Operated Produced Cell Line and Method for Producing and Using the Same

Abstract

To provide engineered producer cell lines and methods of making and using the same.SOLUTION: This application relates to recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines which have been engineered to reduce expression and / or activity of one or more genes and / or proteins to increase rAAV titers. The methods of generating the engineered cell lines are also described herein. Further described herein are methods of identifying one or more genes and / or proteins that are relevant to production of the rAAV.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - References to Related Applications 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, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Sequence Listing This application is electronically filed in ASCII format and contains a sequence listing that is hereby incorporated by reference in its entirety. The ASCII copy created on April 9, 2020, is named ULP - 005WO_SL.txt and is 113 kb in size.

[0003] Field of the Disclosure This application as a whole relates to engineered production and / or packaging cell lines for increasing recombinant adeno - associated virus (rAAV) titers, and methods of generating engineered production and / or packaging cell lines.

Background Art

[0004] Background rAAV-based vectors are one of the most promising vehicles for human gene therapy. rAAV vectors have been investigated for a wide range of gene therapy applications. In particular, rAAV vectors can deliver therapeutic genes to both dividing and non-dividing cells, and these genes can persist for long periods without being integrated into the genome of the target cells. Systems for producing rAAV have evolved over the past 20 years, but several problems remain unsolved. One limitation of rAAV production systems is the low titer yield of rAAV particles. The development of rAAV-based gene products as pharmaceuticals at the preclinical stage requires large amounts of rAAV vectors for studies in larger species in order to enable complete toxicity and biodistribution studies that can help predict dosing in humans. Furthermore, current rAAV production systems result in low titer yields, making it difficult to produce sufficient levels of rAAV for use in human trials and commercial applications. Researchers have explored numerous methods for generating rAAV particles with sufficiently high titers, but there remains a great need to address this problem. In particular, there is a need for efficient cell lines that can produce high-quality rAAV with high titer yields. The production of high-titer rAAV by the engineered cell lines described herein promotes the application of this vector system for in vivo gene therapy use. Summary of the Invention Means for Solving the Problems

[0005] Abstract The present disclosure addresses the need to obtain improved rAAV titers for gene therapy applications by providing rAAV packaging and / or production cell lines that contain 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 and / or production cell lines.

[0006] Compositions and methods for generating rAAV packaging and / or production cell lines that can produce rAAV with a high titer compared to control parental cells are described herein. More specifically, rAAV packaging and / or production cell lines are provided herein that include cells in which the expression of one or more genes and / or proteins is modulated, resulting in a higher rAAV titer compared to control parental cells. In one aspect, the disclosure provides rAAV packaging and / or production cell lines that include cells in which the expression of one or more genes and / or proteins is decreased 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 (calcium-activated potassium channel subfamily N member 2) and / or NALCN-AS1 (NALCN antisense RNA1) is decreased compared to control parental cells.

[0007] In some embodiments, the disclosure provides rAAV packaging and / or production cell lines that include cells in which the expression of KCNN2, LINC00319, RGMA and SPANXN3 is decreased compared to control parental cells.

[0008] In certain embodiments, the present disclosure provides rAAV packaging and / or production cell lines comprising cells engineered to have reduced expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to corresponding unmodified parental cells. In certain embodiments, the present disclosure provides rAAV packaging and / or production cell lines that exhibit reduced expression and / or activity of a polypeptide or polynucleotide expressed from 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.

[0009] In one aspect, the present disclosure provides rAAV packaging and / or production cell lines in which the expression of one or more genes is reduced by the use of a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO).

[0010] In certain embodiments, the expression of one or more genes is reduced by the use of siRNAs comprising a nucleotide sequence selected from any one of the sequences 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 sequence of SEQ ID NO: 42 in the antisense strand.

[0011] In certain embodiments, the nuclease used to reduce 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.

[0012] In certain embodiments, the expression of one or more genes is reduced by the use of CRISPR genome editing. In some embodiments, a guide RNA pair is used to target a gene to reduce and / or eliminate its expression. In certain embodiments, the expression of one or more genes is reduced by the use of a guide RNA pair, and each guide RNA comprises (a) a sequence selected from the nucleotide sequences of SEQ ID NOs: 12-15 and / or (b) targets a target DNA sequence selected from any one 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 comprising the sequence of SEQ ID NO: 12 and a second gRNA molecule comprising the sequence of SEQ ID NO: 13. In some embodiments, a gRNA pair is used to target KCNN2 and comprises a first gRNA molecule comprising the sequence of SEQ ID NO: 14 and a second gRNA molecule comprising the sequence of SEQ ID NO: 15. In some embodiments, each gRNA molecule is a 2'O-methyl analog comprising a 3' phosphorothioate nucleotide internucleotide linkage at the 3 nucleotides at either or both of its 5' and 3' ends.

[0013] In certain embodiments, one guide RNA pair is used to reduce the expression of one gene. In certain other embodiments, multiple guide RNA pairs 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, is decreased and / or removed in the rAAV packaging and / or production cell line as compared to the control parental cell line. In certain embodiments, the gene expression and / or activity is removed in the rAAV packaging and / or production cells as compared to the control parental cells.

[0014] In some embodiments described herein, the rAAV packaging and / or production cell line is a eukaryotic cell line. In certain embodiments, the rAAV packaging and / or production cell line is a human cell line. In certain embodiments, the rAAV packaging and / or production cell line is an insect cell line. In certain embodiments, the rAAV packaging and / or production cell line is a HeLa cell line. In certain other embodiments, the rAAV packaging and / or production cell line is a human embryonic kidney (HEK) 293 cell line.

[0015] In some embodiments described herein, the rAAV packaging and / or production cell line of the present disclosure produces a higher rAAV titer than the control parental cell line. In certain embodiments, the titer of rAAV produced from the cells of the rAAV production cell line of the present disclosure is increased by about 1.5 to about 7 times as compared to the titer of rAAV produced from a cell line including control parental cells. Also described herein is the lysate of the engineered cell line. In certain embodiments, higher titer rAAV is collected from the lysate. Also described herein is the cell culture supernatant from the engineered cell line. In certain embodiments, higher titer rAAV is collected from the cell culture supernatant.

[0016] Also provided herein is a method for generating a producer cell line, the method comprising delivering an rAAV vector to cells of a packaging cell line 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. In certain embodiments, the disclosure provides a method for generating a producer cell line, the method comprising delivering an rAAV vector to cells of a packaging cell line in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to a control parental cell.

[0017] Also provided herein is a method for producing rAAV by infecting cells of a producer cell line generated by 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 is reduced in the packaging cell line compared to a control parental cell. In certain embodiments, the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced in the packaging cell line compared to a control parental cell.

[0018] In one aspect, the disclosure provides a method for producing rAAV by infecting cells of a producer cell line with a helper virus, wherein the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced in the producer cell line compared to a control parental cell. In certain embodiments, the disclosure provides a method for producing rAAV by infecting cells of a producer cell line with a helper virus, wherein the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced in the producer cell line compared to a control parental cell.

[0019] Also described herein is a method for collecting rAAV from a producer cell line 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 line. Also described is a method for collecting rAAV from a producer cell line in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to a control parental cell line. In certain embodiments, the production of rAAV from the producer cell lines of the disclosure is enhanced compared to a control parental cell line.

[0020] Also described herein is a method for identifying one or more genes associated with the production of rAAV, the method comprising: i.) adding one or more supplemental substances that increase rAAV titer to a cell line; ii.) measuring global gene expression across the transcriptomes of the supplemented and non-supplemented cell lines; iii.) obtaining a list of genes differentially expressed between the supplemented and non-supplemented 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 the production of rAAV.

[0021] Also described herein are methods of producing rAAV packaging and / or production cell lines that promote an increase in the production of rAAV. 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 a supplemented rAAV production cell line and an unsupplemented rAAV production cell line. 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 a supplemented rAAV production cell line and an unsupplemented rAAV production cell line. In some embodiments, 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 a cell line that does not undergo modulation. In certain embodiments, modulating the expression is a decrease in the expression of one or more genes. In certain embodiments, modulating the expression includes a decrease in the expression of one or more proteins. In certain embodiments, modulating the expression is a removal of the expression of one or more genes. In certain embodiments, modulating the expression includes a removal of the expression of one or more proteins.

[0022] In some embodiments, the rAAV packaging 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.

[0023] Also described herein are recombinant adeno-associated virus (rAAV) packaging and / or production cell lines comprising cells engineered to have decreased expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to a corresponding non-modified parental cell.

[0024] In some embodiments, the cell line has a permanently or persistently reduced expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0025] In some embodiments, the cell line is engineered to contain 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.

[0026] In some embodiments, the cell line is engineered to contain a gene disruption in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0027] In some embodiments, the cell line is engineered to contain a gene disruption in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1.

[0028] In some embodiments, the cell line is engineered to contain 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.

[0029] In some embodiments, the cell line is engineered to contain partial or complete gene deletions in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1.

[0030] Also provided are recombinant packaging and / or production cell lines that exhibit decreased expression and / or activity of a polypeptide or polynucleotide expressed from 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.

[0031] Other features and advantages of the disclosure will be apparent from the following detailed description and claims.

[0032] Unless otherwise noted, all publications, references, patents, and / or patent applications referred to herein are hereby incorporated by reference in their entirety for all purposes.

[0033] The disclosure will be more fully understood with reference to the following. In embodiments of the invention, for example, the following items are provided. (Item 1) A recombinant adeno-associated virus (rAAV) packaging 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 decreased as compared to a control parental cell. (Item 2) The packaging and / or production cell line according to Item 1, comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is decreased as compared to a control parental cell. (Item 3) The packaging and / or production cell line according to item 1 or 2, wherein the expression is reduced by the use of nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO). (Item 4) The packaging and / or production cell line according to any one of items 1 to 3, wherein the expression is reduced by the use of siRNA containing a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 11. (Item 5) The packaging and / or production cell line according to item 4, wherein the expression of ATP5EP2 is reduced, and the siRNA contains 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. (Item 6) The packaging and / or production cell line according to item 4, wherein the expression of LINC00319 is reduced, and the siRNA contains 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. (Item 7) The packaging and / or production cell line according to item 4, wherein the expression of CYP3A7 is reduced, and the siRNA contains 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. (Item 8) The packaging and / or production cell line according to item 4, wherein the expression of NOG is reduced, and the siRNA contains 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. (Item 9) The packaging and / or production cell line according to item 4, wherein the expression of SPANXN3 is reduced, and the siRNA contains 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. (Item 10) The expression of MYRIP is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 11) The expression of KCNN2 is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 12) The expression of NALCN-AS1 is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 13) The expression of RGMA is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 14) The expression of PGA5 is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 15) The expression of ABCA10 is decreased, 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, and the packaging and / or production cell line according to item 4. (Item 16) 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, and the packaging and / or production cell line according to item 3. (Item 17) The packaging and / or production cell line according to any one of items 1 to 16, wherein the expression is reduced by the use of CRISPR genome editing. (Item 18) The expression is reduced by the use of a guide RNA pair, and each guide RNA (a) contains a sequence selected from the nucleotide sequences of SEQ ID NOs: 12 to 15, and / or (b) targets a target DNA sequence selected from any one of the nucleotide sequences of SEQ ID NOs: 16 to 31, The packaging and / or production cell line according to item 17. (Item 19) The gRNA pair is used to target KCNN2, and the packaging and / or production cell line according to item 18, 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. (Item 20) The gRNA pair is used to target KCNN2, and the packaging and / or production cell line according to item 18, comprising 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. (Item 21) The packaging and / or production cell line according to item 19 or 20, wherein each gRNA molecule is a 2'O-methyl analog containing a 3' phosphorothioate nucleotide internucleotide linkage at the 3 nucleotides at either or both of its 5' and 3' termini. (Item 22) The packaging and / or production cell line according to any one of items 1 to 21, wherein the gene expression is removed as compared to the control parental cells. (Item 23) The packaging and / or production cell line according to any one of items 1 to 22, which is a human cell line. (Item 24) The packaging and / or production cell line according to item 23, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line. (Item 25) The cell line according to any one of Items 1 to 24, which is an rAAV packaging cell line. (Item 26) The cell line according to any one of Items 1 to 24, which is an rAAV-producing cell line. (Item 27) The cell line according to Item 26, wherein the titer of rAAV is increased by about 1.5 to about 7 times as compared with the titer of rAAV produced from the cell line containing the control parental cells. (Item 28) A lysate of the cell line according to any one of Items 1 to 27. (Item 29) A cell culture supernatant from the cell line according to any one of Items 1 to 27. (Item 30) A method for generating a producing cell line, comprising delivering a recombinant adeno-associated virus (rAAV) vector to the cells of the packaging cell line according to Item 25. (Item 31) A method for producing rAAV, comprising infecting the cells of the producing cell line generated by the method according to Item 30 with a helper virus. (Item 32) A method for producing rAAV, comprising infecting the cells of the producing cell line according to Item 26 with a helper virus. (Item 33) The method according to Item 31 or 32, wherein the rAAV is collected from the producing cell line. (Item 34) The method according to any one of Items 31 to 33, wherein the production of rAAV is enhanced as compared with a control parental cell line. (Item 35) A method for identifying one or more genes associated with the production of rAAV, comprising: i. adding one or more supplementary substances that increase the rAAV titer to a cell line; ii. measuring the comprehensive gene expression across the transcriptomes in the supplemented and non-supplemented cell lines; iii. obtaining a list of genes differentially expressed between the supplemented cell line and the non-supplemented cell line; and iv. identifying one or more genes associated with the production of rAAV A method comprising the steps of: (Item 36) The method according to item 35, wherein the one or more supplement substances added to the cell line comprise dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide or triamcinolone. (Item 37) A method for producing an rAAV packaging and / or production cell line to promote an increase in the production of rAAV, the method comprising modulating the expression of one or more genes identified using the method according to item 35. (Item 38) The method according to any one of items 35 to 37, wherein the cell line is an rAAV packaging cell line. (Item 39) The method according to any one of items 35 to 37, wherein the cell line is an rAAV production cell line. (Item 40) The method according to item 39, wherein the rAAV production cell line increases the rAAV titer by at least 1.5-fold greater than the rAAV titer produced by an rAAV production cell line that does not modulate the expression of the corresponding one or more genes. (Item 41) The method according to any one of items 37 to 40, wherein modulating the expression comprises decreasing the expression of one or more genes. (Item 42) The method according to any one of items 37 to 40, wherein modulating the expression comprises eliminating the expression of one or more genes. (Item 43) The method according to any one of items 30 to 42, wherein the cell line is a human cell line. (Item 44) The method according to item 43, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line. (Item 45) A recombinant adeno-associated virus (rAAV) packaging and / or production cell line comprising cells engineered to have reduced expression and / or activity of a gene product expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to a corresponding unmodified parental cell. (Item 46) The rAAV packaging and / or production cell line according to item 45, wherein the expression and / or activity of a gene product expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced indefinitely or permanently. (Item 47) The rAAV packaging and / or production cell line according to item 46, which is engineered to contain 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. (Item 48) The rAAV packaging and / or production cell line according to item 47, which is engineered to contain a gene disruption in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1. (Item 49) The rAAV packaging and / or production cell line according to item 47, which is engineered to contain gene disruptions in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1. (Item 50) The rAAV packaging and / or production cell line according to item 47, which is engineered to contain 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. (Item 51) The rAAV packaging and / or production cell line according to item 47, which is 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 NALCN-AS1. (Item 52) A recombinant adeno-associated virus (rAAV) packaging and / or production cell line that exhibits reduced expression and / or activity of a polypeptide or polynucleotide expressed from 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.

Brief Description of the Drawings

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[0043] DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure describes recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines comprising cells in which the expression of one or more genes and / or proteins is modulated. The modulation of gene expression results in an increased titer yield compared to cell lines in which the expression of one or more genes and / or proteins is not modulated.

[0044] Unless otherwise indicated, technical terms are used according to their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds.), It can be found in 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, Inc., 1995 (ISBN 1-56081-569-8).

[0045] The following definitions are included for the purpose of understanding the subject matter and for constructing the appended claims. Abbreviations used herein have their conventional meanings in the technical fields of chemistry and biology. Definitions

[0046] As used herein, "modulation" or "modulate" refers to a change in the regulation, expression or activity of a gene and / or a protein. Modulation may increase, decrease (reduce) or eliminate the expression and / or activity of one or more genes and / or proteins. When multiple genes and / or proteins are modulated, all of the gene and / or protein expression and / or activity may increase, all of the gene and / or protein expression and / or activity may decrease, or one or more genes and / or proteins may increase while other genes and / or proteins may decrease.

[0047] As used herein, the term "cell" 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, a COS cell, a HEK293 cell, an A549 cell, a BHK cell, or a Vero cell. In other embodiments, the cell is an insect cell, such as an Sf9 cell, an Sf-21 cell, a Tn-368 cell, or a BTI-Tn-5B1-4 (High-Five) cell. The term "cell line" refers to a clonal population of cells that can continue to divide and be free from senescence. Unless otherwise indicated, the terms "cell" or "cell line" are understood to include modified or engineered variants of the indicated cell or cell line.

[0048] As used herein, the term "engineered cell line" refers to a cell line modified to increase the production of rAAV by one or more means that reduce the expression or other properties (such as biological activity) of one or more endogenously expressed genes and / or proteins (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1).

[0049] As used herein, the term "control parental cell" refers to a cell that has not been modified to increase the production of rAAV by one or more means that reduce the expression or other properties (such as biological activity) of one or more endogenously expressed genes and / or proteins (such as ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1).

[0050] As used herein, the term "control parental cell line" refers to a clonal population of control parental cells that can continue to divide and be free from senescence.

[0051] "Lysis" refers to the destruction of cells, often due to viruses, enzymes, or osmotic mechanisms that compromise cell integrity. "Lysed cells" are cells that have undergone substantial lysis. As used herein, the term "lysate" refers to a fluid containing the contents of lysed cells.

[0052] As used herein, the term "higher titer" refers to a titer that is increased in comparison to the titer produced by an unmodified control parental cell line and / or control parental cells.

[0053] As used herein, the term "cell culture supernatant" refers to the cell culture medium in which cells are suspended and / or cultured.

[0054] As used herein, the term "gene" refers to a transcription unit, as well as control regions that flank (e.g., are located upstream and downstream of) the transcription unit and are operably linked. A transcription unit is a series of nucleotides that is transcribed into an RNA molecule. A transcription unit may include a coding region. A "coding region" is a nucleotide sequence that encodes an unprocessed preRNA (i.e., an RNA molecule that includes both exons and introns) that is subsequently processed 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. An example of a non-coding RNA is microRNA. The boundaries of a transcription unit are generally determined by the start site at the 5' end of the transcription unit and the transcription termination factor at the 3' end of the transcription unit. A "control region" is a nucleotide sequence that controls the expression of the transcription unit to which it is operably linked. Non-limiting examples of control sequences include promoters, enhancers, transcription start sites, translation start sites, translation stop sites, transcription termination factors, and poly(A) signals. Control regions located upstream of a transcription unit may be referred to as 5' UTRs, and control regions located downstream of a transcription unit may be referred to as 3' UTRs. Control regions may be transcribed and may be part of an unprocessed preRNA.

[0055] In the context of this document, the term "target" or "target gene" refers to any gene that, when modulated, alters some aspect of virus production and includes genes encoding protein-coding genes and non-coding RNAs (e.g., miRNAs). Target genes include endogenous genes, viral genes, and transgenes.

[0056] With respect to gene notation, a single gene is often represented by multiple symbols. In the context of this document, gene symbols are represented by either uppercase or lowercase letters, regardless of whether they are human or non-human. The use of one particular symbol or the adoption of uppercase or lowercase symbols is not intended to limit the scope of the gene in the context of these disclosures. All gene identification numbers (gene IDs) identified in this specification are from the National Center for Biotechnology Information "Entrez Gene" or the KEGG website, unless otherwise identified.

[0057] The term "about" is used in this document to mean approximately, nearly, roughly, or around. When the term "about" is used with a numerical range, it modifies that range by extending the boundaries above and / or below the recited numerical value within the acceptable range of values.

[0058] As used herein, whether in a transitional phrase or in the body of the claims, the terms "comprise(s)" and "comprising" should be interpreted in a non-limiting sense. That is, these terms should be construed synonymously 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 characteristics or components, but may also include additional characteristics or components.

[0059] For the purpose of promoting an understanding of the embodiments described herein, preferred embodiments and specific terms are referred to and described. The terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present disclosure. Throughout the present disclosure, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. All percentages and ratios used herein are by weight unless otherwise indicated.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Additionally, 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. Adeno-associated virus (AAV)

[0061] AAV is a small non-enveloped replication-defective virus that infects humans and some other primate species. AAV is not known to cause disease and induces a very mild immune response. Gene therapy vectors utilizing AAV can infect both dividing and quiescent cells and can persist episomally without integrating into the host cell genome. These properties make AAV an attractive viral vector for gene therapy. AAV includes a number of serologically distinguishable types, including not only serotypes AAV-1 to AAV-12, but also more than 100 serotypes derived from non-human primates (see, for example, Srivastava, J. Cell Biochem., 105(1): 17-24 (2008), and Gao et al., J. Virol., 78(12), 6381-6388 (2004)). AAV replicates non-autonomously and has a life cycle with a latent and an infectious phase. During the latent phase, after a cell becomes infected with AAV, AAV integrates site-specifically into the host genome as a provirus. The infectious phase does not occur unless a helper virus that enables AAV replication (e.g., adenovirus (AV) or herpes simplex virus) also infects the cell.

[0062] The wild-type AAV genome contains two 145-nucleotide inverted terminal repeats (ITRs) that contain signal sequences directing 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. The two rep promoters, in combination with alternative splicing of a single AAV intron, result in the production of four rep proteins (Rep78, Rep68, Rep52, and Rep40) from the rep gene. The rep proteins are responsible for genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins (VP1, VP2, and VP3) that are splice variants of the cap gene. These proteins form the capsid of the AAV particle.

[0063] Because cis-acting signals for replication, encapsidation, and integration are contained within the ITRs, some or all of the 4.3 kb internal genome can be replaced with 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 produce an AAV vector, a cell line that permits AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and the helper functions provided by a helper virus, such as the adenoviral genes E1a, E1b55K, E2a, E4orf6, and VA (Weitzman et al., Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011). Production of the AAV vector can also result in production of helper virus particles that must be removed or inactivated prior to use of the AAV vector. A number of cell types, including HEK293 cells, COS cells, HeLa cells, BHK cells, Vero cells, and insect cells, are suitable for producing AAV vectors (see, for example, 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. WO00 / 47757, WO00 / 24916, and WO96 / 17947). AAV vectors are typically produced in these cell types with one plasmid containing the ITR-flanked expression cassette and one or more additional plasmids providing additional AAV and helper virus genes.

[0064] Any serotype of AAV can be used in the present disclosure. Similarly, it is contemplated that any AV type can be used, and one of ordinary skill in the art can identify the AAV and AV types suitable for the production of their desired recombinant AAV vector (rAAV). AAV and AV particles can be purified, for example, by affinity chromatography, iodixanol gradient, or CsCl gradient.

[0065] The genome of wild-type AAV is single-stranded DNA and is 4.7 kb. An AAV vector can have a single-stranded genome that is larger than or smaller than 4.7 kb, including a jumbo genome on the order of 5.2 kb or a smaller genome on the order of 3.0 kb. Additionally, the vector genome can be substantially self-complementary such that the genome is substantially double-stranded within the virus. AAV vectors containing all types of genomes are suitable for use in the methods of the present disclosure.

[0066] As discussed above, AAV requires co-infection with a helper virus to enter the infectious phase 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 may also provide helper functions for AAV (see, for example, Hermonat et al., Molecular Therapy 9, S289-S290 (2004)). Helper viruses include any virus that can produce and enable AAV replication. AV is a non-enveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb. AV provides the E1a, E1b55K, E2a, E4orf6, and VA genes and can rescue latent AAV proviruses in cells by enabling AAV replication and capsid encapsidation. HSV is a family of viruses with a relatively large double-stranded linear DNA genome encapsulated in an icosahedral capsid surrounded by a lipid bilayer envelope. HSV is infectious and highly contagious. The following HSV-1 replication proteins: the helicase / primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene have been identified as necessary for AAV replication, and other proteins enhance the helper function. The AAV packaging system serves two purposes, namely, the AAV packaging system avoids problems with the transfection process and enables production techniques based on the use of one or several helper functions. Production of rAAV

[0067] The general principles of rAAV can be reviewed elsewhere (see, for example, Carter, 1992, Current Opinions in Biotechnology, 3:533-539, and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol., 158:97-129). Generally, to enable the production of rAAV, cells must be provided with not only the AAV ITRs (which may flank a heterologous nucleotide sequence of interest), the AAV rep and cap gene functions, but also additional helper functions. These can be provided to the cells using any number of plasmids or vectors. The additional helper functions can be provided, for example, by adenovirus (AV) infection, by a plasmid carrying all of the necessary AV helper function genes, or by a virus such as HSV or baculovirus. Any gene, gene function, or other genetic material necessary for rAAV production by a cell can be transiently present within the cell or stably inserted into the cell genome. Suitable rAAV production methods for use with the methods of the present disclosure include those disclosed in 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 major approaches to AAV production systems are recombinant adeno-associated virus (rAAV) packaging cell lines and adeno-associated virus (rAAV) producer cell lines. Recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines

[0068] rAAV packaging cell lines can be produced by enabling the cellular expression of the AAV gene elements described herein. Stable transfection of a cell line (e.g., HEK293, HeLa) with a plasmid encoding the AAV rep and cap genes can result in the production of a packaging cell line. Co-infection of this rAAV packaging cell line with two different adenoviruses (a helper virus and a hybrid virus containing the AAV gene therapy element) can produce rAAV particles. Alternatively, stable transfection of a packaging cell with a plasmid containing an rAAV vector or infecting a packaging cell with an rAAV vector results in an rAAV-producing cell line. Infection of the producing cells with a helper virus results in the production of rAAV. Figure 1 illustrates the packaging and production cell lines.

[0069] In certain embodiments of the present disclosure, rAAV packaging cell lines comprising AAV rep and cap gene functions are engineered to increase rAAV titers.

[0070] In one aspect, the present disclosure provides an rAAV packaging cell line 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.

[0071] In some embodiments, the present disclosure provides an rAAV packaging cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to a control parental cell.

[0072] In other embodiments, the present disclosure provides an rAAV-producing cell line comprising cells in which the expression of one or more genes and / or proteins is reduced as compared to control parental cells. For example, the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced as compared to control parental cells. In some embodiments, the rAAV-producing cell line of the present disclosure is engineered to reduce the gene expression of KCNN2, LINC00319, RGMA, and SPANXN3.

[0073] In certain embodiments, the cell line of the present disclosure can be in adherent or suspension form.

[0074] In certain embodiments, the cell line of the present disclosure (e.g., an rAAV packaging and / or producing cell line) is a mammalian cell line (e.g., HeLa, human embryonic kidney (HEK) 293, COS, A549, or Vero cell line). In certain embodiments, the cell line is an insect cell line (e.g., Sf9, Sf-21, Tn-368, or BTI-Tn-5B1-4). Method for generating an rAAV-producing cell line

[0075] 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 as compared to control cells.

[0076] 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 as compared to control parental cells. Supplementary substances

[0077] As used herein, the term "supplemental substance" refers to any compound or other substance that can be used in the medium for cell culture to increase the rAAV titer or to assay for an increase in the rAAV titer, regardless of whether it is of chemical or biological origin. Non-limiting examples of supplemental substances 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 supplemental substances added to the rAAV packaging and / or production cell lines of the present disclosure are glucocorticoid analogs. In certain embodiments, examples of one or more supplemental substances added to the rAAV packaging and / or production cell lines include dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, and / or triamcinolone.

[0078] 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.

[0079] As used herein, the term "supplemented cell line" refers to a cell line (e.g., an rAAV packaging and / or production cell line) to which one or more supplementing substances (e.g., glucocorticoid analogs) have been added to increase the rAAV titer. As used herein, the term "unsupplemented cell line" refers to a cell line (e.g., an rAAV packaging and / or production cell line) that has not been exposed to one or more supplementing substances to increase the rAAV titer. As used herein, the terms "unsupplemented" and "non-supplemented" are used interchangeably to refer to culture conditions under which a cell line (e.g., an rAAV packaging and / or production cell line) has not been exposed to one or more supplementing substances to increase the rAAV titer. Method for identifying one or more genes associated with rAAV production

[0080] The present disclosure is directed, in part, to a method for identifying one or more genes associated with the production of rAAV by comparing the comprehensive gene expression patterns in supplemented and unsupplemented cell lines.

[0081] The term "comprehensive gene expression" is well known in the art (see, e.g., Wang Z. et al, Nature Reviews Genetics, 10(1), 57-63 (2009)). The term "comprehensive gene expression" refers to one or more sets of data containing information regarding different situations of gene expression. The data set optionally includes information regarding 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 supplementing substance) to modulate the expression of a particular gene; and the ability of various treatments (e.g., addition of a supplementing substance) to change the expression of a particular gene to different levels.

[0082] The term "differentially expressed" 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)).

[0083] In certain embodiments, the cell lines of the present disclosure (e.g., rAAV packaging and / or production cell lines) are supplemented with one or more supplemental substances that increase rAAV production. In some embodiments, RNA samples are extracted from one or more cell lines (supplemented and non-supplemented) using any of well-known procedures. For example, total RNA can be purified from cells using silica-based isolation in an automated compatible 96-well format such as the Rneasy® purification platform (Qiagen, Inc., Valencia, Calif.).

[0084] The pattern of gene expression 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 level of gene expression relative to other expression products and / or relative to a control sequence. One convenient and widely applicable method for determining relative expression levels is to compare the expression levels of one or more target genes to the expression levels of a control gene such as a housekeeping gene (e.g., HPRT1, HSP70 or β-actin).

[0085] To confirm whether changes in observed expression data, e.g., changes in 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 simply due to experimental noise or product of population heterogeneity, an estimation of the probability distribution can be constructed for each gene and phenotypic endpoint in each biological sample. Construction of the estimated population distribution involves conducting multiple independent experiments for each treatment, e.g., all experiments are conducted in replicates of 2, replicates of 3, replicates of 4, etc. Expression data from multiple biological samples (e.g., supplemented and unsupplemented cell lines) can be grouped or clustered using multivariate analysis. Analysis of the data can generate a list of genes that are differentially expressed, e.g., between a supplemented cell line and an unsupplemented cell line, in response to the treatment. The list of differentially expressed genes can be filtered using various gene filtering methodologies to identify one or more genes useful for increasing the production of rAAV.

[0086] In some embodiments, the present disclosure is directed to a method of identifying one or more genes associated with rAAV production from a list of genes differentially expressed between a supplemented cell line and an unsupplemented cell line. 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, the comprehensive gene expression is measured across different cell lines (e.g., between an unsupplemented HeLa cell line and a supplemented HeLa cell line, between an unsupplemented HEK293 cell line and a supplemented HEK293 cell line, between an unsupplemented HeLa cell line and a supplemented HEK293 cell line, between an unsupplemented HeLa cell line and an unsupplemented HEK293 cell line, between a supplemented HeLa cell line and a supplemented HEK293 cell line) to identify one or more genes associated with rAAV production. In certain embodiments, the comprehensive gene expression data from supplemented HEK293 and supplemented HeLa can be combined and compared with the combined comprehensive gene expression data from unsupplemented HEK293 and unsupplemented HeLa cell lines to identify one or more genes associated with rAAV production.

[0087] In certain embodiments, the present disclosure provides a method of producing an rAAV packaging and / or production cell line that promotes an increase in rAAV production. In some embodiments, the 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 a supplemented rAAV production cell line and an unsupplemented rAAV production cell line. In certain embodiments, the titer of rAAV is increased by modulating the expression of one or more genes and / or proteins identified from a list of differentially expressed genes between a supplemented rAAV production cell line and an unsupplemented rAAV production cell line. In some embodiments, the rAAV titer is 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 titer produced by a cell line that does not modulate the expression of the corresponding genes and / or proteins. Genes and / or proteins to be modulated

[0088] In certain embodiments, the present disclosure provides a list of genes that enhance rAAV production when modulated (individually or in combination) in rAAV packaging and / or production cell lines.

[0089] 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 proton gradient across the membrane generated by the electron transport complexes of the respiratory chain. Examples of the human ATP5EP2 sequence are available under reference sequence NM_006886.4 (SEQ ID NO: 43) or NG_053163.1 (SEQ ID NO: 44) (nucleotide sequence) in the NCBI nucleotide database.

[0090] Long intergenic non-protein coding RNA 319 (also known as LINC00319) is an RNA gene and belongs to the non-coding RNA class. Long non-coding RNAs (lncRNAs) have been shown to play important roles in the regulation of the etiology and progression of multiple cancers. Examples of the LINC00319 sequence are available under reference sequence NM_194309 (SEQ ID NO: 45) or NR_026960.1 (SEQ ID NO: 46) (nucleotide sequence) in the NCBI nucleotide database.

[0091] Cytochrome P450 family 3 subfamily A member 7 (also known as CYP3A7) is a gene that encodes a member of the cytochrome P450 superfamily of enzymes involved in drug metabolism and in the synthesis of cholesterol, steroids, and other lipids. This enzyme hydroxylates testosterone and dehydroepiandrosterone 3-sulfate and is involved in the formation of estriol during pregnancy. This gene is part of a cluster of related genes on chromosome 7q21.1. An example of the CYP3A7 sequence is available under reference sequence NM_000765 (SEQ ID NO:47) (nucleotide sequence) in the NCBI nucleotide database.

[0092] ATP-binding cassette subfamily A member 10 (also known as ABCA10) encodes a membrane-associated protein that belongs to the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. The ABC genes are divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White). ABCA10 is a member of the ABC1 subfamily. Members of the ABC1 subfamily constitute the only major ABC subfamily found only in multicellular eukaryotes. This gene clusters between four other ABC1 family members on 17q24. An example of the ABCA10 sequence is available under reference sequence NM_080282.3 (SEQ ID NO:48) (nucleotide sequence) in the NCBI nucleotide database.

[0093] Noggin (also known as NOG) encodes a secreted polypeptide that binds to and inactivates members of the transforming growth factor-beta (TGF-beta) superfamily of signaling proteins, such as bone morphogenetic protein-4 (BMP4). Although not bound by theory, this protein is thought to have a major role in creating morphogenetic gradients by diffusing more efficiently into the extracellular matrix than members of the TGF-beta superfamily. Noggin appears to have pleiotropic effects both early and late in development. An example of the NOG sequence is available under reference sequence NM_005450.4 (SEQ ID NO:49) (nucleotide sequence) in the NCBI nucleotide database.

[0094] Repulsive guidance molecule BMP co-receptor 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 systems. This protein may also function as a tumor suppressor in some cancers. Examples of the RGMA sequence are available under reference sequence NM_020211.2 (SEQ ID NO:50) or NM_001166283.1 (SEQ ID NO:51) (nucleotide sequence) in the NCBI nucleotide database.

[0095] 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 under reference sequence NM_001009609 (SEQ ID NO:52) (nucleotide sequence) in the NCBI nucleotide database.

[0096] 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 the chief cells of the stomach, undergoes autocatalytic cleavage under acidic conditions to form the active enzyme, and functions in the digestion of food proteins. This gene is found in a cluster of related genes on chromosome 11, each of which encodes one of several pepsinogens. An example of the PGA5 sequence is available under reference sequence NM_014224.4 (SEQ ID NO:53) (nucleotide sequence) in the NCBI nucleotide database.

[0097] Myosin VIIA and Rab-interacting protein (also known as MYRIP) encodes a Rab effector protein involved in melanosome transport that serves as a linker between melanosome-binding RAB27A and the motor proteins MYO5A and MYO7A. This Rab effector protein functions as a protein kinase A anchor protein (AKAP) and can act as a scaffold protein that links PKA to components of the exocytotic machinery, thus facilitating exocytosis, including insulin release. An example of the MYRIP sequence is available under reference sequence NM_015460 (SEQ ID NO:54) or NM_001284423.1 (SEQ ID NO:55) (nucleotide sequence) in the NCBI nucleotide database.

[0098] The calcium-activated potassium 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 results in multiple transcript variants. An example of the KCNN2 sequence is available under reference sequence NM_170775.2 (SEQ ID NO:56) or NM_001278204.1 (SEQ ID NO:57) (nucleotide sequence) in the NCBI nucleotide database.

[0099] NALCN antisense RNA 1 (also known as NALCN-AS1) is an RNA gene and belongs to the non-coding RNA class. Examples of the NALCN-AS1 sequence are available under reference sequence NW_011332700.1 (SEQ ID NO: 58) or NR_047687.1 (SEQ ID NO: 59) (nucleotide sequence) in the NCBI nucleotide database.

[0100] In certain embodiments, the present disclosure provides an rAAV packaging 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 as compared to control parental cells.

[0101] In certain embodiments, the present disclosure provides an rAAV packaging and / or production cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced as compared to control parental cells.

[0102] In certain embodiments, the present disclosure provides a list of genes that, when individually modulated in an rAAV packaging and / or production cell line, enhance rAAV production as compared to a control parental cell line. In some aspects, modulation of different combinations of genes in the rAAV packaging and / or production cell line increases rAAV production. In some aspects, modulating 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 and / or production cell line results in increased rAAV production as compared to a control parental cell line. Methods of modulating one or more genes and / or proteins

[0103] Modulating the expression or activity of a gene (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 or NALCN-AS1), such as reducing it, can be achieved by various mechanisms including, but not limited to: 1) gene copy number, 2) transcription or translation of the gene, 3) stability or lifespan of the transcript, 4) number of copies of mRNA or miRNA, 5) availability of non-coding RNA or non-coding RNA target sites, 6) location or extent of post-translational modification of the protein, or 7) activity of the protein. Means that can be used to modulate gene expression include, but are not limited to, nucleases, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA oligonucleotides (ASO), gene disruption or partial or complete gene deletion. Nuclease

[0104] In certain embodiments, gene modulation is achieved using zinc finger nucleases (ZFNs). Synthetic ZFNs are composed of, for example, zinc finger binding domains fused to FokI DNA cleavage domains. ZFNs can be designed / engineered for genome editing 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 manipulation in a wide variety of cell types. The reagents described can be used to target promoters, protein-coding regions (exons), introns, 5' and 3' UTRs, etc. Double-stranded RNA (dsRNA) molecules for modulation

[0105] 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 and / or production cell lines). The dsRNA molecules can be designed to antagonize one or more genes by targeting the corresponding RNA sequences based on sequence homology. Such dsRNAs can be small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), or microRNAs (miRNAs). The sequences of such dsRNAs can include complementary portions of the mRNA encoding the 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 (e.g., 90% or higher, or 95% or higher) can also be used. Typically, the percent complementarity is determined over a certain length of contiguous nucleic acid residues. The dsRNA molecules of the present disclosure can have at least 80% complementarity with the target portion within the mRNA, measured over, for example, 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 with the target portion within the mRNA over the entire length of the dsRNA molecule.

[0106] Another gene targeting reagent that uses the RNA interference (RNAi) pathway is small hairpin RNA, also referred to as shRNA. For example, shRNA delivered to cells via an expression construct (e.g., plasmid, lentivirus) has the ability to constitutively or controllably achieve long-term reduction of gene expression, 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 that target the gene(s) of interest. Such lentiviruses can infect cells, stably integrate their viral genome into the host genome, and constitutively, controllably, or (if multiple shRNAs are expressed) constitutively and controllably express the shRNA. 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 having similar or overlapping functions or sequence motifs. One of ordinary skill in the art will recognize that lentiviral constructs can also incorporate cloned DNA or ORF expression constructs.

[0107] In multiple 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 variety of chemical modifications, multiple levels of complementarity to the target transcript of interest, and designs to enhance stability, cellular delivery, specificity, and functionality (see U.S. Patent No. 8,188,060). Additionally, such reagents can be designed to target diverse regions of a gene (including the 5’ UTR, open reading frame, 3’ UTR of the mRNA) or, in some cases, the promoter / enhancer regions of genomic DNA encoding the gene of interest. Gene modulation (e.g., reduction of gene expression, i.e., knockdown) can be achieved by introducing a single siRNA or miRNA, or a pool of multiple siRNAs or miRNAs targeting 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 may be referred to as foreign 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.

[0108] siRNAs can be used to reduce the expression of one or more genes (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1). In some embodiments, siRNAs comprising nucleotide sequences selected from SEQ ID NOs: 1-11 or variants thereof are used to reduce the expression of the target gene.

Table 1-1

Table 1-2

Table 1-3

[0109] In some embodiments, the siRNA used to reduce the expression of ATP5EP2 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.

[0110] In some embodiments, the siRNA used to reduce the expression of LINC00319 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.

[0111] In some embodiments, the siRNA used to reduce the expression of CYP3A7 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.

[0112] In some embodiments, the siRNA used to reduce the expression of NOG 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.

[0113] In some embodiments, the siRNA used to reduce the expression of SPANXN3 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.

[0114] In some embodiments, the siRNA used to reduce the expression of MYRIP 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.

[0115] In some embodiments, the siRNA used to reduce the expression of KCNN2 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.

[0116] In some embodiments, the siRNA used to reduce the expression of NALCN-AS1 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.

[0117] In some embodiments, the siRNA used to reduce the expression of RGMA 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.

[0118] In some embodiments, the siRNA used to reduce the expression of PGA5 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.

[0119] In some embodiments, the siRNA used to reduce the expression of ABCA10 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. Antisense RNA oligonucleotide (ASO)

[0120] Antisense RNA oligonucleotide (ASO) can be used to modulate the expression of one or more genes in an rAAV packaging and / or production cell line. Typically, ASO is used to reduce the expression of one or more genes. By using known techniques based on knowledge of the sequence of the one or more genes to be modulated, ASO molecules can be designed to antagonize one or more genes by targeting the corresponding RNA based on sequence homology. The ASO sequence can comprise a nucleotide sequence complementary to the target portion of the mRNA or lncRNA produced from one or more genes. This portion can be 100% complementary to the target portion within the mRNA or lncRNA, but lower levels of complementarity (e.g., 90% or higher, or 95% or higher) can also be used.

[0121] In some embodiments, the ASO can be an antisense RNA oligonucleotide in which at least one nucleoside linkage of the sequence is a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, and an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage or a boranophosphate linkage. In certain embodiments, at least one internucleoside linkage of the antisense RNA oligonucleotide sequence is a phosphorothioate linkage. In some embodiments, all internucleoside linkages of the antisense RNA oligonucleotide sequence are phosphorothioate linkages. CRISPR Genome Editing

[0122] In some embodiments, modulation of gene expression in rAAV packaging and / or production cell lines is performed using CRISPR genome editing. CRISPR genome editing typically involves two distinct components: (1) a guide RNA and (2) an endonuclease, specifically a CRISPR-associated (Cas) nuclease (e.g., Cas9). The guide RNA is a combination of the endogenous bacterial crRNA and tracrRNA that results in a single chimeric guide RNA (gRNA) transcript. 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.

[0123] 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 of a gene related to reduction (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 or NALCN-AS1). For efficient 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 localizes Cas to the genomic target sequence of one or more of the genes of the present disclosure such that wild-type Cas can cleave both strands of the DNA that causes a double-strand break. The double-strand break can be repaired by one of two general repair pathways: (1) the non-homologous end joining DNA repair pathway or (2) the homologous recombination repair pathway. The non-homologous repair pathway can introduce insertions / deletions that can result in frameshifts and / or premature stop codons at the site of the double-strand break, effectively disrupting the open reading frame of the target gene. The homologous recombination repair pathway requires the presence of a repair template to correct the double-strand break.

[0124] Any suitable gRNA pair can be used for CRISPR genome editing. Typically, the gRNA pair is used to reduce the expression of one or more genes (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1). In some embodiments described herein, the gRNA pair is used to modulate (e.g., reduce or eliminate / knock out) the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1.

[0125] The gRNA pair can be designed using known techniques based on knowledge of the sequence of one or more genes to be modulated, typically using any suitable publicly available computer program. Knockout packaging and / or production cells can be generated using any suitable technique for which standard techniques are known in the art and for which suitable kits are commercially available.

[0126] The gRNA pair can be delivered to the production cell line of the present disclosure by any suitable means. Suitable techniques are known in the art and include the use of plasmid, viral, and bacterial vectors to deliver the gRNA pair to the production cell line. Typically, the gRNA pair is delivered using plasmid DNA.

[0127] The gRNA pairs can be used to reduce the expression of one or more genes (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1). Multiple gRNA pairs can be used to modulate the expression of genes. In some embodiments described herein, the gRNA pairs 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 gRNA pairs can be used to modulate the expression of KCNN2, LINC00319, RGMA, and SPANXN3. In some embodiments, the gRNA can be modified to enhance the editing efficiency by increasing its binding to the target site and inhibiting nuclease degradation. In certain embodiments, these modifications can be 2'O-methyl analogs and 3' phosphorothioate nucleotide internucleotide linkages at the 3 nucleotides at both the 5' and 3' ends of the gRNA. Exemplary target DNA sequences targeted by the gRNA pairs used to modulate the gene expression of one or more genes can include any one of the nucleotide sequences selected from SEQ ID NOs: 16-31 listed in Table 2 or variants thereof.

Table 2-1

Table 2-2

[0128] For example, the gRNA pairs used to target KCNN2 can include sequences selected from the nucleotide sequences of SEQ ID NOs: 12-15 (shown in Table 2). In some embodiments, the gRNA pair used to target KCNN2 includes a first gRNA molecule comprising the sequence of SEQ ID NO: 12 and a second gRNA molecule comprising the sequence of SEQ ID NO: 13. In some embodiments, the gRNA pair used to target KCNN2 includes a first gRNA molecule comprising or having the sequence of SEQ ID NO: 14 and a second gRNA molecule comprising or having the sequence of SEQ ID NO: 15.

[0129] In some embodiments, the gRNA molecule targeting KCNN2 is a 2’O-methyl analog comprising a 3’ phosphorothioate internucleotide linkage at the 3 nucleotides at either or both of its 5’ and 3’ ends and comprises the sequence of SEQ ID NO: 12, 13, 14 or 15.

[0130] Variant gRNA sequences can be measured over sequences of any suitable length and can have at least 80% sequence identity with the sequences of the present disclosure. Typically, the percent sequence identity is determined over a continuous nucleic acid of a certain length. The variant gRNA sequences of the present disclosure can have at least 80% sequence identity with the sequences of the present disclosure, for example, when 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 to the gRNA molecule of the present disclosure over the entire length of the variant gRNA molecule. In some embodiments, the variant gRNA molecules of the present disclosure can be variants of one or more gRNA molecules whose target region is complementary to one of the target sequences of SEQ ID NOs: 16-30. The gRNA pairs of the present disclosure can include variants of one or both of the two gRNA sequences that pair to target a gene, for example, a gene selected from the group consisting of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1. For example, variants of a gRNA pair comprising a first gRNA molecule comprising the sequence of SEQ ID NO: 12 and a second gRNA molecule comprising the sequence of SEQ ID NO: 13 can include 1) a first gRNA molecule comprising a variant of the sequence of SEQ ID NO: 12, 2) a second gRNA molecule comprising a variant of the sequence of SEQ ID NO: 13, or 3) both. Protein-level modulation

[0131] In another embodiment, modulation of gene expression and / or activity occurs at the protein (e.g., polypeptide) level. By way of example, reduction of gene function at the protein level can be achieved by methods including, but not limited to, targeting the protein with small molecules, peptides, aptamers, destabilizing domains, or other methods that can, for example, downregulate the activity of the gene product or enhance the rate of degradation of the gene product. Alternatively, the expressed protein can be modified to reduce or eliminate biological activity via site-directed mutagenesis and / or incorporation of missense or nonsense mutations. In some embodiments, for example, small molecules that bind to the active site and inhibit the function of the target protein are added to, for example, the cell culture medium and can thereby be introduced into the packaging and / or producer cells. Alternatively, the target protein function can be modulated, for example, by introducing into the cell (e.g., the packaging and / or producer cell) a peptide that, for example, interferes with protein-protein interactions (see Shangary et. al., (2009) Annual Review of Pharmacology and Toxicology 49:223). Such peptides can be introduced into the cell (e.g., the packaging and / or producer cell) by, for example, transfection or electroporation, or via an expression construct. Alternatively, the peptide can be introduced into the cell (e.g., the packaging and / or producer cell) by adding one or more moieties that facilitate cellular delivery (e.g., via conjugation), or by overfeeding molecules that enhance self-delivery. Techniques for expressing the peptide include, but are not limited to, fusion of the peptide to a scaffold or addition of a signal sequence for stabilizing or directing the peptide to the desired location or compartment, respectively.In certain embodiments, the rAAV packaging and / or production cell line is engineered to reduce the expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 using any of the methods described above. Effect of modulation on the expression of one or more genes and / or proteins

[0132] In certain embodiments, the modulation methods described in the present disclosure can be utilized to generate rAAV packaging and / or production cell lines that produce high-titer 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 (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1) and / or a significant decrease in the activity of proteins expressed by one or more genes (e.g., a decrease of at least 5%, at least 10%, at least 20%, or greater). In certain embodiments, the expression of the target gene is reduced 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%).

[0133] In certain embodiments, the modulation methods described in the present disclosure can result in a significant decrease in the activity of proteins or RNAs expressed by target genes (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 proteins or RNAs expressed by target genes of at least 5%, at least 10%, at least 20%, or greater. In certain embodiments, the target gene protein or RNA activity decreases 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%). Further, modulation of one or more genes can result in modulation of multiple genes (e.g., by miRNA).

[0134] In certain embodiments, the method of modulation 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), such as a decrease of at least 5%, at least 10%, at least 20%, or greater. In certain embodiments, the expression of the gene product decreases 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%).

[0135] In certain embodiments, the methods of modulation described in the present disclosure can result in a significant decrease (e.g., a decrease of at least 5%, at least 10%, at least 20% or greater) in the expression of an expressed polypeptide or polynucleotide (e.g., from at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1). In certain embodiments, the expression of the polypeptide or polynucleotide 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%).

[0136] In certain embodiments, the methods of modulation described in the present disclosure can result in a significant decrease (e.g., at least 5%, at least 10%, at least 20% or greater decrease) in the activity of an expressed polypeptide or polynucleotide (e.g., from at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1). In certain embodiments, the activity of the expressed polypeptide or polynucleotide 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%).

[0137] In certain embodiments, the decrease in the expression and / or activity of one or more genes, proteins or RNAs in an rAAV packaging 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).

[0138] In certain embodiments, the decrease in the expression and / or activity of one or more genes, proteins or RNAs in an rAAV packaging and / or production cell line is intended to be maintained indefinitely or permanently, for example through the use of gene disruption or partial or complete gene deletion.

[0139] In certain embodiments, the reduction in the expression and / or activity of one or more genes, proteins or RNAs in the rAAV packaging and / or production cell line is maintained during at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40 or more passages of the rAAV packaging and / or production cell line in culture. Effect of modulation on rAAV production

[0140] Modulation of one or more genes and / or proteins in the rAAV packaging and / or production cell line can result in an increase in the titer of rAAV. In some embodiments, the modulation results in an increase in the titer of rAAV produced from the rAAV packaging and / or production cell line of 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 from the rAAV packaging and / or production cell line increases 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 the rAAV titer resulting from the modulation of one or more genes and / or proteins can be compared to the rAAV titer produced from the control parental cell line.

[0141] In some embodiments, modulation of one or more genes and / or proteins in an rAAV packaging and / or production cell line can increase rAAV titer production for at least 2 days, at least 5 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days or longer. Method for producing rAAV

[0142] In certain embodiments, the present disclosure describes a method for producing rAAV from an rAAV packaging and / or production cell line engineered to modulate 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 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 is modulated. In certain embodiments, production of rAAV from the engineered rAAV packaging and / or production cell line is enhanced compared to a control parental cell line.

[0143] 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 replication of rAAV. In some embodiments, cells of the engineered production cell line are infected with a helper virus (e.g., adenovirus (AV) or herpes simplex virus). Method for collecting rAAV

[0144] 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 the use of nucleases such as benzonase or DNase, proteases such as trypsin, or detergents or surfactants. Physical disruption such as homogenization or grinding, or pressurization with a microfluidizer pressure cell, or freeze-thaw cycles can also be used. In certain embodiments, the lysate from the engineered rAAV packaging and / or production cells can be used to collect rAAV particles.

[0145] In certain embodiments, the cell culture supernatant can be harvested from the engineered rAAV packaging and / or production cells without the need for cell lysis. In certain embodiments of the present disclosure, the engineered rAAV packaging and / or production cells secrete rAAV particles that can be harvested from the cell culture supernatant without the need for cell lysis. In certain embodiments, the engineered rAAV packaging and / or production cell line has an rAAV titer higher than the rAAV titer of the control parental cell line, such that more rAAV is collected from the engineered rAAV packaging and / or production cell line compared to the control parental cell line.

[0146] After recovering the rAAV particles, it may be necessary to purify the sample containing the rAAV particles, for example, to remove cell debris resulting from cell lysis. Methods for minimal purification of AAV particles are known in the art. Two exemplary purification methods are density gradient purification based on cesium chloride (CsCl) and iodixanol. Both methods are described in Strobel et al., Human Gene Therapy Methods, Vol. 26(4):147-157 (2015). Minimal purification can also be achieved using, for example, affinity chromatography using AVB Sepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). A method for AAV purification using AVB Sepharose affinity resin is described, for example, in Wang et al., Mol Ther Methods Clin Dev., Vol. 2:15040 (2015). After purification, the rAAV particles can be filtered and stored at -60°C or below.

[0147] In certain embodiments, the present disclosure provides a method of collecting rAAV particles produced from an engineered rAAV packaging cell line after the cells have been co-infected with two different adenoviruses.

[0148] In certain embodiments, the present disclosure provides a method of collecting rAAV particles produced after infection of an rAAV-producing cell line generated from an engineered rAAV packaging cell line.

[0149] In certain embodiments, the present disclosure provides a method of collecting rAAV particles produced after infection of an engineered rAAV-producing cell line with a helper virus. Quantification of rAAV Particles

[0150] Quantification of rAAV particles is complicated by the fact that AAV infection does not result in a cytopathic effect in vitro and thus plaque assays cannot be used to determine infectivity titers. However, rAAV particles can be quantified using several methods including quantitative polymerase chain reaction (qPCR) (Clark et al., Hum. Gene Ther., 10:1031-1039 (1999)), dot blot hybridization (Samulski et al., J. Virol., 63:3822-3828 (1989)), and by the optical density of highly purified vector preparations (Sommer et al., Mol. Ther., 7:122-128 (2003)). DNase resistant particles (DRP) can be quantified by real-time quantitative gene expression reduction polymerase chain reaction (qPCR) (DRP-qPCR) in a thermocycler (e.g., iCycler iQ 96 well block format thermocycler (Bio-Rad, Hercules, CA)). Samples containing rAAV particles can be incubated at 37° C. for 60 minutes in the presence of DNaseI (100 U / ml; Promega, Madison, Wis.), followed by proteinase K (Invitrogen, Carlsbad, Calif.) digestion (10 U / ml) for 60 minutes at 50° C., and then denatured at 95° C. for 30 minutes. The primer-probe set used should be specific for the non-native portion of the rAAV vector genome, e.g., the poly(A) sequence of the protein of interest. PCR products can be amplified using any suitable set of cycling parameters based on the length and composition of the primers, probe, and amplified sequence. Alternative protocols are disclosed, for example, in Lock et al., Human Gene Therapy Methods, 25(2):115-125 (2014).

[0151] Viral genome amplification can also be measured using qPCR techniques similar to those described above. However, to quantify total genome amplification in the producer cells, only intracellular samples are taken, and this sample is not treated with DNaseI to measure both the packaged and non-packaged viral genomes. Viral genome amplification can be calculated per host cell by simultaneously measuring host cell housekeeping genes, such as RNase P.

[0152] The infectivity of rAAV particles can be determined, for example, using the TCID 50 (50% tissue culture infective dose) assay described by Zhen et al., Human Gene Therapy, Volume 15: pages 709 - 715 (2004). In this assay, rAAV vector particles are serially diluted and used to co-infect a Rep / Cap-expressing cell line with AV particles in a 96-well plate. At 48 hours post-infection, total intracellular DNA is extracted from the infected and control wells. Then, qPCR is used with a transgene-specific probe and primers to measure the replication of the rAAV vector. The TCID 50 infectivity (TCID 50 / ml) is calculated using the Kaerber equation with the ratio of AAV-positive wells in a 10-fold serial dilution. Therapeutic applications

[0153] The engineered rAAV packaging and / or the rAAV produced from the producer cell lines described herein can be used, for example, for gene therapy in mammals. The rAAV produced from the engineered cells described herein can be used for ex vivo and / or in vivo gene therapy applications. The rAAV produced from the engineered cells described herein can be used, for example, to deliver small molecules (e.g., siRNA or sgRNA), peptides, and / or proteins.

[0154] In some embodiments, the rAAV produced from the engineered cell lines described herein can be used to treat a disease or disorder in a human subject in need thereof. In certain embodiments, the rAAV produced from the engineered cell lines described herein can be administered with a pharmaceutically acceptable carrier.

[0155] Any suitable method or route can be used to administer the rAAV or rAAV-containing composition produced from the engineered packaging and / or production 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 composition comprising rAAV produced from the engineered packaging and / or production cell lines is administered intravenously.

[0156] The practice of the present disclosure will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only and should not be construed as limiting the present disclosure in any way.

Examples

[0157] (Example 1) Development of knockdown protocol siRNA knockdown experiments were optimized and developed for 6-well and 24-well formats by knocking down the housekeeping gene HPRT1. Experiments conducted in 24-well plates were evaluated based on a number of factors such as seeding density, cell culture conditions (e.g., percent carbon dioxide (CO2), percent fetal bovine serum (FBS)), ratio of transfection reagent (Lipofectamine® RNAiMax) to siRNA ("ratio"), incubation time, and siRNA concentration. Commercial siRNAs designed for HPRT1 gene knockdown were used to optimize the experimental conditions. HeLa producer cells were transfected with various concentrations of siRNA using Lipofectamine® RNAiMax according to the manufacturer's instructions. The percent decrease in HPRT1 expression was determined by real-time PCR. The optimized 24-well siRNA knockdown method was able to knockdown HPRT1, a highly expressed gene, by more than 80% compared to the baseline control. As shown in FIGS. 2A - D, cells seeded at 1×10 5 cells per well, a transfection reagent to siRNA ratio of 1:5, and 8 nM siRNA showed the highest knockdown efficiency. FIG. 2A shows the effect of various siRNA concentrations / ratios used on the percent knockdown of HPRT1. FIG. 2B shows the effect of various siRNA concentrations / ratios on the percent expression of HPRT1. For 6-well protocol optimization, two different siRNA concentrations were tested. For the data plotted in FIGS. 2A and 2B, seeding densities of 5×10 4 , 8×10 4 and 1×10 5 were tested. FIG. 2C shows the effect of various siRNA concentrations on the percent knockdown of HPRT1. FIG. 2D shows the effect of various concentrations of siRNA on the percent expression of HPRT1. All experiments were performed in triplicate. (Example 2) RNA Sequencing

[0158] Eight 3-liter bioreactors were operated under supplemented and non-supplemented production conditions for two different HeLa S3-producing cell lines. Two additional bioreactors were operated without using adenovirus 5 (Ad5) as a non-infected control. Table 3 describes the details of the bioreactor conditions and production levels. [Table 3] Abbreviations used in Table 3: Addition of one or more supplementing substances is indicated by (+), and the absence of one or more supplementing substances is indicated by (-); MOI - multiplicity of infection.

[0159] Thirty hours after Ad5 infection, samples were taken for RNA-Seq. The samples were washed once with PBS and the cell pellets were stored at -80 °C until just before shipping. RNA extraction and cDNA synthesis of the extracted RNA were carried out 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 generated reads were mapped to the human genome, Ad5 genome, and AAV2 genome using mapping methods well-known in the art. Any reads mapped to the Ad5 genome were discarded. Sequencing was performed for another round to enrich the reads mapped to the human genome. Differential analysis was performed using the data generated by RNA sequencing (see Table 4). [Table 4] * PCL1 - producing cell line 1; PCL2 - producing cell line 2; non-production - non-infected control cells; production (N.S.) - Ad5-infected cells cultured under non-supplemented conditions; production (S) - Ad5-infected cells cultured under supplemented conditions.

[0160] In this example, differential expression analysis was calculated as the log fold change (LogFC) of the mRNA level under experimental conditions compared to control conditions. Upregulated genes were represented as positive LogFC, and downregulated genes were represented as negative LogFC. Differentially expressed genes with a p-value of 0.05 or less were considered statistically significant. In each differential analysis, hundreds to thousands of genes were significantly upregulated or downregulated. Filtering criteria were established (see, for example, FIG. 6) and applied to reduce the dataset to a manageable number of genes for evaluation. Gene sets were aligned as described in Example 6 and subjected to the filter criteria. (Example 3) Verification by RT-qPCR of results obtained from RNA sequencing

[0161] A small gene set was selected for verification of the RNA sequencing data. The RNA-Seq results were confirmed using an RT-qPCR assay according to methods well known in the art. The data were analyzed using the ΔΔCt method. RT-qPCR independently confirmed the trends observed in the RNA-Seq data. FIGS. 3A - B show the log fold change values of gene expression obtained from the bioinformatics analysis of RNA-Seq data for PGA5 (FIG. 3A) and SPANXN3 (FIG. 3B). The x-axis indicates the conditions under which the producer cell line was grown (supplemented (differential analysis number 5 described in Table 4) vs. non-supplemented (differential analysis number 1 described in Table 4)), and the y-axis indicates the log fold change (LogFC) of gene expression. The log fold change of PGA5 (FIG. 3A) and SPANXN3 (FIG. 3B) expression in cells cultured in non-supplemented cell culture medium was plotted against the corresponding gene expression in non-infected cells (cells not infected with helper virus). The log fold change of PGA5 (FIG. 3A) and SPANXN3 (FIG. 3B) expression in cells cultured in supplemented cell culture medium was plotted against the corresponding gene expression in cells cultured in non-supplemented cell culture medium.

[0162] The expression of the PGA5 and SPANXN3 genes in the producer cell lines grown under supplemented and non-supplemented conditions was also evaluated by RT-qPCR using methods well-known in the art. Figures 3C-D show the RT-qPCR fold change values of the expression of PGA5 (Figure 3C) and SPANXN3 (Figure 3D) in non-infected cells (cells not infected with helper virus) in cells cultured in non-supplemented and supplemented cell culture media. Figures 3A-D show that the data obtained from qPCR and RNA sequencing follow the same trend. (Example 4) Verification of results obtained from RNA sequencing by RT-qPCR in different clones of producer cell lines

[0163] The RNA sequencing results were further verified by performing RT-qPCR experiments on RNA extracted from different clones of the HeLa S3 producer cell line. Figures 4A-B show the fold change values of the expression of PGA5 (Figure 4A) and SPANXN3 (Figure 4B) in non-infected cells (cells not infected with helper virus) in producer cell line clones cultured in non-supplemented and supplemented cell culture media as determined by RT-qPCR. 21C5, 3C6, and 2B6 represent different clones of the HeLa producer cell line. Figures 4C-D show the relative doubling rates of the expression of PGA5 (Figure 4C) and SPANXN3 (Figure 4D) in producer cell line clones 21C5, 3C6, and 2B6 cultured in supplemented cell culture media compared to the clones cultured in non-supplemented cell culture media. These results further validate the bioinformatics RNA sequencing and RT-qPCR data described in Example 3. (Example 5) Effect of gene knockdown on rAAV titer

[0164] Knockdown experiments were performed by individually knocking down genes in the HeLa producer cell line based on the optimized protocol discussed in Example 1. siRNA nucleotide sequences were designed for each gene (see Table 1).

[0165] The conditions were set at a seeding density of 1×10 5 , an 8 nM siRNA, and a siRNA:RNAiMAX ratio of 1:5. AAV production was induced 24 hours after the reduction in gene expression, and rAAV was collected 72 hours after infection. Titers were determined for each sample and compared to a non-targeting nonsense siRNA control. This experiment was performed independently three times, the results were averaged, and statistical analysis was carried out. Figures 5A - 5C show the results of siRNA of individual genes in production cell lines 1 - 3 respectively by absolute rAAV titers (GC / mL, GC = genomic copy). Figures 5D - 5F show the fold increase in rAAV titers by siRNA of individual genes in different production cell lines 1 - 3 respectively.

[0166] As shown in Figures 5A - 5F, a decrease in the expression of KCNN2, LINC00319, RGMA, or SPANXN3 in the production cell lines resulted in statistically significant rAAV titers that were 2 - 4 times higher than the nonsense control. Across the three production cell lines, these four genes showed a statistically relevant positive effect on titer when knocked down. These results indicate that these genes are excellent targets for more permanent modifications such as CRISPR / Cas9 knockout. (Example 6) Gene filtering methodology

[0167] For Filter 1, genes from differential analyses 1 and 7 (described in Table 3) were aligned. Differential analyses 1 and 7 defined genes that were up - or down - regulated upon addition of adenovirus 5 under serum - free conditions. Analysis 1 examined cells from the 21C5 production cell line (production cell line 1, PCL1). Analysis 7 examined cells from 2B6 (production cell line 2, PCL2). The list of genes after this Filter 1 identified genes that were not cell - line specific, and this alignment provided a total of 9149 genes common between the two production cell lines.

[0168] Regarding Filter 2, the genes from Filter 1 were aligned against the genes present in Differential Analysis 5. Differential Analysis 5 examined the genes that were upregulated and downregulated in cells derived from the 21C5-producing cell line (PCL1) under supplemented conditions compared to non-supplemented conditions. The purpose of this differential analysis was to define the effect of production under supplemented conditions on production under non-supplemented conditions. The purpose of aligning the gene set from Filter 1 against Differential Analysis 5 was to identify genes that 1) were not by-products of the production improvement conditions and 2) might be related to two different cell lines under production improvement conditions. After alignment, 374 genes were advanced forward.

[0169] Regarding Filter 3, only the genes with a large LogFc threshold of LogFC greater than 2 in absolute value were advanced forward. This was done to ensure a high level of upregulation / downregulation in the genes advanced forward and to obtain a certain degree of confidence that the selected genes were not RNA-Seq artifacts. After filtering, 77 genes were advanced forward.

[0170] Regarding Filter 4, only the genes that showed upregulation in both Differential Analysis 1 and Differential Analysis 5, or downregulation in both Differential Analysis 1 and Differential Analysis 5, were retained. For example, one of the 77 genes had to show upregulation from Differential Analysis 1 and also upregulation in Differential Analysis 5, or downregulation in Differential Analysis 1 and also downregulation in Differential Analysis 5. The purpose of this filter was to ensure that for the genes being evaluated, the high-titer condition did not have an antagonistic effect on the regulation of that specific gene compared to the low-titer condition. After filtering, 11 genes remained and were evaluated. An exemplary flowchart showing an exemplary gene filtering methodology is shown in Figure 6 (abbreviations used: LogFC = log fold change).

[0171] Table 5 provides the Log2FC data from each comparison during the process of filtering genes important for productivity.

Table 5

[0172] In this example, two existing highly optimized monoclonal HeLa-producing cell lines (PCL)-2H5 and 7B12-were genetically modified to knock out the KCNN2 gene encoding the calcium-activated potassium channel protein SK2 (previously identified in the RNA-seq screening described herein).

[0173] KCNN2 was knocked out in 2H5 or 7B12 HeLa cells using an eGFP selection marker. Cells that were thought to have KCNN2 knocked out were enriched for eGFP expression and seeded into 96-well plates. Cell colonies were formed, genomic DNA was collected, and PCR was performed to amplify the region containing the knockout. The PCR products were Sanger sequenced, and the sequence files were analyzed for the presence of insertions / deletions. 2H5 and 7B12 clones with a high likelihood of knockout were scaled up for further testing.

[0174] The top clones were transferred to serum-free suspension culture. Clone productivity compared to the parental strain was evaluated by 24 deep-well rAAV production. Clones were seeded at 2×10 5 cells / mL in 3 mL of culture and infected with Ad5 at a multiplicity of infection (MOI) of 50. Four days after infection, rAAV was collected and evaluated for titer. The doubling rate of titer was normalized to the parental control. The best clones exhibited a doubling rate of titer of 1.5 - 2.7 compared to the control sample. The 2H5 titer ranged from 2.46×10 9 to 4.98×10 10 GC / mL (Figure 7A). When the titer was normalized to the parental control, the doubling rate was in the range of 1.2 - 2.7-fold (Figure 7B). The 7B12 titer ranged from 4.33×10 8 to 1.88×10 10It was in the range of GC / mL (Figure 7C). When the titer was normalized against the parental control, the doubling rate was in the range of 1.5 - 2.6-fold (Figure 7D). Next, the clone with the minimum doubling rate of 1.5 was scaled up to shake flask culture and seeded into ambr® 15 for high seeding density-supplemented rAAV production. The cells were seeded at 1.5×10 6 cells / mL and infected with Ad5 at an MOI of 50. rAAV was collected 4 days after infection and evaluated for titer. The doubling rate of the titer was normalized against the parental control. The best clone exhibited a doubling rate of titer of 1.5 - 2.3-fold compared to the control sample. The 2H5 titer was in the range of 1.5×10 11 ~3.82×10 11 GC / mL (Figure 8A). When the titer was normalized against the parental control, the doubling rate was in the range of 1.3 - 2.3-fold (Figure 8B). The 7B12 titer was in the range of 2.62×10 10 ~1.35×10 11 GC / mL (Figure 8C). When the titer was normalized against the parental control, the doubling rate was in the range of 1.2 - 1.5-fold (Figure 8D).

[0175] These data demonstrate that reducing or removing the expression of one or more of the genes described herein in AAV-producing cells (e.g., via gene knockout) can be used to increase the production of rAAV from the engineered cells. (Example 8) Multi-combinatorial siRNA knockdown

[0176] In this example, multiple combinatorial knockdowns of the genes previously identified in the RNA-seq screening described herein using siRNA were performed to determine whether targeting multiple genes simultaneously would have an additive effect on the titer.

[0177] Multiple combinatorial knockdowns were performed using a modified method of the method described in Example 5. Briefly, cells were transfected using each siRNA at 8 nM, maintaining a siRNA:RNAiMAX ratio of 1:5. AAV production was induced 24 hours after the reduction in gene expression, and rAAV was harvested 72 hours after infection. Titers were determined for each sample and compared to a non-targeting missense siRNA control.

[0178] In this example, KCNN2 was knocked down in combination with a panel of other siRNAs described previously. Additionally, RGMA and SPANXN3 were knocked down in combination with each other. In 2H5, the combinatorial knockdown exhibited a titer increase in the range of 4.6 - 11.4 fold compared to the missense control (Figure 9A). In 7B12, the combinatorial knockdown exhibited a titer increase in the range of 3.4 - 9.7 fold compared to the missense control (Figure 9B). All combinations exhibited an increase in titer, but not all combinations were an improvement over knocking down KCNN2 alone. KCNN2 knockdown resulted in a doubling rate of 5.3 in 2H5 (Figure 9A) and 5.1 in 7B12 (Figure 9B).

[0179] These data demonstrate that additional increases in rAAV production can be obtained by direct targeting of multiple genomic regions in an established high rAAV titer-producing monoclonal PCL. Numbered Embodiments 1. A recombinant adeno-associated virus (rAAV) packaging 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 control parental cells. 2. The packaging and / or production cell line according to embodiment 1, comprising cells in which the expression of KCNN2, LINC00319, RGMA and SPANXN3 is reduced compared to control parental cells. 3. The packaging and / or production cell line according to embodiment 1 or 2, wherein the expression is reduced by the use of nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO). 4. The packaging and / or production cell line according to any one of embodiments 1 to 3, wherein the expression is reduced by the use of siRNA comprising a nucleotide sequence selected from any one of SEQ ID NOs: 1 to 11. 5. The packaging 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. 6. The packaging 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. 7. The packaging 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. 8. The packaging 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. 9. The packaging 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. 10. The packaging 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. 11. A packaging and / or production cell line according to embodiment 4, wherein the expression of KCNN2 is decreased, 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. 12. A packaging and / or production cell line according to embodiment 4, wherein the expression of NALCN-AS1 is decreased, 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. A packaging and / or production cell line according to embodiment 4, wherein the expression of RGMA is decreased, 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. 14. A packaging and / or production cell line according to embodiment 4, wherein the expression of PGA5 is decreased, 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. 15. A packaging and / or production cell line according to embodiment 4, wherein the expression of ABCA10 is decreased, 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. 16. The packaging 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. 17. The packaging and / or production cell line according to any one of embodiments 1 to 16, wherein the expression is decreased by the use of CRISPR genome editing. 18. The expression is decreased by the use of a guide RNA pair, and each guide RNA (a) comprises a sequence selected from the nucleotide sequences of SEQ ID NOs: 12 to 15, and / or (b) targets a target DNA sequence selected from any one of the nucleotide sequences of SEQ ID NOs: 16 to 31. The packaging and / or production cell line according to Embodiment 17. 19. The packaging and / or production cell line according to Embodiment 18, wherein the pair of gRNAs is used to target KCNN2 and comprises a first gRNA molecule comprising the sequence of SEQ ID NO: 12 and a second gRNA molecule comprising the sequence of SEQ ID NO: 13. 20. The packaging and / or production cell line according to Embodiment 18, wherein the pair of gRNAs is used to target KCNN2 and comprises a first gRNA molecule comprising the sequence of SEQ ID NO: 14 and a second gRNA molecule comprising the sequence of SEQ ID NO: 15. 21. The packaging and / or production cell line according to Embodiment 19 or 20, wherein each gRNA molecule is a 2'O-methyl analog comprising a 3' phosphorothioate internucleotide linkage at the 3 nucleotides at either or both of its 5' and 3' termini. 22. The packaging and / or production cell line according to any one of Embodiments 1 to 21, wherein the gene expression is removed as compared to the control parental cells. 23. The packaging and / or production cell line according to any one of Embodiments 1 to 22, which is a human cell line. 24. The packaging 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. 25. The cell line according to any one of Embodiments 1 to 24, which is an rAAV packaging cell line. 26. The cell line according to any one of Embodiments 1 to 24, which is an rAAV production cell line. 27. The cell line according to Embodiment 26, wherein the titer of rAAV is increased by about 1.5 to about 7 times as compared to the titer of rAAV produced from the cell line containing the control parental cells. 28. A lysate of the cell line according to any one of Embodiments 1 to 27. 29. A cell culture supernatant from the cell line according to any one of Embodiments 1 to 27. 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 described in Embodiment 25. 31. A method of producing rAAV, the method comprising infecting cells of a production cell line generated by the method described in Embodiment 30 with a helper virus. 32. A method of producing rAAV, the method comprising infecting cells of a production cell line described in Embodiment 26 with a helper virus. 33. The method according to Embodiment 31 or 32, wherein the rAAV is collected from the production cell line. 34. The method according to any one of Embodiments 31 to 33, wherein the production of rAAV is enhanced as compared to a control parental cell line. 35. A method of identifying one or more genes associated with the production of rAAV, adding one or more supplementary substances that increase the rAAV titer to a cell line; measuring global gene expression across the transcriptomes in the supplemented and non-supplemented cell lines; obtaining a list of genes differentially expressed between the supplemented cell line and the non-supplemented cell line; and identifying one or more genes associated with the production of rAAV comprising the method. 36. The method according to Embodiment 35, wherein the one or more supplementary substances added to the cell line comprise dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide or triamcinolone. 37. A method of producing an rAAV packaging and / or production cell line to promote an increase in the production of rAAV, the method comprising modulating the expression of one or more genes identified using the method described in Embodiment 35. 38. The method according to any one of Embodiments 35 to 37, wherein the cell line is an rAAV packaging cell line. 39. The method according to any one of embodiments 35 to 37, wherein the cell line is an rAAV-producing cell line. 40. The method according to embodiment 39, wherein the rAAV-producing cell line increases the rAAV titer by at least 1.5-fold greater than the rAAV titer produced by an rAAV-producing cell line that does not modulate the expression of one or more corresponding genes. 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. 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. 43. The method according to any one of embodiments 30 to 42, wherein the cell line is a human cell line. 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. 45. A recombinant adeno-associated virus (rAAV) packaging and / or production cell line comprising cells engineered to reduce the expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 as compared to corresponding unmodified parental cells. 46. The rAAV packaging and / or production cell line according to embodiment 45, wherein the expression and / or activity of gene products expressed from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1 is reduced indefinitely or permanently. 47. The rAAV packaging and / or production cell line according to embodiment 46, which is engineered to contain gene disruption or partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1. 48. The rAAV packaging and / or production cell line according to embodiment 47, which is engineered to contain gene disruption in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1. 49. The rAAV packaging and / or production cell line according to embodiment 47, which is engineered to contain gene disruption in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1. 50. The rAAV packaging and / or production cell line according to embodiment 47, which is engineered to contain partial or complete gene deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1. 51. The rAAV packaging and / or production cell line according to embodiment 47, which is 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 NALCN-AS1. 52. A recombinant adeno-associated virus (rAAV) packaging and / or production cell line that exhibits reduced expression and / or activity of a polypeptide or polynucleotide expressed from 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. Incorporation by reference

[0180] The entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes. Equivalents

[0181] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative rather than restrictive of the invention described herein. The scope of the invention is, accordingly, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

**Claim 1** A cell line that packages and / or produces recombinant adeno-associated virus (rAAV), in which the expression and / or activity of the gene product expressed from RGMA is reduced compared to the corresponding unmodified parental cells, or the expression and / or activity of the gene product expressed from RGMA and one or more genes selected from the group consisting of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1 is reduced, the cell line comprising a plurality of engineered cells. **Claim 2** The cell line according to claim 1, wherein the expression and / or activity of the gene product expressed from RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and / or NALCN-AS1 is reduced indefinitely or permanently. **Claim 3** The cell line according to claim 2, wherein the plurality of engineered cells comprises a gene disruption or partial or complete gene deletion in at least one of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and / or NALCN-AS1. **Claim 4** The cell line according to claim 3, wherein the plurality of engineered cells comprises a gene disruption in at least one of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and / or NALCN-AS1. **Claim 5** The cell line according to claim 3, wherein the plurality of engineered cells comprises gene disruptions in at least two selected from RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1. **Claim 6** The cell line according to claim 3, wherein the plurality of engineered cells comprises a partial or complete gene deletion in at least one of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and / or NALCN-AS1. **Claim 7** The cell line according to claim 3, wherein the plurality of engineered cells comprise a partial or complete gene deletion in at least two selected from RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1.

8. The cell line according to any one of claims 1 to 7, wherein the expression and / or activity is reduced by the use of clustered regularly interspaced short palindromic repeats (CRISPR) genome editing.

9. The cell line according to claim 8, wherein the CRISPR genome editing uses a guide RNA (gRNA) pair, and each gRNA targets a target DNA sequence selected from any one of the nucleotide sequences of SEQ ID NOs: 20 to 31.

10. The cell line according to claim 9, wherein each gRNA molecule is a 2'-O-methyl analog comprising a 3'-phosphorothioate nucleotide internucleotide linkage at the 3 nucleotides at either or both of its 5' and 3' ends.

11. The cell line according to any one of claims 1 to 10, wherein the cell line is a human cell line, and optionally, the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

12. A cell line that packages and / or produces recombinant adeno-associated virus (rAAV), which exhibits a reduced expression and / or activity of a polypeptide or polynucleotide expressed from RGMA, or a reduced expression and / or activity of a polypeptide or polynucleotide expressed from RGMA and one or more genes selected from the group consisting of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1, as compared to the corresponding parental cells, and comprises a plurality of engineered cells.

13. The cell line according to claim 12, wherein the expression and / or activity is reduced by the use of a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO).

14. The cell line according to claim 12, wherein (a) The expression and / or activity of ATP5EP2 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (b) The expression and / or activity of LINCO00319 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (c) The expression and / or activity of CYP3A7 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (d) The expression and / or activity of NOG in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (e) The expression and / or activity of SPANXN3 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (f) The expression and / or activity of MYRIP in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (g) The expression and / or activity of NALCN-AS1 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (h) The expression and / or activity of RGMA in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; (i) The expression and / or activity of PGA5 in the plurality of cells is / are reduced by the use of siRNA, and the siRNA includes 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; and / or (j) The expression and / or activity of ABCA10 in the plurality of cells is reduced by the use of siRNA, and the siRNA contains 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. Cell line.

15. A lysate or cell culture supernatant of the cell line according to any one of Claims 1 to 14.

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