in supplemented cell medium relative to cells cultured in unsupplemented cell medium. Figures 3C - 3D Shows RT-qPCR fold change values of the expression of PGA5 ( Figure 3C ) and SPANXN3 ( Figure 3D ) in cells cultured in unsupplemented and supplemented cell medium relative to uninfected cells.

[0039] Figures 4A - 4B Shows the fold change values of the expression of PGA5 ( Figure 4A ) and SPANXN3 ( Figure 4B ) in producer cell line clones cultured in unsupplemented and supplemented cell medium relative to uninfected cells (cells not infected with helper virus), as determined by RT-qPCR. 21C5, 3C6, 2B6 represent different clones of the HeLa producer cell line. Figures 4C - 4D Shows the relative fold increase in the expression of PGA5 ( Figure 4C ) and SPANXN3 ( Figure 4D ) in producer cell line clones 21C5, 3C6, 2B6 cultured in supplemented cell medium compared to those cultured in unsupplemented cell medium.

[0040] Figures 5A - 5F Shows the effect of reducing the expression of individual genes in different producer cell lines on the rAAV titer. These graphs show the titer of rAAV genomes produced by producer cell line #1 ( Figure 5A ), producer cell line #2 ( Figure 5B ), and producer cell line #3 ( Figure 5C ) in genome copies (GC) / milliliter (mL). Figures 5D - 5F Shows the fold change in the titer of rAAV produced by producer cell line #1 ( Figure 5D ), producer cell line #2 ( Figure 5E ), and producer cell line #3 ( Figure 5F ). Figures 5A - 5B Shows the mean of 3 biological replicates. Figures 5C - 5F Shows the mean of 4 biological replicates.

[0041] Figure 6 Is an illustrative flow chart showing an exemplary gene filtering method.

[0042] Figure 7AShows the 24-deep well titers of the top 19 2H5 knockout clones. Titers are reported as genome copies / mL. The control sample is unmodified 2H5. Figure 7B Shows the fold change in titer compared to the 2H5 control. The 2H5 titer is set to 1 and other titers are shown as fold increases over the 2H5 control. Figure 7C Shows the 24-deep well titers of the top 19 7B12 knockout clones. Titers are reported as genome copies / mL. The control sample is unmodified 7B12. Figure 7D Shows the fold change in titer compared to the 7B12 control. The 7B12 titer is set to 1 and other titers are shown as fold increases over the 7B12 control.

[0043] Figure 8A Shows the 15-titers of the top five 2H5 knockout clones. Titers are reported as genome copies / mL. The control sample is unmodified 2H5. Figure 8B Shows the fold change in titer compared to the 2H5 control. The 2H5 titer is set to 1 and other titers are shown as fold increases over the 2H5 control. Figure 8C Shows the 15-titers of the top four 7B12 knockout clones. Titers are reported as genome copies / mL. The control sample is unmodified 7B12. Figure 8D Shows the fold change in titer compared to the 7B12 control. The 7B12 titer is set to 1 and other titers are shown as fold increases over the 7B12 control.

[0044] Figures 9A - 9B Shows the effect on rAAV titer resulting from reducing the expression of various gene combinations in two production cell lines. The figure shows the fold change in rAAV titer compared to a control treated with a missense siRNA. Figure 9A Shows the fold change in titer compared to the 2H5 missense control. The 2H5 missense titer is set to 1 and other titers are shown as fold increases over the 2H5 missense control. Figure 9B Shows the fold change in titer compared to the 7B12 missense control. The 7B12 missense titer is set to 1 and other titers are shown as fold increases over the 7B12 missense control. Detailed Description

[0045] The present disclosure describes recombinant adeno-associated virus (rAAV) packaging cell lines and / or production cell lines that comprise cells in which the expression of one or more genes and / or proteins is regulated. Regulation of gene expression results in increased titer yields compared to cell lines in which the expression of one or more genes and / or proteins is not regulated.

[0046] Unless otherwise indicated, technical terms are used in accordance with convention. Definitions of common terms in molecular biology can be found in the following: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995 (ISBN 1-56081-569-8).

[0047] The following definitions are included to aid in understanding the subject matter and constructing the appended patent claims. Abbreviations used herein have their conventional meanings within the chemical and biological arts.

[0048] Definitions

[0049] As used herein, "modulation" or "modulate" refers to an alteration in the regulation, expression, or activity of a gene and / or protein. Modulation can be an increase, decrease (reduction), or elimination of the expression and / or activity of one or more genes and / or proteins. In the case of modulating multiple genes and / or proteins, all of the expression and / or activity of the genes and / or proteins can be increased, or all of the expression and / or activity of the genes and / or proteins can be decreased, or the expression and / or activity of one or more genes and / or proteins can be increased while the expression and / or activity of other genes and / or proteins can be decreased.

[0050] The term "cell" as used herein refers to any one or more cells capable of producing recombinant adeno-associated virus (rAAV). In some embodiments, the cell is a mammalian cell such as a HeLa cell, COS cell, HEK293 cell, A549 cell, BHK cell, or Vero cell. In other embodiments, the cell is an insect cell such as an Sf9 cell, Sf-21 cell, Tn-368 cell, or BTI-Tn-5B1-4 (High-Five) cell. The term "cell line" refers to a clonal population of cells capable of continued division without undergoing senescence. Unless otherwise indicated, the terms "cell" or "cell line" shall be understood to include modified or engineered variants of the indicated cell or cell line.

[0051] As used herein, the term "engineered cell line" refers to a cell line that has been modified in one or more ways to reduce the expression or other properties (such as biological activity) of one or more endogenous expressed genes and / or proteins (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1) so as to increase rAAV production.

[0052] As used herein, the term "control parental cell" refers to a cell that has not been modified in one or more ways to reduce the expression or other properties (such as biological activity) of one or more endogenous expressed genes and / or proteins (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1) so as to increase rAAV production.

[0053] As used herein, the term "control parental cell line" refers to a clonal population of control parental cells that are capable of continued division and do not undergo senescence.

[0054] "Lysis" refers to the breakdown of cells, typically by a virus, enzyme, or osmotic mechanism that compromises their integrity. A "lysed cell" is a cell that has undergone substantial lysis. As used herein, the term "lysate" refers to a fluid containing the contents of lysed cells.

[0055] As used herein, the term "higher titer" indicates a titer that is increased compared to the titer produced by an unmodified control parental cell line and / or control parental cell.

[0056] As used herein, the term "cell culture supernatant" refers to the cell culture medium in which cells are suspended and / or cultured.

[0057] As used herein, the term "gene" refers to a transcription unit and regulatory regions adjacent (e.g., upstream and downstream) to the transcription unit and operably linked thereto. A transcription unit is a series of nucleotides that are transcribed into an RNA molecule. A transcription unit may include a coding region. A "coding region" is a nucleotide sequence that encodes an unprocessed precursor RNA (i.e., an RNA molecule that includes exons and introns), which is subsequently processed into mRNA. A transcription unit may encode a non-coding RNA. A non-coding RNA is an RNA molecule that is not translated into a protein. Examples of non-coding RNAs include microRNAs. The boundaries of a transcription unit are typically determined by the start site at its 5' end and the transcription terminator at its 3' end. A "regulatory region" is a nucleotide sequence that regulates the expression of a transcription unit operably linked thereto. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription start sites, translation start sites, translation termination sites, transcription terminators, and poly(A) signals. A regulatory region located upstream of a transcription unit may be referred to as a 5' UTR, and a regulatory region located downstream of a transcription unit may be referred to as a 3' UTR. A regulatory region may be transcribed and is part of the unprocessed precursor RNA.

[0058] In the context of this disclosure, the term "target" or "target gene" refers to any gene, including genes encoding proteins and genes encoding non-coding RNAs (e.g., miRNAs), that when modulated alters some aspect of virus production. Target genes include endogenous genes, viral genes, and transgenes.

[0059] Regarding gene names, a single gene is typically represented by multiple symbols. In the context of this disclosure, gene symbols, whether human or non-human, may be represented by uppercase or lowercase letters. In these disclosed contexts, using a particular symbol or adopting lowercase or uppercase symbols is not intended to limit the scope of the gene. Unless otherwise indicated, all gene identification numbers (GeneID) identified herein are from the National Center for Biotechnology Information "Entrez Gene" or the KEGG website.

[0060] The term "about" as used herein means approximately, within a region, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and / or below the stated numerical values within the permitted numerical range.

[0061] As used in this disclosure, whether in a transitional phrase or in the body of a claim, the terms "comprise(s)" and "comprising" shall be interpreted to have an open-ended meaning. That is, the terms shall be interpreted as synonymous with the phrase "having at least" or "including at least." When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term "comprising" means that the composition includes at least the recited features or components, but may also include additional features or components.

[0062] To facilitate understanding of the embodiments described herein, references to preferred embodiments and specific language are used to describe them. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the disclosure. As used throughout this disclosure, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. All percentages and ratios used herein are by weight unless otherwise indicated.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the following describes suitable methods and materials. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

[0064] Adeno-associated virus (AAV):

[0065] AAV is a small, replication-defective, non-enveloped virus that infects humans and some other primate species. AAV is known not to cause disease and elicits a very mild immune response. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist in an episomal state without integrating into the host cell genome. These features make AAV an attractive viral vector for gene therapy. AAV includes many serologically distinguishable types, including serotypes AAV-1 to AAV-12, and over 100 serotypes from non-human primates (see, e.g., Srivastava, J. Cell Biochem. [Journal of Cellular Biochemistry], 105(1):17-24 (2008), and Gao et al., J. Virol. [Journal of Virology], 78(12), 6381-6388 (2004)). AAV is non-autonomous and has a life cycle that includes a latent and an infectious phase. During the latent phase, after a cell is infected with AAV, AAV integrates site-specifically into the host genome as a provirus. The infectious phase does not occur unless the cell is also infected with a helper virus that permits AAV replication, such as adenovirus (AV) or herpes simplex virus.

[0066] The wild-type AAV genome contains two inverted terminal repeats (ITRs) of 145 nucleotides that contain signal sequences that direct AAV replication, genome encapsidation, and integration. In addition to the ITRs, three AAV promoters, p5, p19, and p40, drive the expression of two open reading frames encoding the rep and cap genes. Two rep promoters, coupled with differential splicing of a single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. The Rep proteins are responsible for genome replication. The cap gene is expressed by the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3), which are splicing variants of the cap gene. These proteins form the capsid of the AAV particle.

[0067] Because the cis-acting signals for replication, encapsidation, and integration are contained within the ITR, some or all of the 4.3 kb internal genome can be replaced by foreign DNA, such as an expression cassette for a foreign protein of interest. In this case, the rep and cap proteins are provided in trans, for example, on a plasmid. To generate an AAV vector, a cell line that permits AAV replication must express the rep and cap genes, an ITR-flanked expression cassette, and the helper functions provided by a helper virus, such as the AV genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011). Generation of AAV vectors can also result in the production of helper virus particles, which must be removed or inactivated before using the AAV vector. Many cell types are suitable for generating AAV vectors, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells (see, e.g., U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, 8,163,543, U.S. Publication No. 20020081721, PCT Publication Nos. WO 00 / 47757, WO00 / 24916, and WO 96 / 17947). AAV vectors are typically generated in these cell types from a plasmid containing an ITR-flanked expression cassette and one or more additional plasmids that provide the additional AAV and helper virus genes.

[0068] Any serotype of AAV can be used in the present invention. Similarly, it is contemplated that any type of AV can be used, and those skilled in the art will be able to identify AAV and AV types suitable for generating their desired recombinant AAV vector (rAAV). AAV and AV particles can be purified, for example, by affinity chromatography, iodixanol gradient, or CsCl gradient.

[0069] The genome of wild-type AAV is single-stranded DNA, 4.7 kb in size. AAV vectors can have a single-stranded genome that is 4.7 kb or larger or smaller than 4.7 kb, including oversized genomes up to 5.2 kb or as small as 3.0 kb. In addition, the vector genome can be substantially self-complementary such that the genome within the virus is substantially double-stranded. AAV vectors containing all types of genomes are suitable for the methods of the present disclosure.

[0070] As described above, AAV requires co - infection with a helper virus to enter the infectious stage of its life cycle. Helper viruses include adenovirus (AV) and herpes simplex virus (HSV), and there are systems for producing AAV in insect cells using baculovirus. It has also been proposed that papillomavirus can also provide helper functions for AAV (see, e.g., Hermonat et al., Molecular Therapy 9, S289 - S290 (2004)). A helper virus includes any virus capable of producing and allowing AAV replication. AV is an unenveloped nuclear DNA virus with a double - stranded DNA genome of approximately 36 kb. AV is able to rescue latent AAV provirus in cells by providing the E1a, E1b55K, E2a, E4orf6, and VA genes and allowing AAV replication and encapsidation. HSV is a family of viruses with a relatively large double - stranded linear DNA genome enclosed in an icosahedral capsid, which is in turn enclosed in a lipid bilayer envelope. HSV is infectious and highly contagious. The following HSV - 1 replication proteins have been identified as necessary for AAV replication: the helicase / primase complex (UL5, UL8, and UL52) and the DNA - binding protein ICP8 encoded by the UL29 gene, as well as other proteins that enhance helper functions. The AAV packaging system has two purposes: it avoids the problems of transfection methods and provides a production technology based on the use of one or several helper functions.

[0071] Production of rAAV

[0072] The general principles of rAAV can be reviewed elsewhere (see, e.g., Carter, 1992, Current Opinions in Biotechnology, 3:533-539; and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158:97-129). Generally, in order to permit the production of rAAV, cells must be provided with AAV ITRs (which can flank, for example, a heterologous nucleotide sequence of interest), AAV rep and cap gene functions, and additional helper functions. These can be provided to the cells using any number of suitable plasmids or vectors. The additional helper functions can be provided by, for example, adenovirus (AV) infection, a plasmid carrying all the required AV helper function genes, or other viruses such as HSV or baculovirus. Any gene, gene function, or genetic material necessary for a cell to produce rAAV can be transiently present in the cell or stably inserted into the cell genome. rAAV production methods suitable for use in the methods of the present disclosure include those disclosed in the following: Clark et al., Human Gene Therapy 6:1329-1341 (1995), Martin et al., Human Gene Therapy Methods 24:253-269 (2013), Thorne et al., Human Gene Therapy 20:707-714 (2009), Fraser Wright, Human Gene Therapy 20:698-706 (2009), and Virag et al., Human Gene Therapy 20:807-817 (2009). Two main methods of AAV production systems are recombinant adeno-associated virus (rAAV) packaging cell lines and adeno-associated virus (rAAV) producer cell lines.

[0073] Recombinant adeno-associated virus (rAAV) packaging cell lines and / or producer cell lines

[0074] rAAV packaging cell lines can be generated by allowing cellular expression of the AAV genetic elements described herein. Stable transfection of a cell line (e.g., HEK293, HeLa) with plasmids encoding the AAV rep and cap genes can generate a packaging cell line. The rAAV packaging cell line can be co-infected with two different adenoviruses (a helper virus and a hybrid virus containing the AAV gene therapy element) to produce rAAV particles. Alternatively, stable transfection of the packaging cells with a plasmid containing the rAAV vector or infection of them with the rAAV vector generates an rAAV producer cell line. Infection of the producer cells with the helper virus results in the production of rAAV. Figure 1 Describe the packaging cell line and the producer cell line.

[0075] In certain embodiments of the present disclosure, rAAV packaging cell lines comprising the AAV rep and cap gene functions are engineered to increase rAAV titers.

[0076] In one aspect, the present disclosure provides rAAV packaging cell lines comprising cells in which the expression of one or more genes and / or proteins is reduced compared to a control parental cell. For example, the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to a control parental cell.

[0077] In some embodiments, the present disclosure provides rAAV packaging cell lines comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to a control parental cell.

[0078] In other embodiments, the present disclosure provides rAAV producer cell lines comprising cells in which the expression of one or more genes and / or proteins is reduced compared to a control parental cell. For example, the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to a control parental cell. In some embodiments, the rAAV producer cell lines of the present disclosure have been engineered to reduce the gene expression of KCNN2, LINC00319, RGMA, and SPANXN3.

[0079] In certain embodiments, the cell lines of the present disclosure can be in adherent or suspension form.

[0080] In certain embodiments, the cell lines of the present disclosure (e.g., rAAV packaging cell lines and / or production cell lines) are mammalian cell lines (e.g., HeLa, human embryonic kidney (HEK) 293, COS, A549, or Vero cell lines). In certain embodiments, the cell line is an insect cell line (e.g., Sf9, Sf-21, Tn-368, or BTI-Tn-5B1-4).

[0081] Method for generating rAAV Production cell line

[0082] In some embodiments, the present disclosure provides a method for generating a production cell line by delivering an rAAV vector to an engineered rAAV packaging cell line comprising cells in which the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to control cells.

[0083] In certain embodiments, the present disclosure provides a method for generating a production cell line by delivering an rAAV vector to an engineered rAAV packaging cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parental cells.

[0084] Supplement

[0085] As used herein, the term "supplement" refers to any compound or other material of chemical or biological origin that can be used in the culture medium of cell culture to increase the rAAV titer or to assay for an increase in the rAAV titer. Non-limiting examples of supplements include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, enzymes, nucleosides, metabolites, surfactants, emulsifiers, inorganic salts, and polymers. In certain embodiments, one or more supplements added to the rAAV packaging cell lines and / or production cell lines of the present disclosure are glucocorticoid analogs. In certain embodiments, one or more supplements added to the rAAV packaging cell lines and / or production cell lines include dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, and / or triamcinolone.

[0086] In certain embodiments, the concentration of the glucocorticoid analog in the solution for increasing the rAAV titer can be greater than or equal to 1 μM, greater than or equal to 0.1 μM, greater than or equal to 0.01 μM, between 0 and 1 μM, between 0 and 0.1 μM, between 0 and 0.01 μM, between 0.01 and 1 μM, or between 0.01 and 0.1 μM.

[0087] As used herein, a "supplemented cell line" refers to a cell line (e.g., an rAAV packaging cell line and / or a production cell line) in which one or more supplements (e.g., a glucocorticoid analog) have been added to increase the rAAV titer. As used herein, an "unsupplemented cell line" refers to a cell line (e.g., an rAAV packaging cell line and / or a production cell line) that has not been exposed to one or more supplements used to increase the rAAV titer. As used herein, the terms "unsupplemented" and "non-supplemented" are used interchangeably and refer to culture conditions in which a cell line (e.g., an rAAV packaging cell line and / or a production cell line) is not exposed to one or more supplements used to increase the rAAV titer.

[0088] Method for identifying one or more genes associated with rAAV production

[0089] The present disclosure relates in part to a method for identifying one or more genes associated with rAAV production by comparing global gene expression patterns in supplemented and unsupplemented cell lines.

[0090] The term "global gene expression" is well known in the art (see Wang Z et al., Nature Reviews Genetics, 10(1), 57-63 (2009)). The term "global gene expression" refers to one or more sets of data that contain information about different aspects of gene expression. The data set optionally includes information about: the presence of a target transcript in a cell or cell-derived sample; the relative and absolute abundance levels of the target transcript; the ability of various treatments (e.g., addition of a supplement) to regulate the expression of a particular gene; and the ability of various treatments (e.g., addition of a supplement) to alter the expression of a particular gene to different levels.

[0091] The term "differential expression" is well known in the art (see Wang Z. et al., Nature Reviews Genetics, 10(1), 57-63 (2009), Ozsolak, F. et al Nature Reviews Genetics, 12(2), 87-98 (2011), Han, Y. et al Bioinformatics and Biology Insights, 9, 29-46 (2015)).

[0092] In certain embodiments, the cell lines of the present disclosure (e.g., rAAV packaging cell lines and / or production cell lines) are supplemented with one or more supplements that increase rAAV production. In some embodiments, RNA samples are extracted from one or more cell lines (supplemented and unsupplemented) using any known procedure. For example, silica-based isolation can be used to purify total RNA from cells in an automation-compatible 96-well format, such as the purification platform (Qiagen, Inc.; Valencia, California).

[0093] The gene expression patterns in the expressed RNA samples can be evaluated by either (or both) qualitative and quantitative measurements. In some embodiments, it is useful to quantify the expression levels of genes relative to other expressed products and / or relative to a control sequence. A convenient and widely applicable method for determining relative expression is to compare the expression of one or more genes of interest to the expression of a control gene (e.g., a housekeeping gene such as HPRT1, HSP70, or β-actin).

[0094] To determine whether the observed expression data, e.g., changes in the gene expression profiles in response to one or more treatments (e.g., addition of a supplement) of a biological sample (e.g., supplemented and unsupplemented cell lines), are significant, e.g., not just the product of experimental noise or population heterogeneity, an estimate of the probability distribution can be constructed for each genetic and phenotypic endpoint in each biological sample. Construction of the estimated population distribution includes performing multiple independent experiments for each treatment, e.g., all experiments are performed in duplicate, triplicate, quadruplicate, etc. Multivariate statistics can be used to group or cluster the expression data from multiple biological samples (e.g., supplemented and unsupplemented cell lines). Analysis of the data can yield a list of genes, for example, that are differentially expressed in response to treatment between supplemented and unsupplemented cell lines. Various gene filtering methods can be used to filter the list of differentially expressed genes to identify one or more genes that can be used to increase rAAV production.

[0095] In some embodiments, the present disclosure relates to methods of identifying one or more genes associated with rAAV production from a list of genes differentially expressed between supplemented and unsupplemented cell lines. In certain embodiments, the cell line is a eukaryotic cell line. In certain embodiments, the cell line is a human cell line. In certain embodiments, the cell line is a HeLa cell line or a HEK293 cell line. In certain embodiments, global gene expression is measured in different cell lines (e.g., between unsupplemented and supplemented HeLa cell lines, between unsupplemented and supplemented HEK293 cell lines, between unsupplemented HeLa and supplemented HEK293 cell lines, between unsupplemented HeLa and unsupplemented HEK293 cell lines, between supplemented HeLa and supplemented HEK293 cell lines) to identify one or more genes associated with rAAV production. In certain embodiments, global gene expression data from supplemented HEK293 and supplemented HeLa can be combined and compared with combined global gene expression data from unsupplemented HEK293 and unsupplemented HeLa cell lines to identify one or more genes associated with rAAV production.

[0096] In certain embodiments, the present disclosure provides methods of generating rAAV packaging cell lines and / or production cell lines to facilitate increased rAAV production. In some embodiments, rAAV production is increased by modulating the expression of one or more genes and / or proteins identified from a list of genes differentially expressed between supplemented and unsupplemented rAAV production cell lines. In certain embodiments, rAAV titers are increased by modulating the expression of one or more genes and / or proteins identified from a list of genes differentially expressed between supplemented and unsupplemented rAAV production cell lines. In some embodiments, the rAAV titers are increased by at least 1.5-fold (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, or 30-fold) compared to the rAAV titers produced by cell lines that do not modulate the expression of the corresponding one or more genes and / or one or more proteins.

[0097] Regulated genes and / or proteins

[0098] In certain embodiments, the present disclosure provides a list of genes that enhance rAAV production when modulated (alone or in combination) in an rAAV packaging cell line and / or production cell line.

[0099] ATP synthase F1 subunit epsilon pseudogene 2 (also known as ATP5EP2) encodes a mitochondrial ATP synthase subunit epsilon-like protein. ATP5EP2 is a mitochondrial membrane ATP synthase that produces ATP from ADP in the presence of a transmembrane proton gradient generated by the electron transport complexes of the respiratory chain. Examples of the human ATP5EP2 sequence can be obtained in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_006886.4 (SEQ ID NO: 43) or NG_053163.1 (SEQ ID NO: 44).

[0100] Long intergenic non-protein coding RNA 319 (also known as LINC00319) is an RNA gene and is related to the class of non-coding RNAs. Long non-coding RNAs (lncRNAs) have been shown to play important regulatory roles in the pathogenesis and progression of various cancers. Examples of the LINC00319 sequence can be obtained in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_194309 (SEQ ID NO: 45) or NR_026960.1 (SEQ ID NO: 46).

[0101] Cytochrome P450 family 3 subfamily A member 7 (also known as CYP3A7) is a gene encoding a member of the cytochrome P450 superfamily of enzymes involved in drug metabolism and cholesterol, steroid, and other lipid synthesis. The enzyme hydroxylates testosterone and dehydroepiandrosterone 3-sulfate, which is involved in the formation of estriol during pregnancy. This gene is part of a cluster of related genes on chromosome 7q21.1. Examples of the CYP3A7 sequence can be obtained in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_000765 (SEQ ID NO: 47).

[0102] ATP-binding cassette subfamily A member 10 (also known as ABCA10) encodes a membrane-associated protein belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport various molecules across the extracellular and intracellular membranes. The ABC genes are divided into 7 distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White). ABCA10 is a member of the ABC1 subfamily. Members of the ABC1 subfamily include the only major ABC subfamily found in multicellular eukaryotes. This gene clusters among four other ABC1 family members on 17q24. Examples of the ABCA10 sequence can be obtained in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_080282.3 (SEQ ID NO: 48).

[0103] Noggin (also known as NOG) encodes a secreted polypeptide that binds to and inactivates members of the transforming growth factor-β (TGF-β) superfamily of signaling proteins, such as bone morphogenetic protein-4 (BMP4). Without being bound by theory, it is believed that by diffusing more efficiently through the extracellular matrix than members of the TGF-β superfamily, this protein may have a major role in generating morphogenetic gradients. NOG appears to have pleiotropic effects both early and late in development. An example of the NOG sequence is available in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_005450.4 (SEQ ID NO: 49).

[0104] Repulsive guidance molecule BMP coreceptor A (also known as RGMA) is a gene that encodes a member of the repulsive guidance molecule family. The encoded protein is a glycosylphosphatidylinositol-anchored glycoprotein that functions as an axon guidance protein in the developing and adult central nervous system. The protein may also function as a tumor suppressor in some cancers. An example of the RGMA sequence is available in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_020211.2 (SEQ ID NO: 50) or NM_001166283.1 (SEQ ID NO: 51).

[0105] SPANX (sperm protein associated with the nucleus on the X chromosome) family member N3 (also known as SPANXN3) is a protein-coding gene. An example of the SPANXN3 sequence is available in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_001009609 (SEQ ID NO: 52).

[0106] Pepsinogen-5, group I (also known as PGA5 or pepsinogen A) encodes the protein precursor of the digestive enzyme pepsin, which is a member of the peptidase A1 family of endopeptidases. The encoded precursor is secreted by gastric chief cells and undergoes autocatalytic cleavage under acidic conditions to form the active enzyme, which functions in the digestion of dietary proteins. This gene is present in a related gene cluster on chromosome 11, and each gene encodes one of several pepsinogens. An example of the PGA5 sequence is available in the NCBI nucleotide database (nucleotide sequence) as reference sequence NM_014224.4 (SEQ ID NO: 53).

[0107] Myosin VIIA and Rab interacting protein (also known as MYRIP) encodes a Rab effector involved in melanosome trafficking, which serves as a link between melanosome-bound RAB27A and the motor proteins MYO5A and MYO7A. This Rab effector functions as a protein kinase A anchoring protein (AKAP) and can act as a scaffold protein that links PKA to components of the exocytosis machinery, thereby promoting exocytosis, including insulin release. Examples of MYRIP sequences are available in the NCBI nucleotide database (nucleotide sequences) as reference sequences NM_015460 (SEQ ID NO: 54) or NM_001284423.1 (SEQ ID NO: 55).

[0108] Potassium calcium-activated channel subfamily N member 2 (also known as KCNN2) gene is a member of the KCNN family of potassium channel genes. The encoded protein is an integral membrane protein that forms a voltage-independent calcium-activated channel with three other calmodulin-binding subunits. Alternative splicing of this gene generates multiple transcript variants. Examples of KCNN2 sequences are available in the NCBI nucleotide database (nucleotide sequences) as reference sequences NM_170775.2 (SEQ ID NO: 56) or NM_001278204.1 (SEQ ID NO: 57).

[0109] NALCN antisense RNA 1 (also known as NALCN-AS1) is an RNA gene and is related to the non-coding RNA class. Examples of NALCN-AS1 sequences are available in the NCBI nucleotide database (nucleotide sequences) as reference sequences NW_011332700.1 (SEQID NO: 58) or NR_047687.1 (SEQ ID NO: 59).

[0110] In certain embodiments, the present disclosure provides rAAV packaging cell lines and / or production cell lines that contain cells in which the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to control parental cells.

[0111] In certain embodiments, the present disclosure provides rAAV packaging cell lines and / or production cell lines that contain cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parental cells.

[0112] In certain embodiments, the present disclosure provides a list of genes that, when individually regulated in an rAAV packaging cell line and / or a production cell line, increase rAAV production compared to a control parental cell line. In some aspects, the regulation of different gene combinations in the rAAV packaging cell line and / or the production cell line increases rAAV production. In some aspects, regulating the expression of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 genes in the rAAV packaging cell line and / or the production cell line results in increased rAAV production compared to a control parental cell line.

[0113] Methods for regulating one or more genes and / or proteins

[0114] Regulating (e.g., decreasing) the expression or activity of a gene (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, or NALCN-AS1) can be achieved by different mechanisms, including but not limited to altering one or more of the following: 1) gene copy number, 2) transcription or translation of the gene, 3) transcript stability or lifespan, 4) copy number of mRNA or miRNA, 5) availability of non-coding RNA or non-coding RNA target sites, 6) location or extent of post-translational modification on the protein, or 7) activity of the protein. Tools that can be used to regulate gene expression include but are not limited to nucleases, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), antisense RNA oligonucleotides (ASO), gene disruption, or partial or complete gene deletion.

[0115] Nuclease

[0116] In certain embodiments, gene regulation is achieved using zinc finger nucleases (ZFNs). Synthetic ZFNs are composed of zinc finger binding domains fused to, for example, the FokI DNA cleavage domain. ZFNs can be designed / engineered for editing the genome of cells, including but not limited to knocking out or knocking in gene expression in a variety of organisms. Meganucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (e.g., Cas nucleases) and triplexes can also be used for genome engineering of a variety of cell types. The reagents can be used to target promoters, protein-coding regions (exons), introns, 5′ and 3′ UTRs, etc.

[0117] Double-stranded RNA (dsRNA) molecules for regulation

[0118] In certain embodiments, double-stranded RNA (dsRNA) molecules can be used to modulate the expression of one or more genes in the cell lines described herein (e.g., rAAV packaging cell lines and / or production cell lines). The dsRNA molecules can be designed to antagonize one or more genes through sequence homology-based targeting of the corresponding RNA sequences. Such dsRNA can be small interfering RNA (siRNA), small hairpin RNA (shRNA), or microRNA (miRNA). The sequence of such dsRNA will contain a complementary portion of the mRNA encoding one or more genes to be modulated. This portion can be 100% complementary to the target portion within the mRNA, but lower levels of complementarity can also be used (e.g., 90% or higher or 95% or higher). Typically, the percentage of complementarity is determined over a continuous stretch of nucleic acid residues. The dsRNA molecules of the present disclosure can, for example, have at least 80% complementarity to the target portion within the mRNA, which is measured over at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or more nucleic acid residues. In some cases, the dsRNA molecule has at least 80% complementarity to the target portion of the mRNA over the entire length of the dsRNA molecule.

[0119] Another gene targeting reagent that uses the RNA interference (RNAi) pathway is small hairpin RNA, also known as shRNA. shRNA delivered to cells, for example, by an expression construct (e.g., plasmid, lentivirus), has the ability to provide a long-term reduction in gene expression in a constitutive or regulatable manner, depending on the type of promoter used. In one embodiment, the genome of the lentiviral particle is modified to include one or more shRNA expression cassettes targeting one or more genes of interest. Such lentivirus can infect cells, stably integrate its viral genome into the host genome, and express the shRNA in a constitutive, regulatable, or (in the case of expressing multiple shRNAs) constitutive and regulatable manner. Thus, in some embodiments, shRNAs can be designed to target individual variants of a single gene or multiple closely related gene family members. Individual shRNAs can modulate a set of targets with similar or redundant functions or sequence motifs. Those skilled in the art will recognize that lentiviral constructs can also incorporate cloned DNA or ORF expression constructs.

[0120] In the embodiments described herein, gene targeting reagents, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), can be used to modulate gene function. siRNAs and miRNAs can incorporate a wide range of chemical modifications, complementary levels, and designs to the target transcript of interest (see U.S. Patent No. 8,188,060) to enhance stability, cellular delivery, specificity, and functionality. Additionally, such reagents can be designed to target different regions of a gene (including the 5′ UTR, open reading frame, 3′ UTR of mRNA) or, in some cases, the promoter / enhancer region of genomic DNA encoding the gene of interest. Gene regulation (e.g., reducing gene expression, knockout) can be achieved by introducing individual siRNAs or miRNAs or libraries of multiple siRNAs or miRNAs that target different regions of the same mRNA transcript (into cells). Synthetic siRNA / miRNA delivery can be achieved by many methods, including but not limited to 1) self-delivery, 2) lipid-mediated delivery, 3) electroporation, or 4) vector / plasmid-based expression systems. The introduced RNA molecules can be referred to as exogenous nucleotide sequences or polynucleotides. In some embodiments, siRNAs can be designed to target individual variants of a single gene or multiple closely related gene family members.

[0121] siRNAs can be used to reduce the expression of one or more genes (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1). In some embodiments, siRNAs comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 11 or variants thereof are used to reduce the expression of a target gene.

[0122] Table 1: siRNA sequences for reducing gene expression.

[0123]

[0124]

[0125]

[0126] * siRNA sequences (sense and antisense) for reducing gene expression. Lowercase nucleotides in the sequences represent 3` overhangs.

[0127] In some embodiments, the siRNA for reducing ATP5EP2 expression comprises the nucleotide sequence of SEQ ID NO: 1 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 1 in the sense strand and the nucleotide sequence of SEQ ID NO: 32 in the antisense strand.

[0128] In some embodiments, the siRNA for reducing LINC00319 expression comprises the nucleotide sequence of SEQ ID NO: 2 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 2 in the sense strand and the nucleotide sequence of SEQ ID NO: 33 in the antisense strand.

[0129] In some embodiments, the siRNA for reducing CYP3A7 expression comprises the nucleotide sequence of SEQ ID NO: 3 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 3 in the sense strand and the nucleotide sequence of SEQ ID NO: 34 in the antisense strand.

[0130] In some embodiments, the siRNA for reducing NOG expression comprises the nucleotide sequence of SEQ ID NO: 4 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 4 in the sense strand and the nucleotide sequence of SEQ ID NO: 35 in the antisense strand.

[0131] In some embodiments, the siRNA for reducing SPANXN3 expression comprises the nucleotide sequence of SEQ ID NO: 5 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 5 in the sense strand and the nucleotide sequence of SEQ ID NO: 36 in the antisense strand.

[0132] In some embodiments, the siRNA for reducing MYRIP expression comprises the nucleotide sequence of SEQ ID NO: 6 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 6 in the sense strand and the nucleotide sequence of SEQ ID NO: 37 in the antisense strand.

[0133] In some embodiments, the siRNA for reducing KCNN2 expression comprises the nucleotide sequence of SEQ ID NO: 7 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 7 in the sense strand and the nucleotide sequence of SEQ ID NO: 38 in the antisense strand.

[0134] In some embodiments, the siRNA for reducing NALCN-AS1 expression comprises the nucleotide sequence of SEQ ID NO: 8 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 8 in the sense strand and the nucleotide sequence of SEQ ID NO: 39 in the antisense strand.

[0135] In some embodiments, the siRNA for reducing RGMA expression comprises the nucleotide sequence of SEQ ID NO: 9 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 9 in the sense strand and the nucleotide sequence of SEQ ID NO: 40 in the antisense strand.

[0136] In some embodiments, the siRNA for reducing PGA5 expression comprises the nucleotide sequence of SEQ ID NO: 10 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 10 in the sense strand and the nucleotide sequence of SEQ ID NO: 41 in the antisense strand.

[0137] In some embodiments, the siRNA for reducing ABCA10 expression comprises the nucleotide sequence of SEQ ID NO: 11 or a variant thereof. For example, in some embodiments, the siRNA comprises the nucleotide sequence of SEQ ID NO: 11 in the sense strand and the nucleotide sequence of SEQ ID NO: 42 in the antisense strand.

[0138] Antisense RNA oligonucleotide (ASO)

[0139] Antisense RNA oligonucleotide (ASO) can be used to regulate the expression of one or more genes in rAAV packaging cell lines and / or production cell lines. Generally, ASO is used to reduce the expression of one or more genes. Using known techniques and based on the knowledge of the sequence of the one or more genes to be regulated, ASO molecules can be designed to antagonize one or more genes by sequence homology-based targeting of the corresponding RNA. The ASO sequence can comprise a nucleotide sequence complementary to the target portion of the mRNA or lncRNA produced by the one or more genes. This portion can be 100% complementary to the target portion within the mRNA or lncRNA, but lower levels of complementarity can also be used (e.g., 90% or higher or 95% or higher).

[0140] In some embodiments, the ASO can be an antisense RNA oligonucleotide, wherein at least one internucleoside bond of the sequence is a phosphorothioate bond, a dithiophosphonate bond, a phosphotriester bond, an alkylphosphonate bond, an aminoalkylphosphotriester bond, an alkylidene phosphonate bond, a phosphonite bond, an aminophosphate bond, and an aminoalkylaminophosphate bond, a phosphorothioamidate bond, a thioalkylphosphonate bond, a thioalkylphosphotriester bond, a phosphorothioate bond, a selenophosphate bond, or a boranophosphate bond. In certain embodiments, at least one internucleoside bond of the antisense RNA oligonucleotide sequence is a phosphorothioate bond. In some embodiments, all internucleoside bonds of the antisense RNA oligonucleotide sequence are phosphorothioate bonds.

[0141] CRISPR genome editing

[0142] In some embodiments, CRISPR genome editing is used to regulate gene expression in rAAV packaging cell lines and / or production cell lines. CRISPR genome editing generally involves two different components: (1) guide RNA and (2) an endonuclease, particularly a CRISPR-associated (Cas) nuclease (e.g., Cas9). The guide RNA is a transcript of the endogenous bacterial crRNA and tracrRNA combined into a single chimeric guide RNA (gRNA). Without being bound by theory, it is believed that when the gRNA and Cas are expressed in a cell, the genomic target sequence can be modified or permanently disrupted.

[0143] The gRNA / Cas complex is recruited to the target sequence by base pairing between the gRNA sequence and the complementary sequence of the target DNA sequence in the gene to be downregulated (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, or NALCN-AS1). For successful binding of Cas, the genomic target sequence must also contain the correct protospacer adjacent motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas complex positions Cas at the genomic target sequence in one or more of the genes of the present disclosure such that wild-type Cas can cleave both strands of the DNA, causing a double-strand break. This can be repaired by one of two general repair pathways: (1) the non-homologous end joining DNA repair pathway or (2) the homologous directed repair pathway. The non-homologous repair pathway can result in insertions / deletions at the double-strand break, which can lead to frameshifts and / or premature stop codons, effectively disrupting the open reading frame of the target gene. The homologous directed repair pathway requires the presence of a repair template, which is used to repair the double-strand break.

[0144] Any suitable pair of gRNAs can be used for CRISPR genome editing. Generally, pairs of gRNAs are used to reduce the expression of one or more genes (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1). In some embodiments described herein, pairs of gRNAs are used to regulate (e.g., reduce or eliminate / knock out) the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0145] Pairs of gRNAs can be designed using known techniques and knowledge of the sequences of the one or more genes to be regulated, typically using any suitable computer program available to the public. Knockout packaging cells and / or producer cells can be generated using any suitable technique, where standard techniques are known in the art and suitable kits are commercially available.

[0146] Pairs of gRNAs can be delivered to the producer cell lines of the present disclosure in any suitable manner. Suitable techniques are known in the art, including using plasmid, viral, and bacterial vectors to deliver the pairs of gRNAs to the producer cell lines. Generally, plasmid DNA is used to deliver pairs of gRNAs.

[0147] Pairs of gRNAs can be used to reduce the expression of one or more genes (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1). Multiple pairs of gRNAs can be used to regulate gene expression. In some embodiments described herein, pairs of gRNAs are used to reduce the expression of at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, or NALCN-AS1. Multiple pairs of gRNAs can be used to regulate the expression of KCNN2, LINC00319, RGMA, and SPANXN3. In some embodiments, the gRNAs can be modified to enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation. In certain embodiments, these modifications can be 2′-O-methyl analogs of the terminal three nucleotides at the 5′ and 3′ ends of the gRNA and 3′ phosphorothioate internucleotide linkages. Exemplary target DNA sequences targeted by pairs of gRNAs for regulating the gene expression of one or more genes can comprise any nucleotide sequence selected from SEQ ID NOs: 16-31 listed in Table 2 or variants thereof.

[0148] Table 2: Exemplary target region sequences (SEQ ID NO: 12-15) and target DNA sequences (SEQ ID NO: 16-31) of gRNA pairs

[0149]

[0150]

[0151] For example, the gRNA pair for targeting KCNN2 can comprise a sequence selected from the nucleotide sequences of SEQ ID NO: 12-15 (shown in Table 2). In some embodiments, the gRNA pair for targeting KCNN2 comprises a first gRNA molecule containing the sequence of SEQ ID NO: 12 and a second gRNA molecule containing the sequence of SEQ ID NO: 13. In some embodiments, the gRNA pair for targeting KCNN2 comprises a first gRNA molecule containing or having the sequence of SEQ ID NO: 14 and a second gRNA molecule containing or having the sequence of SEQ ID NO: 15.

[0152] In some embodiments, the gRNA molecule for targeting KCNN2 is a 2′O-methyl analogue that contains a 3′ phosphorothioate internucleotide bond in the terminal three nucleotides at either or both of its 5′ and 3′ ends and contains the sequence of SEQ ID NO: 12, 13, 14, or 15.

[0153] Variant gRNA sequences can have at least 80% sequence identity with the sequences of the present disclosure, as measured over any suitable length of sequence. Typically, the percent sequence identity is determined over a contiguous stretch of nucleic acids. Variant gRNA sequences of the present disclosure can, for example, have at least 80% sequence identity with the sequences of the present disclosure, as measured over at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or more nucleic acid residues. In some embodiments, the variant gRNA molecule has at least 80% sequence identity with the gRNA molecule of the present disclosure over the entire length of the variant gRNA molecule. In some embodiments, variant gRNA molecules of the present disclosure can be variants of one or more gRNA molecules whose target regions are complementary to the target sequences of one of SEQ ID NOs: 16 to 30. The gRNA pairs of the present disclosure can include variants of one or both of the two gRNA sequences in a pair that target a gene (e.g., a gene selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1). For example, a variant of a gRNA pair comprising a first gRNA molecule containing the sequence of SEQ ID NO: 12 and a second gRNA molecule containing the sequence of SEQ ID NO: 13 can include 1) a first gRNA molecule containing a variant of the sequence of SEQ ID NO: 12, 2) a second gRNA molecule containing a variant of the sequence of SEQ ID NO: 13, or 3) both.

[0154] Regulation at the protein level

[0155] In another embodiment, the regulation of gene expression and / or activity occurs at the protein (e.g., polypeptide) level. For example, a reduction in gene function at the protein level can be achieved by methods including, but not limited to, targeting the protein with small molecules, peptides, aptamers, destabilizing domains, or other methods that can, for example, downregulate the activity of the gene product or increase the rate of gene product degradation. Alternatively, the expressed protein can be modified by site-directed mutagenesis and / or introduction of missense or nonsense mutations to reduce or eliminate biological activity. In some embodiments, small molecules that bind, for example, to the active site and inhibit the function of the target protein can be added to, for example, the cell culture medium and thereby introduced into the packaging cells and / or production cells. Alternatively, the function of the target protein can be regulated by introducing, for example, peptides into the cells (e.g., packaging cells and / or production cells), such peptides preventing, for example, protein-protein interactions (see Shangary et al., (2009) Annual Review of Pharmacology and Toxicology 49:223). Such peptides can be introduced into the cells (e.g., packaging and / or production cells) by, for example, transfection or electroporation or via an expression construct. Alternatively, the peptides can be introduced into the cells (e.g., packaging cells and / or production cells) by adding (e.g., by conjugation) one or more moieties or booster molecules that facilitate cellular delivery to enhance their own delivery. Techniques for expressing peptides include, but are not limited to, fusion of the peptide to a scaffold, or attachment of a signal sequence, to stabilize or direct the peptide to the location or compartment of interest, respectively. In certain embodiments, the rAAV packaging cell line and / or production cell line comprises cells that have been engineered using any of the foregoing methods to reduce the expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0156] Modulation of the effect on the expression of one or more genes and / or proteins

[0157] In certain embodiments, the modulation methods described in the present disclosure can be used to generate rAAV packaging cell lines and / or production cell lines that produce high titers of rAAV. In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the expression of one or more genes (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1) and / or a significant decrease in the activity of the proteins expressed by one or more genes (such as a decrease of at least 5%, at least 10%, at least 20%, or greater). In certain embodiments, the expression of the target gene is decreased by about 40% to about 100% (such as, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0158] In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the activity of a protein or RNA expressed by a target gene (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1). For example, the methods described herein can result in a decrease in the activity of a protein or RNA expressed by a target gene of at least 5%, at least 10%, at least 20%, or more. In certain embodiments, the target gene protein or RNA activity is decreased by about 40% to about 100% (e.g., about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%). In addition, the modulation of one or more genes can result in the modulation of multiple genes (e.g., via miRNA).

[0159] In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the expression of a gene product (e.g., the gene product of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1) (e.g., a decrease of at least 5%, at least 10%, at least 20%, or more). In certain embodiments, the expression of the gene product is decreased by about 40% to about 100% (e.g., about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0160] In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the expression of a polypeptide or polyribonucleotide expressed by at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 (e.g., a decrease of at least 5%, at least 10%, at least 20%, or more). In certain embodiments, the expression of the polypeptide or polyribonucleotide is decreased by about 40% to about 100% (e.g., about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0161] In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the activity of a polypeptide or polyribonucleotide expressed by at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 (e.g., a decrease of at least 5%, at least 10%, at least 20%, or more). In certain embodiments, the activity of the expressed polypeptide or polyribonucleotide is decreased by about 40% to about 100% (e.g., about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%; or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%).

[0162] In certain embodiments, the reduction in the expression and / or activity of one or more genes, proteins, or RNAs in the rAAV packaging cell line and / or production cell line is maintained for about 5 days (e.g., about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer).

[0163] In certain embodiments, the reduction in the expression and / or activity of one or more genes, proteins, or RNAs in the rAAV packaging cell line and / or production cell line is intended to be maintained indefinitely or permanently, e.g., by using gene disruption or partial or complete gene deletion.

[0164] In certain embodiments, the reduction in the expression and / or activity of one or more genes, proteins, or RNAs in the rAAV packaging cell line and / or production cell line is maintained in culture for at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty, at least thirty, at least forty, or more generations of the rAAV packaging cell line and / or production cell line.

[0165] Modulating the effect on rAAV production

[0166] Modulation of one or more genes and / or proteins in the rAAV packaging cell line and / or production cell line can result in an increase in rAAV titer. In some embodiments, the modulation results in an increase in the titer of rAAV produced by the rAAV packaging cell line and / or production cell line to about 1.5 to about 7-fold (e.g., about 1.5 to about 6.5, about 1.5 to about 6, about 1.5 to about 5.5, about 1.5 to about 5, about 1.5 to about 4.5, about 1.5 to about 4, about 1.5 to about 3.5, about 1.5 to about 3.0, about 1.5 to about 2.5, about 1.5 to about 2.0, about 2 to about 7, about 2.5 to about 7, about 3 to about 7, about 3.5 to about 7, about 4 to about 7, about 4.5 to about 7, about 5 to about 7, about 5.5 to about 7, about 6 to about 7, about 6.5 to about 7, or about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, or about 7.0). In some embodiments, the titer of rAAV produced by the rAAV packaging cell line and / or production cell line is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, or more. Any increase in rAAV titer caused by the modulation of one or more genes and / or proteins can be compared to the rAAV titer produced by a control parental cell line.

[0167] In some embodiments, the regulation of one or more genes and / or proteins in the rAAV packaging cell line and / or production cell line can increase rAAV titers for at least 2 days, at least 5 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, or longer.

[0168] Method for producing rAAV

[0169] In certain embodiments, the present disclosure describes methods for producing rAAV from rAAV packaging cell lines and / or production cell lines that have been engineered to regulate the expression of one or more genes, proteins, or non-coding RNAs. In certain embodiments, rAAV is produced by infecting cells of an rAAV production cell line that is generated by delivering an rAAV vector to an engineered rAAV packaging cell line. In certain embodiments, rAAV is produced by infecting cells of an rAAV production cell line in which the expression of one or more genes, proteins, or non-coding RNAs has been regulated. In certain embodiments, the production of rAAV from engineered rAAV packaging cell lines and / or production cell lines is enhanced compared to a control parental cell line.

[0170] In certain embodiments, cells of the engineered packaging cell line are infected with a helper virus (e.g., adenovirus (AV) or herpes simplex virus) that permits rAAV replication. In some embodiments, cells of the engineered production cell line are infected with a helper virus (e.g., adenovirus (AV) or herpes simplex virus).

[0171] Method for harvesting rAAV

[0172] rAAV particles can be obtained from engineered rAAV packaging and / or production cells by lysing the cells. Lysis of the engineered rAAV packaging and / or production cells can be achieved by chemically or enzymatically treating the cells to release infectious virus particles. These methods include using nucleases (e.g., benzonase or DNase), proteases (e.g., trypsin), or detergents or surfactants. Physical disruption, such as homogenization or grinding, or applying pressure through a microfluidizer pressure unit, or freeze-thaw cycles can also be used. In certain embodiments, lysates from engineered rAAV packaging and / or production cells can be used to harvest rAAV particles.

[0173] In certain embodiments, cell culture supernatants can be harvested from engineered rAAV packaging and / or production cells without cell lysis. In certain embodiments of the present disclosure, engineered rAAV packaging and / or production cells secrete rAAV particles, which can be harvested from the cell culture supernatant without cell lysis. In certain embodiments, engineered rAAV packaging cell lines and / or production cell lines have higher rAAV titers than control parental cell lines, such that more rAAV is harvested from the engineered rAAV packaging cell lines and / or production cell lines compared to the control parental cell lines.

[0174] After harvesting the rAAV particles, it may be necessary to purify the sample containing rAAV to remove, for example, cell debris generated by cell lysis. Methods for minimal purification of AAV particles are known in the art. Two exemplary purification methods are cesium chloride (CsCl)-based and iodixanol-based density gradient purification. These two methods are described in Strobel et al., Human Gene Therapy Methods, 26(4):147-157 (2015). Affinity chromatography can also be used to achieve minimal purification, for example, using AVB Sepharose gel affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The AAV purification method using AVB Sepharose gel affinity resin is described in, for example, Wang et al., Mol Ther Methods Clin Dev., 2:15040 (2015). After purification, the rAAV particles can be filtered and stored at ≤ -60 °C.

[0175] In certain embodiments, the present disclosure provides a method for harvesting rAAV particles produced from an engineered rAAV packaging cell line after co-infection of the cells with two different adenoviruses.

[0176] In certain embodiments, the present disclosure provides a method for harvesting rAAV particles produced after infection of an rAAV production cell line generated from an engineered rAAV packaging cell line.

[0177] In certain embodiments, the present disclosure provides a method for harvesting rAAV particles produced after infection of an engineered rAAV production cell line with a helper virus.

[0178] Quantification of rAAV Particles

[0179] Since AAV infection does not result in a cytopathic effect in vitro, plaque assays cannot be used to determine the infectivity titer, and thus quantification of rAAV particles is complex. However, rAAV particles can be quantified using a variety of methods, including quantitative polymerase chain reaction (qPCR) (Clark et al., Hum. Gene Ther. [Human Gene Therapy] 10, 1031 - 1039 (1999)), dot blot hybridization (Samulski et al., J. Virol. [Journal of Virology] 63, 3822 - 3828 (1989)), and by the optical density of highly purified vector preparations (Sommer et al., Mol. Ther. [Molecular Therapy] 7, 122 - 128 (2003)). DNase-resistant particles (DRP) can be quantified by quantitative polymerase chain reaction with reduced gene expression (DRP-qPCR) in a thermal cycler (e.g., iCycler iQ 96-well modular thermal cycler (Bio-Rad, Hercules, Calif.)) in real time. In the presence of DNase I (100 U / ml; Promega, Madison, Wis.), samples containing rAAV particles can be incubated at 37 °C for 60 minutes, then protease K (Invitrogen, Carlsbad, Calif.) digestion (10 U / ml) is carried out at 50 °C for 60 minutes, and then denaturation is carried out at 95 °C for 30 minutes. The primer-probe set used should be specific to the non-native portion of the rAAV vector genome, such as the poly(A) sequence of the protein of interest. Based on the length and composition of the primers, probes, and amplification sequences, any suitable set of cycling parameters can be used to amplify the PCR product. Alternatives are disclosed, for example, in Lock et al., Human Gene Therapy Methods 25(2):115 - 125 (2014).

[0180] Viral genome amplification can also be measured using qPCR techniques similar to those described above. However, to quantify the total genomic amplification in producer cells, only intracellular samples are collected and the samples are not treated with DNase I to measure the packaged and unpackaged viral genomes. Viral genome amplification can be calculated on a per host cell basis by simultaneously measuring a host cell housekeeping gene such as RNase P.

[0181] The infectivity of rAAV particles can be determined using the TCID50 (50% tissue culture infective dose) assay, as described, for example, in Zhen et al., Human Gene Therapy 15:709-715 (2004). In this assay, rAAV vector particles are serially diluted and used to co-infect a cell line expressing Rep / Cap together with AV particles in a 96-well plate. Forty-eight hours post-infection, total cellular DNA is extracted from the infected and control wells. Then, using qPCR, rAAV vector replication is measured with a transgene-specific probe and primers. The TCID50 infectivity / ml is calculated using the equation, using the ratio of AAV-positive wells with 10-fold serial dilutions.

[0182] Therapeutic applications

[0183] The rAAV produced by the engineered rAAV packaging cell lines and / or producer cell lines described herein can be used, for example, in gene therapy of mammals. The rAAV produced by the engineered cells described herein can be used in ex vivo and / or in vivo gene therapy applications. The rAAV produced by the engineered cells described herein can be used to deliver, for example, small molecules (such as siRNA or sgRNA), peptides, and / or proteins.

[0184] In some embodiments, the rAAV produced by the engineered cell lines described herein can be used to treat diseases or disorders in human subjects in need thereof. In certain embodiments, the rAAV produced by the engineered cell lines described herein can be administered in combination with a pharmaceutically acceptable carrier.

[0185] Any suitable method or route can be used to administer the rAAV or a composition containing rAAV produced by the engineered packaging cell lines and / or producer cell lines described herein. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration. In some embodiments, the rAAV or a composition containing rAAV produced by the engineered packaging cell lines and / or producer cell lines is administered intravenously.

[0186] The practice of the present disclosure will be more fully understood from the foregoing examples, which are given herein for illustrative purposes only and should not be construed as limiting the present disclosure in any way.

[0187] Examples

[0188] Example 1: Development of a knockdown protocol

[0189] Optimized and developed siRNA knockdown experiments for 6-well and 24-well formats by knocking down the housekeeping gene HPRT1. Experiments conducted in 24-well plates were evaluated based on multiple factors such as seeding density, cell culture conditions (e.g., percentage of carbon dioxide (CO2)), percentage of fetal bovine serum (FBS), ratio between transfection reagent ( RNAiMax) and siRNA (the "ratio"), incubation time, and siRNA concentration. Commercially available siRNAs designed for HPRT1 gene knockdown were used to optimize experimental conditions. According to the manufacturer's instructions, HeLa producer cells were transfected with different concentrations of siRNA using RNAiMax. The percentage reduction in HPRT1 expression was determined by real-time PCR. Compared to the baseline control, the optimized 24-well siRNA knockdown method was able to knock down the highly expressed gene HPRT1 by more than 80%. As Figure 2A shown in -D, cells were seeded at 1 x 105 cells / well, the ratio between transfection reagent and siRNA was 1:5, and 8 nM siRNA showed the highest knockdown efficiency. Figure 2A The effect of different siRNA concentrations / ratios used on the percentage of HPRT1 knockdown is shown. Figure 2B The effect of different siRNA concentrations / ratios on the percentage of HPRT1 expression is shown. For optimization of the 6-well protocol, two different siRNA concentrations were tested. For the data plotted in Figure 2A and 2B , seeding densities of 5 x 10 4 , 8 x 10 4 and 1 x 105 were tested. Figure 2C The effect of different siRNA concentrations on the percentage of HPRT1 knockdown is shown. Figure 2D The effect of different concentrations of siRNA on the percentage of HPRT1 expression is shown. All experiments were repeated three times.

[0190] Example 2: RNA Sequencing

[0191] Eight three-liter bioreactors were run in two different HeLa S3 producer cell lines under supplemented and non-supplemented production conditions. Two additional bioreactors were run without adenovirus 5 (Ad5) as uninfected controls. Table 3 lists the detailed information regarding bioreactor conditions and production levels.

[0192] Table 3: Bioreactor Conditions and Production Levels.

[0193]

[0194]

[0195] Abbreviations used in Table 3: The addition of one or more supplements is indicated by (+); the absence of one or more supplements is indicated by (-); MOI - multiplicity of infection.

[0196] Thirty hours after infection with Ad5, samples were taken for RNA-Seq. The samples were washed once with PBS and the cell pellet was stored at -80 °C until ready for shipment. RNA extraction and cDNA synthesis of the extracted RNA were performed by methods well known in the art. Prior to sequencing, library preparation was performed using a commercially available RNA-Seq library preparation kit. RNA sequencing was performed using a commercially available Illumina sequencing platform. The resulting reads were mapped to the human genome, the Ad5 genome, and the AAV2 genome using mapping methods well known in the art. Any reads mapped to the Ad5 genome were discarded. Another round of sequencing was performed to enrich the reads mapped to the human genome. Differential analysis was performed using the data generated by RNA sequencing (see Table 4).

[0197] Table 4: Differential analysis

[0198] Differential analysis# Control conditions Experimental conditions 1 PCL1; No production PCL1*; Production (N.S.) 2 PCL1; No production PCL2; No production 3 PCL1; No production PCL1; Production (S) 4 PCL1; Production (N.S.) PCL1; Production (S) 5 PCL1; Production (N.S.) PCL1; Production (S) 6 PCL1; Production (N.S.) PCL2; Production (N.S.) 7 PCL2; No production PCL2; Production (N.S.) 8 PCL2; No production PCL2; Production (S) 9 PCL2; No production PCL2; Production (S) 10 PCL1; Production (S) PCL2; Production (S) 11 PCL2; Production (S) PCL2; Production (S) 12 PCL1; Production (S) PCL1; Production (S) 13 PCL1; Production (S) PCL1; Production (S)

[0199] *PCL1 - Production cell line 1; PCL2 - Production cell line 2; No production - uninfected control cells; Production (N.S.) - Ad5-infected cells cultured under unsupplemented conditions; Production (S) - Ad5-infected cells cultured under supplemented conditions.

[0200] In this example, differential expression analysis was calculated as the log fold change (LogFC) of the mRNA level compared to the control condition for the experimental condition. Upregulated gene expression was a positive LogFC, and downregulated gene expression was a negative LogFC. Differentially expressed genes with a p-value ≤ 0.05 were considered statistically significant. In each differential analysis, hundreds or thousands of genes were significantly upregulated or downregulated. Filtering criteria were established (see, for example Figure 6 ), and these were used to reduce the dataset to a manageable number of genes for evaluation. As described in Example 6, gene sets were aligned and moved towards the filtering criteria.

[0201] Example 3: Validation of results obtained from RNA sequencing by RT-qPCR

[0202] A small set of genes was selected for validation of the RNA sequencing data. Using methods well known in the art, RT-qPCR assays were used to confirm the RNA-Seq results. The data were analyzed using the ΔΔCt method. RT-qPCR independently confirmed the trends observed in the RNA-Seq data. Figure 3A -B shows from PGA5 ( Figure 3A ) and SPANXN3 ( Figure 3BThe log fold change values of gene expression obtained from bioinformatics analysis of RNA-Seq data. The x-axis shows the conditions under which the production cell line was grown (supplemented (differential analysis #5 as described in Table 4) versus unsupplemented (differential analysis #1 as described in Table 4)), and the y-axis shows the log fold change (LogFC) of gene expression. The log fold change of PGA5 ( Figure 3A ) and SPANXN3 ( Figure 3B ) expression in cells grown in unsupplemented cell medium was plotted relative to the corresponding gene expression in uninfected cells (cells not infected with the helper virus). The log fold change of PGA5 ( Figure 3A ) and SPANXN3 ( Figure 3B ) expression in cells grown in supplemented cell medium was plotted relative to the corresponding gene expression in cells grown in unsupplemented cell medium.

[0203] The expression of PGA5 and SPANXN3 genes in the production cell line grown under supplemented and unsupplemented conditions was also evaluated by RT-qPCR using methods well known in the art. Figure 3C -D shows the RT-qPCR fold change values of PGA5 ( Figure 3C ) and SPANXN3 ( Figure 3D ) expression in cells grown in unsupplemented and supplemented cell media relative to uninfected cells (cells not infected with the helper virus). Figure 3A -D shows that the data obtained from qPCR and RNA sequencing follow the same trend.

[0204] Example 4: Verification of Results Obtained from RNA Sequencing by RT-qPCR in Different Clones of the Production Cell Line

[0205] The RNA sequencing results were further verified by RT-qPCR experiments on RNA extracted from different clones of the HeLa S3 production cell line. Figure 4A -B shows the fold change values of PGA5 ( Figure 4A ) and SPANXN3 ( Figure 4B ) expression in clones of the production cell line grown in unsupplemented and supplemented cell media relative to uninfected cells (cells not infected with the helper virus), as determined by RT-qPCR. 21C5, 3C6, and 2B6 represent different clones of the HeLa production cell line. Figure 4C -D shows that in clones 21C5, 3C6, and 2B6 of the production cell line grown in supplemented cell medium, the expression of PGA5 ( Figure 4C ) and SPANXN3 ( Figure 4D)Relative fold increase in expression. These results further validate the bioinformatics RNA sequencing and RT-qPCR data described in Example 3.

[0206] Example 5: Effect of gene knockdown on rAAV titer

[0207] Based on the optimization scheme discussed in Example 1, knockdown experiments were performed by individually knocking down genes in the HeLa production cell line. siRNA nucleotide sequences were designed for each gene (see Table 1).

[0208] The conditions were determined to be 1x10 5 seeding density, 8 nM siRNA and siRNA:RNAiMAX ratio of 1:5. AAV production was induced 24 hours after gene expression was reduced, and rAAV was harvested 72 hours after infection. The titer of each sample was measured and compared to a non-targeting missense siRNA control. This experiment was performed independently three times, the results were averaged, and statistical analysis was performed. Figures 5A - 5C Results of siRNAs for individual genes in production cell lines 1-3 on absolute rAAV titer (GC / mL; GC = genomic copy) are shown separately. Figures 5D - 5F Fold increase in rAAV titer of siRNAs for individual genes in different production cell lines 1-3 are shown separately.

[0209] As Figures 5A - 5F shown, reduction of KCNN2, LINC00319, RGMA or SPANXN3 expression in the production cell line resulted in a statistically significantly 2- to 4-fold higher rAAV titer than the missense control. In the three production cell lines, these four genes showed a statistically relevant positive effect on titer when knocked down. These results suggest that these genes are excellent targets for more permanent modifications such as CRISPR / Cas9 knockout.

[0210] Example 6: Gene filtering method

[0211] For Filter 1, genes from differential analyses 1 and 7 (as described in Table 3) were aligned. Differential analyses 1 and 7 identified genes that were upregulated or downregulated after addition of adenovirus 5 under non-supplemented conditions. Analysis 1 observed cells from the 21C5 production cell line (production cell line 1, PCL1). Analysis 7 observed cells from 2B6 (production cell line 2, PCL2). The gene list after this Filter 1 identified non-cell line-specific genes, and this alignment provided a total of 9149 genes common between the two production cell lines.

[0212] For Filter 2, the genes from Filter 1 are aligned with the genes present in Differential Analysis 5. Analysis 5 observes the genes that are upregulated and downregulated in cells from the 21C5 production cell line (PCL1) under supplemented conditions compared to non-supplemented conditions. The purpose of this differential analysis is to determine the impact of production under supplemented conditions relative to production under non-supplemented conditions. The purpose of aligning the gene set from Filter 1 with Differential Analysis 5 is to identify genes under improved productivity conditions that 1) are not by-products of the improved production conditions and 2) may be related to two different cell lines. After alignment, 374 genes move forward.

[0213] For Filter 3, only genes with a large LogFc threshold of >2LogFC± move forward. This is done to ensure a high level of upregulation / downregulation of the genes moving forward and to give confidence that the selected genes are not artifacts of RNA-Seq. After filtering, 77 genes move forward.

[0214] For Filter 4, only genes that show upregulation in both Differential Analysis 1 and Differential Analysis 5 or genes that show downregulation in both Differential Analysis 1 and Differential Analysis 5 are retained. For example, one of the 77 genes must show upregulation in Differential Analysis 1 and further upregulation in Differential Analysis 5 or downregulation in Differential Analysis 1 and further downregulation in Differential Analysis 5. The purpose of this filter is to ensure that for the genes being evaluated, the high-titer condition does not antagonize the regulation of this particular gene compared to the low-titer condition. After filtering, 11 genes are left for evaluation. An illustrative flow chart showing an exemplary gene filtering method is shown in Figure 6 shown (using the abbreviation: LogFC = log fold change).

[0215] Table 5 provides the Log2FC data from each comparison during the process of filtering important genes for productivity.

[0216] Table 5: Log2FC Data

[0217]

[0218] Example 7: Gene Knockout of KCNN2

[0219] In this example, two existing highly optimized monoclonal HeLa production cell lines (PCLs) - 2H5 and 7B12 - were genetically modified to knockout the KCNN2 gene (previously identified in the RNA-seq screen described herein), which encodes the calcium-activated potassium channel protein SK2.

[0220] Knockout of KCNN2 in 2H5 or 7B12 HeLa cells using the eGFP selection marker. Putative KCNN2 knockout enriched for eGFP expression was seeded in 96-well plates. Cell colonies were allowed to form, genomic DNA was harvested, and PCR was performed to amplify the region containing the knockout. The PCR products were Sanger sequenced and analyzed for the presence of insertions / deletions in the sequencing files. 2H5 and 7B12 clones with high knockout likelihood were expanded for further testing.

[0221] The top clones were transferred to serum-free suspension cultures. Clone productivity compared to the parental line was evaluated by 24-deep well rAAV production. The clones were seeded at 2x10 5 cells / mL in 3 mL cultures and infected with Ad5 at a multiplicity of infection (MOI) of 50. Four days post-infection, rAAV was harvested and titers were evaluated. The fold increase in titer was normalized to the parental control. Compared to the control samples, the best clones showed a 1.5 - 2.7-fold increase in titer. The 2H5 titer was 2.46x10 9 -4.98x10 10 GC / mL ( Figure 7A ). When the titer was normalized to the parental control, a fold increase ranging from 1.2 - 2.7-fold ( Figure 7B ) was observed. The 7B12 titer ranged from 4.33x10 8 -1.88x10 10 GC / mL ( Figure 7C ). When the titer was normalized to the parental control, a fold increase ranging from 1.5 - 2.6-fold ( Figure 7D ) was observed. Then the clones with a minimum 1.5-fold increase were expanded to shake flask cultures and seeded into 15 for high-seeding density supplemented rAAV production. The cells were seeded at 1.5x10 6 cells / mL and infected with Ad5 at an MOI of 50. Four days post-infection, rAAV was harvested and titers were evaluated. The fold increase in titer was normalized to the parental control. Compared to the control samples, the best clones showed a 1.5 - 2.3-fold increase in titer. The 2H5 titer was 1.5x10 11 -3.82x10 11 GC / mL ( Figure 8A ). When the titer was normalized to the parental control, a fold increase ranging from 1.3 - 2.3-fold ( Figure 8B ) was observed. The 7B12 titer ranged from 2.62x10 10 -1.35x10 11 GC / mL ( Figure 8C ). When the titer was normalized to the parental control, a fold increase ranging from 1.2 - 1.5-fold ( Figure 8D ) was observed.

[0222] These data indicate that reducing or eliminating the expression of one or more of the genes described herein in AAV-producing cells (e.g., by gene knockout) can be used to increase the production of rAAV in engineered cells.

[0223] Example 8: Multiplex siRNA knockdown

[0224] In this example, multiplex knockdown of genes identified previously in the RNA-seq screens described herein was performed using siRNAs to determine whether simultaneous targeting of multiple genes would have an additive effect on titer.

[0225] Multiplex knockdown was performed using a modification of the method described in Example 5. Briefly, cells were transfected with 8 nM of each siRNA and maintaining the siRNA:RNAiMAX ratio at 1:5. AAV production was induced 24 hours after gene expression was reduced, and rAAV was harvested 72 hours after infection. The titer of each sample was determined and compared to a nontargeting sense siRNA control.

[0226] In this example, KCNN2 was knocked down in combination with a group of other siRNAs described previously. Additionally, RGMA and SPANXN3 were knocked down in combination with each other. In 2H5, multiplex knockdown showed a 4.6 - 11.4-fold increase in titer compared to the sense control ( Figure 9A ). In 7B12, multiplex knockdown showed a 3.4 - 9.7-fold increase in titer compared to the sense control ( Figure 9B ). Each combination showed an increase in titer; however, not all combinations were improved over knockdown of KCNN2 alone. Knockdown of KCNN2 resulted in a 5.3-fold increase in 2H5 ( Figure 9A ) and a 5.1-fold increase in 7B12 ( Figure 9B ).

[0227] These data indicate that additional increases in rAAV production can be obtained by directly targeting multiple genomic regions in established monoclonal PCLs that produce high rAAV titers.

[0228] Numbered embodiments

[0229] 1. A recombinant adeno-associated virus (rAAV) packaging cell line and / or production cell line comprising cells in which the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced compared to a control parental cell.

[0230] 2. The packaging cell line and / or production cell line according to embodiment 1, which comprises cells in which the expression of KCNN2, LINC00319, RGMA and SPANXN3 is reduced compared to the control parental cells.

[0231] 3. The packaging cell line and / or production cell line according to embodiment 1 or 2, wherein the expression is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA) or antisense RNA oligonucleotide (ASO).

[0232] 4. The packaging cell line and / or production cell line according to any one of embodiments 1 to 3, wherein the expression is reduced using an siRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1-11.

[0233] 5. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of ATP5EP2 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 1 in the sense strand and the nucleotide sequence of SEQ ID NO: 32 in the antisense strand.

[0234] 6. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of LINC00319 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 2 in the sense strand and the nucleotide sequence of SEQ ID NO: 33 in the antisense strand.

[0235] 7. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of CYP3A7 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 3 in the sense strand and the nucleotide sequence of SEQ ID NO: 34 in the antisense strand.

[0236] 8. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of NOG is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 4 in the sense strand and the nucleotide sequence of SEQ ID NO: 35 in the antisense strand.

[0237] 9. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of SPANXN3 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 5 in the sense strand and the nucleotide sequence of SEQ ID NO: 36 in the antisense strand.

[0238] 10. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of MYRIP is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 6 in the sense strand and the nucleotide sequence of SEQ ID NO: 37 in the antisense strand.

[0239] 11. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of KCNN2 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 7 in the sense strand and the nucleotide sequence of SEQ ID NO: 38 in the antisense strand.

[0240] 12. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of NALCN-AS1 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 8 in the sense strand and the nucleotide sequence of SEQ ID NO: 39 in the antisense strand.

[0241] 13. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of RGMA is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 9 in the sense strand and the nucleotide sequence of SEQ ID NO: 40 in the antisense strand.

[0242] 14. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of PGA5 is reduced, and the siRNA comprises the sequence of SEQ ID NO: 10 in the sense strand and the sequence of SEQ ID NO: 41 in the antisense strand.

[0243] 15. The packaging cell line and / or production cell line according to embodiment 4, wherein the expression of ABCA10 is reduced, and the siRNA comprises the sequence of SEQ ID NO: 11 in the sense strand and the sequence of SEQ ID NO: 42 in the antisense strand.

[0244] 16. The packaging cell line and / or production cell line according to embodiment 3, wherein the nuclease is selected from the group consisting of zinc finger nuclease (ZFN), meganuclease, transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein.

[0245] 17. The packaging cell line and / or production cell line according to any one of embodiments 1 to 16, wherein the expression is reduced by CRISPR genome editing.

[0246] 18. The packaging cell line and / or production cell line according to embodiment 17, wherein the expression is reduced using a guide RNA pair, and wherein each guide RNA:

[0247] (a) comprises a sequence selected from the nucleotide sequences of SEQ ID NOs: 12 - 15, and / or

[0248] (b) targets a target DNA sequence selected from any of the nucleotide sequences of SEQ ID NOs: 16 - 31.

[0249] 19. The packaging cell line and / or production cell line according to embodiment 18, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 12 and a second gRNA molecule containing the sequence of SEQ ID NO: 13.

[0250] 20. The packaging cell line and / or production cell line according to embodiment 18, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 14 and a second gRNA molecule containing the sequence of SEQ ID NO: 15.

[0251] 21. The packaging cell line and / or production cell line according to embodiment 19 or 20, wherein each gRNA molecule is a 2′O - methyl analogue that contains a 3′ phosphorothioate internucleotide bond in the terminal three nucleotides at either or both of its 5′ and 3′ ends.

[0252] 22. The packaging cell line and / or production cell line according to any one of embodiments 1 to 21, wherein the gene expression is eliminated as compared to a control parental cell.

[0253] 23. The packaging cell line and / or production cell line according to any one of embodiments 1 to 22, wherein the cell line is a human cell line.

[0254] 24. The packaging cell line and / or production cell line according to embodiment 23, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

[0255] 25. The cell line according to any one of embodiments 1 to 24, wherein the cell line is an rAAV packaging cell line.

[0256] 26. The cell line according to any one of embodiments 1 to 24, wherein the cell line is an rAAV production cell line.

[0257] 27. The cell line according to embodiment 26, wherein the titer of rAAV is increased by about 1.5 to about 7-fold compared to the titer of rAAV produced from a cell line comprising the control parental cells.

[0258] 28. A lysate of the cell line according to any one of embodiments 1 to 27.

[0259] 29. A cell culture supernatant from the cell line according to any one of embodiments 1 to 27.

[0260] 30. A method of generating a production cell line, the method comprising delivering a recombinant adeno-associated virus (rAAV) vector to cells of the packaging cell line according to embodiment 25.

[0261] 31. A method of generating rAAV, the method comprising infecting cells of a production cell line generated by the method according to embodiment 30 with a helper virus.

[0262] 32. A method of generating rAAV, the method comprising infecting cells of the production cell line according to embodiment 26 with a helper virus.

[0263] 33. The method according to embodiment 31 or 32, wherein the rAAV is harvested from the production cell line.

[0264] 34. The method according to any one of embodiments 31 to 33, wherein the production of rAAV is enhanced compared to a control parental cell line.

[0265] 35. A method of identifying one or more genes associated with the production of rAAV, the method comprising:

[0266] adding one or more supplements that increase the titer of rAAV in the cell line;

[0267] measuring global gene expression across the transcriptome in the supplemented and unsupplemented cell lines;

[0268] obtaining a list of genes differentially expressed between the supplemented and unsupplemented cell lines; and

[0269] identifying one or more genes associated with the production of rAAV.

[0270] 36. The method according to embodiment 35, wherein the one or more supplements added to the cell line comprise dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, or triamcinolone.

[0271] 37. A method of generating an rAAV packaging cell line and / or a production cell line to facilitate increased rAAV production, the method comprising modulating the expression of one or more genes identified by the method according to embodiment 35.

[0272] 38. The method according to any one of embodiments 35 to 37, wherein the cell line is an rAAV packaging cell line.

[0273] 39. The method according to any one of embodiments 35 to 37, wherein the cell line is an rAAV production cell line.

[0274] 40. The method according to embodiment 39, wherein the rAAV production cell line increases the rAAV titer by at least 1.5-fold compared to the rAAV titer produced by an rAAV production cell line that does not modulate the expression of the corresponding one or more genes.

[0275] 41. The method according to any one of embodiments 37 to 40, wherein modulating the expression comprises reducing the expression of one or more genes.

[0276] 42. The method according to any one of embodiments 37 to 40, wherein modulating the expression comprises eliminating the expression of one or more genes.

[0277] 43. The method according to any one of embodiments 30 to 42, wherein the cell line is a human cell line.

[0278] 44. The method according to embodiment 43, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

[0279] 45. A recombinant adeno-associated virus (rAAV) packaging cell line and / or a production cell line comprising cells engineered to have reduced expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 compared to the corresponding unmodified parental cells.

[0280] 46. The rAAV packaging cell line and / or production cell line according to embodiment 45, wherein the expression and / or activity of gene products expressed by ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced indefinitely or permanently.

[0281] 47. The rAAV packaging cell line and / or production cell line according to embodiment 46, wherein the cell line has been engineered to comprise a gene disruption or a partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0282] 48. The rAAV packaging cell line and / or production cell line according to embodiment 47, wherein the cell line has been engineered to comprise a gene disruption in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0283] 49. The rAAV packaging cell line and / or production cell line according to embodiment 47, wherein the cell line has been engineered to comprise a gene disruption in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1.

[0284] 50. The rAAV packaging cell line and / or production cell line according to embodiment 47, wherein the cell line has been engineered to comprise a partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0285] 51. The rAAV packaging cell line and / or production cell line according to embodiment 47, wherein the cell line has been engineered to comprise a partial or complete gene deletion in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1.

[0286] 52. A recombinant adeno-associated virus (rAAV) packaging cell line and / or production cell line, wherein the cell line exhibits a reduced expression and / or activity of a polypeptide or polynucleotide expressed by at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1 as compared to the corresponding parental cell line.

[0287] Incorporated by reference

[0288] The entire disclosure of each patent document and scientific article mentioned herein is incorporated by reference for all purposes.

[0289] Equivalents

[0290] Without departing from the spirit or essential characteristics of the present disclosure, the invention may be embodied in other specific forms. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Thus, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes within the meaning and range of equivalents of the claims are intended to be embraced therein.

Claims

1. A recombinant adeno-associated virus (rAAV) packaging cell line and / or production cell line, which, compared with the corresponding unmodified parental cells, comprises cells with reduced expression of KCNN2, or cells with reduced expression of KCNN2 and reduced expression of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1.

2. The rAAV packaging cell line and / or production cell line according to claim 1, wherein the expression is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA) or antisense RNA oligonucleotide (ASO).

3. The rAAV packaging cell line and / or production cell line according to claim 1, wherein the expression is reduced using an siRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1-11.

4. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of KCNN2 is reduced using an siRNA comprising the nucleotide sequence of SEQ ID NO: 7 in the sense strand and the nucleotide sequence of SEQ ID NO: 38 in the antisense strand.

5. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of RGMA is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 9 in the sense strand and the nucleotide sequence of SEQ ID NO: 40 in the antisense strand.

6. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of ATP5EP2 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 1 in the sense strand and the nucleotide sequence of SEQ ID NO: 32 in the antisense strand.

7. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of LINC00319 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 2 in the sense strand and the nucleotide sequence of SEQ ID NO: 33 in the antisense strand.

8. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of CYP3A7 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 3 in the sense strand and the nucleotide sequence of SEQ ID NO: 34 in the antisense strand.

9. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of NOG is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO: 4 in the sense strand and the nucleotide sequence of SEQ ID NO: 35 in the antisense strand.

10. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of SPANXN3 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO:5 in the sense strand and the nucleotide sequence of SEQ ID NO:36 in the antisense strand.

11. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of MYRIP is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO:6 in the sense strand and the nucleotide sequence of SEQ ID NO:37 in the antisense strand.

12. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of NALCN-AS1 is reduced, and the siRNA comprises the nucleotide sequence of SEQ ID NO:8 in the sense strand and the nucleotide sequence of SEQ ID NO:39 in the antisense strand.

13. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of PGA5 is reduced, and the siRNA comprises the sequence of SEQ ID NO:10 in the sense strand and the sequence of SEQ ID NO:41 in the antisense strand.

14. The rAAV packaging cell line and / or production cell line according to claim 3, wherein the expression of ABCA10 is reduced, and the siRNA comprises the sequence of SEQ ID NO:11 in the sense strand and the sequence of SEQ ID NO:42 in the antisense strand.

15. The rAAV packaging cell line and / or production cell line according to claim 2, wherein the nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins.

16. The rAAV packaging cell line and / or production cell line according to claim 1, wherein the expression is reduced by CRISPR genome editing.

17. The rAAV packaging cell line and / or production cell line according to claim 16, wherein the CRISPR genome editing uses a guide RNA (gRNA) pair, wherein each gRNA: (a) comprises a sequence selected from the nucleotide sequences of SEQ ID NOs: 12-15, and / or (b) targets a target DNA sequence selected from any of the nucleotide sequences of SEQ ID NOs: 16-31.

18. The rAAV packaging cell line and / or production cell line according to claim 17, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO:12 and a second gRNA molecule containing the sequence of SEQ ID NO:

13.

19. The rAAV packaging cell line and / or production cell line according to claim 17, wherein the gRNA pair is for targeting KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 14 and a second gRNA molecule containing the sequence of SEQ ID NO:

15.

20. The rAAV packaging cell line and / or production cell line according to claim 18 or 19, wherein each gRNA molecule is a 2'O-methyl analogue that contains a 3'-thiolated phosphorothioate internucleotide bond in the terminal three nucleotides at either or both of its 5' and 3' ends.

21. The rAAV packaging cell line and / or production cell line according to claim 1, wherein gene expression is eliminated as compared to the corresponding unmodified parental cells.

22. The rAAV packaging cell line and / or production cell line according to claim 1, wherein the cell line is a human cell line.

23. The rAAV packaging cell line and / or production cell line according to claim 22, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

24. The rAAV packaging cell line and / or production cell line according to claim 23, wherein the cell line is an rAAV packaging cell line.

25. The packaging cell line and / or production cell line according to claim 23, wherein the cell line is an rAAV production cell line.

26. A method for generating an rAAV production cell line, the method comprising delivering an rAAV vector to the cells of the rAAV packaging cell line according to claim 24.

27. A method for generating rAAV, the method comprising infecting the cells of the rAAV production cell line generated by the method according to claim 26 with a helper virus.

28. A method for generating rAAV, the method comprising infecting the cells of the rAAV production cell line according to claim 25 with a helper virus.

29. The method according to claim 27, wherein the rAAV is harvested from the rAAV production cell line.

30. The method according to claim 28, wherein the rAAV is harvested from the rAAV production cell line.

31. The method according to any one of claims 27 to 30, wherein the production of rAAV is enhanced as compared to the corresponding unmodified parental cell line.

32. A recombinant adeno-associated virus (rAAV) packaging cell line and / or production cell line, which, as compared to the corresponding unmodified parental cells, comprises cells engineered to have reduced expression of KCNN2, or cells having reduced expression of KCNN2 and reduced expression of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1.

33. The rAAV packaging cell line and / or production cell line according to claim 32, wherein the expression of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1 is reduced indefinitely or permanently.

34. The rAAV packaging cell line and / or production cell line according to claim 33, which comprises cells engineered to contain gene disruption in at least one of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1.

35. The rAAV packaging cell line and / or production cell line according to claim 33, which comprises cells engineered to contain gene disruption in at least two genes selected from KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and NALCN-AS1.

36. The rAAV packaging cell line and / or production cell line according to claim 33, which comprises cells engineered to contain partial or complete gene deletion in at least one of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1.

37. The rAAV packaging cell line and / or production cell line according to claim 33, which comprises cells engineered to contain partial or complete gene deletion in at least two genes selected from KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and NALCN-AS1.

38. The rAAV packaging cell line and / or production cell line according to any one of claims 34-37, wherein the expression is reduced by clustered regularly interspaced short palindromic repeats (CRISPR) genome editing.

39. The rAAV packaging cell line and / or production cell line according to claim 38, wherein the CRISPR genome editing uses guide RNA (gRNA) pairs, wherein each gRNA: (a) comprises a sequence containing a nucleotide sequence selected from SEQ ID NO: 12-15, and / or (b) targets a target DNA sequence selected from any one of the nucleotide sequences of SEQ ID NO: 16-31.

40. The rAAV packaging cell line and / or production cell line according to claim 39, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 12 and a second gRNA molecule containing the sequence of SEQ ID NO: 13, or wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule containing the sequence of SEQ ID NO: 14 and a second gRNA molecule containing the sequence of SEQ ID NO:

15.

41. The rAAV packaging cell line and / or production cell line according to claim 40, wherein each gRNA molecule is a 2'O-methyl analogue that contains a 3'-thio-phosphate internucleotide bond in the terminal three nucleotides at either or both of its 5' and 3' ends.

42. A recombinant adeno-associated virus (rAAV) packaging cell line and / or production cell line, wherein, compared with the corresponding unmodified parental cell line, the cell line comprises cells that exhibit a reduced expression of a polypeptide or polyribonucleotide expressed by KCNN2, or cells that exhibit a reduced expression of a polypeptide or polyribonucleotide expressed by KCNN2 and a reduced expression of a polypeptide or polyribonucleotide expressed by RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP or NALCN-AS1.

43. The rAAV packaging cell line and / or production cell line according to any one of claims 32-42, wherein the cell line is a human cell line.

44. The rAAV packaging cell line and / or production cell line according to claim 43, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

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