Engineered producer cell line and method of preparation and use thereof

Abstract

The present application relates to rAAV packaging and / or producer cell lines that are engineered to reduce the expression and / or activity of one or more genes and / or proteins in order to increase the titer of recombinant adeno-associated virus (rAAV). A method for producing said engineered cell lines is also described herein.

Description

Technology Field

[0001] Cross-reference of related applications

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

[0003] Sequence list

[0004] The present application comprises a list of sequences electronically submitted in ASCII format, the full text of which is incorporated by reference. The said ASCII copy, created on April 9, 2020, is named ULP-005WO_SL.txt and has a size of 113 kb bytes.

[0005] Field of disclosure

[0006] The present application generally relates to engineered producer and / or packaging cell lines for increasing recombinant adeno-associated virus (rAAV) titers and a method for producing engineered producer and / or packaging cell lines. Background Technology

[0007] rAAV-based vectors are one of the most promising vehicles for human gene therapy. rAAV vectors are being considered for a wide variety of gene therapy applications. In particular, rAAV vectors can deliver therapeutic genes to dividing and non-dividing cells, and these genes can persist for long periods without being integrated into the target cell genome. Although systems for producing rAAV have advanced over the past two decades, several issues remain unresolved. One limitation of rAAV production systems is the low titer yield of rAAV particles. The pharmaceutical development of rAAV-based gene products at the preclinical stage requires large quantities of rAAV vectors for studies in larger species, which enable complete toxicology and biodistribution studies that help predict human dosages. Furthermore, because current rAAV production systems result in low titer yields, it is challenging to manufacture levels of rAAV sufficient for use in human trials and commercial applications. Although researchers are exploring numerous methods to generate rAAV particles of appropriately high titers, there is still a significant need to address this issue. In particular, there is a demand for efficient cell lines capable of producing high-quality rAAV with high titer yields. The production of high-titer rAAV by the engineered cell lines described herein facilitates the application of the corresponding vector system for in vivo gene therapy.

[0008] The present disclosure addresses the need to obtain improved rAAV titers for gene therapy applications by providing rAAV packaging and / or producer cell lines comprising cells in which one or more genes and / or proteins have been modified. Also described herein are a method for identifying one or more genes and / or proteins associated with the production of rAAV, and a method for producing engineered rAAV packaging and / or producer cell lines.

[0009] Described herein are compositions and methods for generating rAAV packaging and / or producer cell lines comprising cells capable of producing higher rAAV titers compared to control parent cells. More specifically, provided herein are rAAV packaging and / or producer cell lines comprising cells in which the expression of one or more genes and / or proteins is modulated to result in higher rAAV titers compared to control parent cells. In one aspect, the present disclosure provides rAAV packaging and / or producer cell lines comprising cells in which the expression of one or more genes and / or proteins is reduced compared to control parent cells. For example, the expression of ATP5EP2 (ATP synthase F1 subunit epsilon pseudogene 2), LINC00319 (long intergene 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 (repulsion-inducing molecule BMP co-receptor A), SPANXN3 (SPANX family member N3), PGA5 (pepsinogen A5), MYRIP (myosin VIIA and Rab interacting protein), KCNN2 (potassium calcium-activated channel subfamily N member 2) and / or NALCN-AS1 (NALCN antisense RNA 1) is reduced compared to control parental cells.

[0010] In some embodiments, the present disclosure provides rAAV packaging and / or producer cell lines comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parent cells.

[0011] In certain embodiments, the present disclosure provides an rAAV package and / or producer cell line comprising a cell engineered to reduce 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 compared to a corresponding unmodified parent cell. In certain embodiments, the present disclosure provides an rAAV package and / or producer cell line exhibiting reduced expression and / or activity of a polypeptide or polyribonucleotide expressed from at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1 compared to a corresponding parent cell line.

[0012] In one aspect, the present disclosure provides an rAAV package and / or producer cell line in which the expression of one or more genes is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA) or antisense RNA oligonucleotide (ASO).

[0013] In certain embodiments, the expression of one or more genes is reduced using siRNA containing a nucleotide sequence selected from any one of the sequences of SEQ ID NO: 1-11. For example, in some embodiments, 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 anti-sense strand. In some embodiments, 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 anti-sense strand. In some embodiments, 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 anti-sense strand. In some embodiments, NOG expression 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 anti-sense strand. In some embodiments, SPANXN3 expression 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 anti-sense strand. In some embodiments, MYRIP expression is reduced, and the siRNA contains the nucleotide sequence of SEQ ID No. 6 in the sense strand and the nucleotide sequence of SEQ ID No. 37 in the anti-sense strand. In some embodiments, KCNN2 expression is reduced, and the siRNA contains the nucleotide sequence of SEQ ID No. 7 in the sense strand and the nucleotide sequence of SEQ ID No. 38 in the anti-sense strand. In some embodiments, the expression of NALCN-AS1 is reduced, and the siRNA contains the nucleotide sequence of sequence identification number: 8 in the sense strand and the nucleotide sequence of sequence identification number: 39 in the anti-sense strand.In some embodiments, the expression of RGMA is reduced, and the siRNA contains the nucleotide sequence of sequence identification number: 9 in the sense strand and the nucleotide sequence of sequence identification number: 40 in the anti-sense strand. In some embodiments, the expression of PGA5 is reduced, and the siRNA contains the sequence of sequence identification number: 10 in the sense strand and the sequence of sequence identification number: 41 in the anti-sense strand. In some embodiments, the expression of ABCA10 is reduced, and the siRNA contains the sequence of sequence identification number: 11 in the sense strand and the sequence of sequence identification number: 42 in the anti-sense strand.

[0014] 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 (ZFN), meganucleases, transcriptional activator-like effector nucleases (TALENs), and short palindromic repeats of regularly spaced clustering (CRISPR) associated proteins.

[0015] In certain embodiments, the expression of one or more genes is reduced using CRISPR genome editing. In some embodiments, a guide RNA pair is used to target a gene to reduce its expression and / or remove it. In certain embodiments, the expression of one or more genes is reduced using a guide RNA pair, wherein each guide RNA (a) comprises a sequence selected from the nucleotide sequences of SEQ ID NOs: 12-15 and / or (b) targets a target DNA sequence selected from any 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 linkage at the terminal three nucleotides on either or both of its 5' and 3' ends.

[0016] In certain embodiments, one pair of guide RNAs is used to reduce the expression of one gene. In other specific embodiments, multiple pairs of guide RNAs are used to reduce the expression of one or more genes. In certain embodiments, the gene expression of one or more genes and / or the activity of one or more genes and / or proteins is reduced and / or eliminated in rAAV packaging and / or producer cell lines compared to control parent cell lines. In certain embodiments, gene expression and / or activity is eliminated in rAAV packaging and / or producer cells compared to control parent cells.

[0017] In some embodiments described herein, the rAAV packaging and / or producer cell line is a eukaryotic cell line. In certain embodiments, the rAAV packaging and / or producer cell line is a human cell line. In certain embodiments, the rAAV packaging and / or producer cell line is an insect cell line. In certain embodiments, the rAAV packaging and / or producer cell line is a HeLa cell line. In other specific embodiments, the rAAV packaging and / or producer cell line is a human embryonic kidney (HEK) 293 cell line.

[0018] In some embodiments described herein, the rAAV packaging and / or producer cell line of the present disclosure produces a higher rAAV titer compared to a control parent cell line. In certain embodiments, the rAAV titer produced from cells of the rAAV producer cell line of the present disclosure is increased by about 1.5 to about 7 times compared to the rAAV titer produced from a cell line containing control parent cells. Also described herein is a lysate of the engineered cell line. In certain embodiments, rAAV of higher titer is harvested from the lysate. Also described herein is the cell culture supernatant from the engineered cell line. In certain embodiments, rAAV of higher titer is harvested from the cell culture supernatant.

[0019] Also described herein is a method for generating a producer cell line, said 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 control parent cells. In certain embodiments, the present disclosure provides a method for generating a producer cell line, said 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 control parent cells.

[0020] Also described herein is a method for producing rAAV by infecting cells of a producer cell line produced 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 control parent cells. In certain embodiments, the expression of KCNN2, LINC00319, RGMA and SPANXN3 is reduced in the packaging cell line compared to control parent cells.

[0021] In one aspect, the present 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 control parent cells. In a specific embodiment, the present 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 control parent cells.

[0022] Also described herein is a method for harvesting 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 parent cell line. Also described herein is a method for harvesting rAAV from a producer cell line in which the expression of KCNN2, LINC00319, RGMA and SPANXN3 is reduced compared to a control parent cell line. In certain embodiments, the production of rAAV from the producer cell line of the present disclosure is enhanced compared to the control parent cell line.

[0023] Also described herein is a method for identifying one or more genes associated with the production of rAAV, wherein the method comprises: i.) adding one or more supplements that increase rAAV titers in cell lines; ii.) measuring total gene expression in transcriptomes in supplemented and non-supplemented cell lines; iii.) obtaining a list of genes that are differentially expressed between supplemented and non-supplemented cell lines; and iv.) identifying one or more genes associated with the production of rAAV. In some embodiments, the one or more identified gene(s) are responsible for reducing the production of rAAV.

[0024] Also described herein is a method for producing rAAV packaging and / or producer cell lines that promote increased 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 supplementary and non-supplementary rAAV producer cell lines. In certain embodiments, rAAV titers are increased by modulating the expression of one or more genes and / or proteins identified from a list of genes that are differentially expressed between supplementary and non-supplementary rAAV producer cell lines. In some embodiments, said modulation of one or more genes and / or proteins increases rAAV titers by at least 1.5-fold compared to the rAAV titers of cell lines without modulation. In certain embodiments, modulating expression is a reduction in the expression of one or more genes. In certain embodiments, modulating expression includes a reduction in the expression of one or more proteins. In certain embodiments, modulating expression is the removal of one or more gene expressions. In certain embodiments, adjusting expression includes removing one or more protein expressions.

[0025] In some embodiments, the rAAV packaging and / or producer 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.

[0026] Also described herein are recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines 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 compared to corresponding non-modified parent cells.

[0027] In some embodiments, 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.

[0028] In some embodiments, the cell line is engineered to include a genetic disruption or partial or complete genetic 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 include genetic disruption of at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1.

[0030] In some embodiments, the cell line is engineered to include genetic disruption in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1.

[0031] In some embodiments, the cell line is engineered to include 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.

[0032] In some embodiments, the cell line is engineered to include 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.

[0033] Also provided is a packaging and / or producer cell line, said cell line exhibiting reduced expression and / or activity of a polypeptide or polyribonucleotide expressed from at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1 compared to a corresponding parent cell line.

[0034] Other features and advantages of the present disclosure will become apparent from the following detailed description and claims.

[0035] Conversely, unless otherwise noted, all disclosures, references, patents and / or patent application references of this Appendix are incorporated by reference in their entirety for all purposes. Brief explanation of the drawing

[0036] The present disclosure can be more fully understood by referring to the following.Fig. 1 ...is a schematic diagram illustrating the rAAV packaging and the method for producing producer cells described herein. Figs. 2A-2D This presents experimental data generated from the optimization and development of HPRT1 siRNA knockdown experiments. do 2A-2B is the percentage knockdown generated from HPRT1 siRNA knockdown experiments performed in 24 wells ( Fig. 2A ) and protein expression ( Fig. 2B ) presents data. do 2C-2D is the percentage knockdown generated from HPRT1 siRNA knockdown experiments performed in 6 wells ( Fig. 2C ) and protein expression ( Figure 2D ) presents data. Figs. 3A-3B PGA5, expressed as the log-fold change in gene expression in cells cultured in unsupplemented cell culture medium compared to non-infected cells (cells not infected with helper virus), and the log-fold change in gene expression in cells cultured in supplemented cell culture medium compared to unsupplemented cell culture medium ( Fig. 3A ) and SPANXN3 ( Fig. 3B We present the log-fold change values ​​of gene expression obtained from bioinformatics analysis of RNA-Seq data for ). do 3C-3D PGA5 in cells cultured in unsupplemented and supplemented cell culture media compared to non-infected cells ( Fig. 3C ) and SPANXN3 ( Figure 3D Presents the RT-qPCR multiple change values ​​of ) expression. Figs. 4A-4B PGA5 in producer cell line clones cultured in unsupplemented and supplemented cell culture media relative to non-infected cells (cells not infected with helper virus) when determined by RT-qPCR ( Fig. 4A ) and SPANXN3 ( Fig. 4B) presents the multiple change values ​​of expression. 21C5, 3C6, and 2B6 represent different clones of the HeLa producer cell line. do 4C-4D PGA5 in producer cell line clones 21C5, 3C6, and 2B6 cultured in supplemental cell culture medium compared to clones cultured in non-supplemental cell culture medium ( Fig. 4C ) and SPANXN3 ( Figure 4D ) suggests a relative multiple increase in expression. Figs. 5A-5F This presents the effect of reducing the expression of individual genes in different producer cell lines on rAAV titers. The figure shows producer cell line #1 ( Fig. 5A ), Producer cell line #2 ( Fig. 5B ) and producer cell line #3 ( Fig. 5C Presents the titer of produced rAAV expressed as genome copy (GC) per milliliter (mL) in ). do 5D-5F is producer cell line #1 ( Fig. 5D ), Producer cell line #2 ( Fig. 5E ) and producer cell line #3 ( Fig. 5F Presents the multiple change in rAAV titer produced from ). do 5A-5B represents the average across three biological replicates. Fig. 5C-5F represents the average across 4 biological replicates. Fig. 6 This is an exemplary flowchart presenting a representative gene filtering methodology. Fig. 7A This presents the 24-deep-well titers of the top 19 2H5 knockout clones. Titers were recorded as genome copies per mL. The control sample is unmodified 2H5. do 7B This presents the multiple change in potency compared to the 2H5 control group. The 2H5 potency was set to 1, and other potencies were shown as multiple increases exceeding the 2H5 control group. do 7CThis presents the 24-deep-well titers of the top 19 7B12 knockout clones. Titers were recorded as genome copies per mL. The control sample is the unmodified 7B12. do 7D This presents the multiple change in potency compared to the 7B12 control group. The 7B12 potency was set to 1, and other potencies were shown as multiple increases exceeding the 7B12 control group. Fig. 8A This presents the ambr® 15 titers of the top 5 2H5 knockout clones. Titers were recorded as genome copies per mL. The control sample is unmodified 2H5. do 8B This presents the multiple change in potency compared to the 2H5 control group. The 2H5 potency was set to 1, and other potencies were shown as multiple increases exceeding the 2H5 control group. do 8C This presents the ambr® 15 titers of the top four 7B12 knockout clones. Titers were recorded as genome copies per mL. The control sample is the unmodified 7B12. do 8D This presents the multiple change in potency compared to the 7B12 control group. The 7B12 potency was set to 1, and other potencies were shown as multiple increases exceeding the 7B12 control group. Figs. 9A-9B This presents the effect on the generated rAAV titer by reducing the expression of various gene combinations in two types of producer cell lines. The figure shows the multiple change in rAAV titer compared to a control group treated with missense siRNA. do 9A This presents the multiple change in titer compared to the 2H5 missense control group. The 2H5 missense titer was set to 1, and other titers were shown as multiple increases exceeding the 2H5 missense control group. Fig. 9B This presents the multiple change in potency compared to the 7B12 missense control group. The 7B12 missense potency was set to 1, and other potencies were shown as multiple increases exceeding the 7B12 missense control group. Specific details for implementing the invention

[0037] The present disclosure describes a recombinant adeno-associated virus (rAAV) package and / or producer cell line comprising a cell in which the expression of one or more genes and / or proteins is regulated. The regulation of gene expression results in an increased titer yield compared to a cell line in which the expression of one or more genes and / or proteins is not regulated.

[0038] Unless otherwise noted, technical terms are used according to common usage. Definitions of general molecular biology terms are in the literature [Benjamin Lewin, Genes V , published by Oxford University Press, 1994 (ISBN 0-19-854287-9)]; [Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9)]; and [Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference It can be found in [ISBN 1-56081-569-8], published by VCH Publishers, Inc., 1995.

[0039] The following definitions are included for the purpose of understanding the subject matter of the invention and for establishing the appended claims. Abbreviations used herein have their ordinary meanings within the fields of chemical and biological technology.

[0040] definition

[0041] As used herein, “adjustment” or “adjust” refers to the regulation, expression, or alteration of the activity of a gene and / or protein. Adjustment may involve increasing, decreasing (reducing), or eliminating the expression and / or activity of one or more genes and / or proteins. Where multiple genes and / or proteins are adjusted, the expression and / or activity of all genes and / or proteins may be increased, the expression and / or activity of all genes and / or proteins may be decreased, or one or more genes and / or proteins may be increased and other genes and / or proteins may be decreased.

[0042] As used herein, the term “cell” refers to any cell or cells capable of producing recombinant adeno-associated virus (rAAV). In some embodiments, the cell is a mammalian cell, e.g., HeLa cell, COS cell, HEK293 cell, A549 cell, BHK cell, or Vero cell. In other embodiments, the cell is an insect cell, e.g., Sf9 cell, Sf-21 cell, Tn-368 cell, or BTI-Tn-5B1-4 (High-Five) cell. The term “cell line” refers to a clonal population of cells that can continue to divide and does not undergo senescence. Unless otherwise indicated, the terms “cell” or “cell line” are understood to include modified or engineered variants of the cell or cell line described.

[0043] As used herein, the term “manipulated cell line” refers to a cell line modified by one or more means of reducing the expression of one or more endogenously expressed genes and / or proteins (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1) or other characteristics (e.g., biological activity) in order to increase the production of rAAV.

[0044] As used herein, the term “control parental cell” refers to a cell that has not been modified by one or more means of reducing the expression of one or more endogenously expressed genes and / or proteins (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1) or other characteristics (e.g., biological activity) in order to increase the production of rAAV.

[0045] As used herein, the term "control parent cell line" refers to a clonal population of control parent cells that can continue to divide and does not undergo aging.

[0046] "Lysis" often refers to the breakdown of a cell by viruses, enzymes, or osmotic mechanisms that impair its integrity. "Lysed cell" is a cell that has undergone substantial lysis. As used herein, the term "lysate" refers to a fluid containing the contents of a lysed cell.

[0047] As used herein, the term "higher potency" refers to an increased potency compared to the potency produced by the unmodified control parent cell line and / or the control parent cells.

[0048] As used herein, the term "cell culture supernatant" refers to a cell culture medium in which cells are suspended or cultured.

[0049] As used herein, the term “gene” refers to a transcription unit and a regulatory region adjacent to the transcription unit (e.g., located upstream and downstream thereof) and functionally connected to it. 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 sequence of nucleotides that codes for unprocessed free RNA (i.e., an RNA molecule containing both exons and introns) that is subsequently processed into mRNA. A transcription unit may code for non-coding RNA. Non-coding RNA is an RNA molecule that is not translated into a protein. Examples of non-coding RNA include microRNA. The boundaries of a transcription unit are generally determined by an initiation site at its 5’ end and a transcription terminator at its 3’ end. A “regulatory region” is a sequence of nucleotides that regulates the expression of the transcription unit to which it is functionally connected. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation initiation sites, translation stop sites, transcription termination factors, and poly(A) signals. A regulatory region located upstream of a transcription unit may be referred to as a 5' UTR, and a regulatory region located downstream of a transcription unit may be referred to as a 3' UTR. The regulatory region may be transcribed and become part of unprocessed free RNA.

[0050] In the context of this document, the terms “target” or “target gene” refer to any gene, including protein-coding genes and genes encoding non-coding RNAs (e.g., miRNAs) that, when regulated, alter some aspect of viral production. Target genes include endogenous genes, viral genes, and transgenes.

[0051] Regarding gene designations, a single gene is often represented by multiple symbols. In the context of this document, gene symbols may be designated as either uppercase or lowercase letters, regardless of whether they are human or non-human. In the context of this disclosure, neither the use of a specific symbol nor the adoption of lowercase or uppercase symbols is intended to limit the category of genes. Unless otherwise noted, all gene identification numbers (GeneIDs) identified herein are derived from the National Center for Biotechnology Information “Entrez Gene” or the KEGG website.

[0052] The term "approximately" is used herein to mean roughly, to an extent, nearly, or about. When the term "approximately" is used in conjunction with a numerical range, it modifies the range by extending the boundary beyond and / or below the numerical value presented within the acceptable value range.

[0053] As used in this disclosure, the terms “comprising” and “comprising” should be interpreted as having an open-closed meaning, whether they are connecting phrases or part of the claims. That is, the terms should be interpreted as synonyms for the phrases “at least having” or “at least including.” When used in the context of a method, the term “comprising” means that the method includes at least the mentioned steps but may also include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the mentioned features or components but may also include additional features or components.

[0054] For the purpose of facilitating understanding of the embodiments described herein, specific language is used to describe preferred embodiments by reference. The technical terms used herein are intended to describe specific embodiments only and are not intended to limit the scope of this disclosure. As used throughout this disclosure, the singular form includes the plural unless the context clearly indicates otherwise. All percentages and ratios used herein are by weight unless otherwise indicated.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein may be used to practice or test this disclosure, suitable methods and materials are described below. Furthermore, materials, methods, and embodiments are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

[0056] Adeno -Related Viruses ( AAV )

[0057] AAV is a small, replication-deficient, non-enveloped virus that infects humans and some other primate species. AAV does not cause disease and is known to elicit a very weak immune response. Gene therapy vectors utilizing AAV can infect both dividing and resting cells and can persist extrachromosomally without being integrated into the host cell genome. These characteristics make AAV an attractive viral vector for gene therapy. AAV encompasses numerous serologically distinguishable types, including serotypes AAV-1 through AAV-12 as well as over 100 serotypes derived from non-human primates (e.g., [Srivastava, J. Cell Biochem., 105(1): 17-24 (2008)] and [Gao et al. [See , J. Virol., 78(12), 6381-6388 (2004)]). AAV is non-autoreplicative and has a life cycle that includes a latent period and an infective period. After a cell is infected with AAV, during the latent period, AAV integrates site-specifically into the host genome as a provirus. The infective period does not occur unless the cell is also infected with a helper virus (e.g., adenovirus (AV) or herpes simplex virus) that enables AAV replication.

[0058] The wild-type AAV genome contains two 145-nucleotide inversion terminal repeats (ITRs) that contain signal sequences inducing AAV replication, genomic capsidization, 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, associated with differential splicing of a single AAV intron, result in the production of four rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. The rep proteins are responsible for genomic replication. The cap gene is expressed from the p40 promoter and codes for three capsid proteins (VP1, VP2, and VP3), which are spliced ​​variants of the cap gene. These proteins form the capsid of the AAV particle.

[0059] Because cis-action signals for replication, capsidization, and integration are contained within the ITR, part or all of the 4.3 kb internal genome may be replaced with foreign DNA, e.g., an expression cassette for the exogenous protein of interest. In such cases, the rep and cap proteins are provided trans, e.g., on a plasmid. To produce an AAV vector, a cell line permissible to AAV replication must express the rep and cap genes, the ITR-flanked expression cassette, and helper functions provided by the helper virus, e.g., AV genes E1a, E1b55K, E2a, E4orf6, and VA (Reference [Weitzman et al.[Adeno-associated virus biology. Adeno-Associated Virus: Methods and Protocols, pp. 1-23, 2011]). The production of AAV vectors may also result in the production of helper virus particles, which must be removed or inactivated before the use of the AAV vector. Numerous cell types are suitable for producing AAV vectors, including insect cells as well as HEK293 cells, COS cells, HeLa cells, BHK cells, and Vero cells (see, e.g., U.S. Patent Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, 5,688,676, 8,163,543; U.S. Publication No. 20020081721; PCT Publications Nos. WO00 / 47757, WO00 / 24916, and WO96 / 17947). In these cell types, the AAV vector is typically produced by one plasmid containing an ITR-flanked expression cassette and one or more additional plasmids providing additional AAV and helper virus genes.

[0060] Any serotype of AAV may be used in the present disclosure. Likewise, any type of AV may be used, and a person skilled in the art will be able to identify the types of AAV and AV suitable for the production of his desired recombinant AAV vector (rAAV). AAV and AV particles may be purified, for example, by affinity chromatography, an iodixanol gradient, or a CsCl gradient.

[0061] The genome of wild-type AAV is single-stranded DNA and is 4.7 kb. AAV vectors may have single-stranded genomes larger or smaller than 4.7 kb, including genomes larger than 5.2 kb or smaller than 3.0 kb. Additionally, vector genomes may be substantially self-complementary, so the genome within the virus is substantially double-stranded. AAV vectors containing all types of genomes are suitable for use in the methods of the present disclosure.

[0062] As discussed above, AAV requires co-infection with a helper virus to enter the infectious phase of its life cycle. Helper viruses include adenoviruses (AV) and herpes simplex virus (HSV), and systems exist for producing AAV in insect cells using baculoviruses. Additionally, it has been suggested that papillomaviruses may also provide a helper function for AAV (e.g., the literature [Hermonat et al.[Refer to , Molecular Therapy 9, S289-S290 (2004)]). Helper viruses include all viruses capable of generating and enabling AAV replication. AV is an unenveloped nuclear DNA virus with a double-stranded DNA genome of approximately 36 kb. AV can rescue latent AAV proviruses within cells by providing the E1a, E1b55K, E2a, E4orf6, and VA genes to enable AAV replication and capsidization. HSV is a family of viruses with a relatively large double-stranded linear DNA genome that is capsidized into an icosahedral capsid wrapped within a lipid bilayer envelope. HSV is infectious and highly transmissible. The following HSV-1 replication proteins have been identified as necessary for AAV replication: the helicase / primase complex (UL5, UL8, and UL52) and the DNA binding protein ICP8 encoded by the UL29 gene, along with other proteins that enhance helper function. The AAV packaging system is used for the following two purposes: it bypasses the problems of the transfection process and provides a production technology based on the use of one or more helper functions.

[0063] rAAV's production

[0064] The general principles of rAAV can be discussed 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 speaking, to enable the production of rAAV, additional helper functions must be provided to the cell, for example, as well as the functions of AAV ITR, AAV rep, and cap genes capable of flanking the xeno-derived nucleotide sequence of interest. These can be provided to the cell using any number of appropriate plasmids or vectors. Additional helper functions can be provided, for example, by adenovirus (AV) infection, by a plasmid containing all the necessary AV helper function genes, or by other viruses such as HSV or baculovirus. Any gene, gene function, or genetic material required for rAAV production by cells may exist transiently within the cell or be stably inserted into the cell genome. Methods for producing rAAV suitable for use with the methods of the present disclosure include the literature [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.These include those disclosed in [Human Gene Therapy 20:807-817 (2009)]. Two major approaches for AAV production systems are recombinant adeno-associated virus (rAAV) packaging cell lines and adeno-associated virus (rAAV) producer cell lines.

[0065] recombination Adeno -Related Viruses ( rAAV ) Packaging and / or producer cell lines

[0066] rAAV packaging cell lines can be produced by enabling cellular expression of the AAV gene elements described herein. Stable transfection of cell lines (e.g., HEK293, HeLa) with plasmids encoding the AAV rep and cap genes can result in the production of packaging cell lines. Such rAAV packaging cell lines can be co-infected with two different adenoviruses (a helper virus and a hybrid virus containing an AAV gene-therapy element) to produce rAAV particles. Alternatively, stable transfection of packaging cells with a plasmid containing an rAAV vector or their infection with an rAAV vector leads to rAAV producer cell lines. Infection of producer cells with a helper virus leads to the production of rAAV. do 1 It illustrates packaging and producer cell lines.

[0067] In a specific embodiment of the present disclosure, an rAAV packaging cell line comprising AAV rep and cap gene functions is engineered to increase rAAV titer.

[0068] In one aspect, the present disclosure provides an rAAV-packaged cell line comprising cells in which the expression of one or more genes and / or proteins is reduced compared to control parent cells. For example, the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1 is reduced compared to control parent cells.

[0069] In some embodiments, the present disclosure provides an rAAV-packaged cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parent cells.

[0070] In another embodiment, the present disclosure provides an rAAV producer cell line comprising cells in which the expression of one or more genes and / or proteins is reduced compared to control parent cells. For example, the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1 is reduced compared to control parent cells. In some embodiments, the rAAV producer cell line of the present disclosure is engineered to reduce the gene expression of KCNN2, LINC00319, RGMA, and SPANXN3.

[0071] In certain embodiments, the cell lines of the present disclosure may exist in an attached or suspended form.

[0072] In certain embodiments, the cell line of the present disclosure (e.g., rAAV packaging and / or producer 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).

[0073] rAAV Method for generating producer cell lines

[0074] In some embodiments, the present disclosure provides a method for generating a producer cell line by delivering an rAAV vector to an engineered rAAV-packaged cell line comprising cells in which the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1 is reduced compared to control cells.

[0075] In certain embodiments, the present disclosure provides a method for generating a producer cell line by delivering an rAAV vector to an engineered rAAV-packaged cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parent cells.

[0076] supplements

[0077] As used herein, the term “supplement” refers to any compound or other substance that may be used in a cell culture medium to increase rAAV titer or to test for an increase in rAAV titer, regardless of whether its origin is chemical or biological. Non-limiting examples of supplements include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, enzymes, nucleosides, metabolites, surfactants, emulsifiers, inorganic salts, and polymers. In certain embodiments, one or more supplements added to the rAAV packaging and / or producer cell lines of this disclosure are glucocorticoid analogs. In certain embodiments, one or more supplements added to the rAAV packaging and / or producer cell lines include dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, and / or triamcinolone.

[0078] In certain embodiments, the concentration of a glucocorticoid analog in a solution for increasing rAAV potency may be 1 μM or more, 0.1 μM or more, 0.01 μM or more, 0 to 1 μM, 0 to 0.1 μM, 0 to 0.01 μM, 0.01 to 1 μM, or 0.01 to 0.1 μM.

[0079] As used herein, “supplemented cell line” refers to a cell line (e.g., rAAV packaging and / or producer cell line) to which one or more supplements (e.g., glucocorticoid analogs) have been added to increase rAAV titer. As used herein, “non-supplemented cell line” refers to a cell line (e.g., rAAV packaging and / or producer cell line) that has not been exposed to supplements or supplements to increase rAAV titer. As used herein, the terms “non-supplemented” and “non-supplemented” are used interchangeably to refer to culture conditions in which a cell line (e.g., rAAV packaging and / or producer cell line) has not been exposed to supplements or supplements to increase rAAV titer.

[0080] rAAV A method for identifying one or more genes associated with production

[0081] The present disclosure relates to a method for identifying one or more genes associated with the production of rAAV by comparing whole gene expression patterns in partially supplemented and non-supplemented cell lines.

[0082] The term "total gene expression" is well known in the relevant technical field (Reference [Wang Z. et al.[See Nature Reviews Genetics, 10(1), 57-63 (2009)]). The term “total gene expression” refers to one or more datasets containing information regarding different aspects of gene expression. Such datasets optionally include information regarding: the presence of a target transcript in cells or cell-derived samples; the relative and absolute abundance levels of the target transcript; the ability of various treatments (e.g., addition of a supplement) to modulate the expression of a specific gene; and the ability of various treatments (e.g., addition of a supplement) to alter the expression of a specific gene to different levels.

[0083] The term "differentially manifested" is well known in the relevant technical field (Reference [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)].

[0084] In certain embodiments, the cell lines of this disclosure (e.g., rAAV packaging and / or producer cell lines) are supplemented with one or more supplements that increase the production of rAAV. In some embodiments, RNA samples are extracted from one or more cell lines (supplemented and non-supplemented) using any of the widely known procedures. For example, total RNA from the cells can be purified using silica-based isolation in an automated-compatible 96-well format, such as the Rneasy® purification platform (Qiagen, Inc.; Valencia, California).

[0085] The gene expression pattern in the expressed RNA sample can be evaluated by either qualitative or quantitative means (or both). In some embodiments, it is useful to quantify the expression level of a gene against other expression products and / or against a control sequence. One common and widely applicable method for determining relative expression is to compare the expression of one or more genes of interest with the expression of a control gene, such as a housekeeping gene (e.g., HPRT1, HSP70, or β-actin).

[0086] To determine whether observed expression data, e.g., changes in gene expression profiles in response to one or more treatments (e.g., addition of supplement) of biological samples (e.g., supplemented and non-supplemented cell lines), are significant and not merely the product of experimental noise or cluster heterogeneity, estimates of likelihood distributions may be constructed for each genetic and phenotypic endpoint in each biological sample. The construction of estimated cluster distributions involves conducting multiple independent experiments for each treatment, e.g., all experiments are conducted in double, triple, quadruple, etc. Expression data from multiple biological samples (e.g., supplemented and non-supplemented cell lines) may be grouped or clustered using multivariate statistics. Analysis of the data may produce a list of genes that are differentially expressed in response to treatment between supplemented and non-supplemented cell lines, e.g. The list of differentially expressed genes may be filtered using various gene filtering methodologies to identify one or more genes useful for increasing rAAV production.

[0087] In some embodiments, the present disclosure relates to a method for identifying one or more genes from a list of genes that are differentially expressed between supplementary and non-supplementary cell lines associated with the production of rAAV. 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 an HEK293 cell line. In certain embodiments, to identify one or more genes associated with the production of rAAV, different cell lines are crossed (e.g., between non-supplementary HeLa and supplementary HeLa cell lines, between non-supplementary HEK 293 and supplementary HEK 293 cell lines, between non-supplementary HeLa and supplementary HEK 293 cell lines, between non-supplementary HeLa and non-supplementary HEK 293 cell lines, between supplementary HeLa and supplementary HEK 293 cell lines) and total gene expression is measured. In certain embodiments, to identify one or more genes associated with the production of rAAV, combined whole gene expression data from supplemental HEK 293 and supplemental HeLa may be combined and compared with whole gene expression data from non-supplemental HEK 293 and non-supplemental HeLa cell lines.

[0088] In certain embodiments, the present disclosure provides a method for producing rAAV packaging and / or producer cell lines that promote increased production of rAAV. In some embodiments, rAAV production is increased by adjusting the expression of one or more genes and / or proteins identified in a list of genes that are differentially expressed between supplementary and non-supplementary rAAV producer cell lines. In certain embodiments, the rAAV titer is increased by adjusting the expression of one or more genes and / or proteins identified in a list of genes that are differentially expressed between supplementary and non-supplementary rAAV producer cell lines. In some embodiments, the rAAV titer is increased by at least 1.5 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 30 times) compared to the rAAV titer produced by a cell line without adjustment of the expression of the corresponding gene(s) and / or protein(s).

[0089] Modulated genes and / or proteins

[0090] In certain embodiments, the present disclosure provides a list of genes that enhance the production of rAAV when modified (individually or in combination) in rAAV packaging and / or producer cell lines.

[0091] ATP synthase F1 subunit epsilon pseudogene 2 (also known as ATP5EP2) codes for the 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 complex of the respiratory chain. Examples of human ATP5EP2 sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequences NM_006886.4 (sequence identification number: 43) or NG_053163.1 (sequence identification number: 44).

[0092] Long intergene non-protein coding RNA 319 (also known as LINC00319) is an RNA gene and is related to the non-coding RNA class. Long non-coding RNAs (lncRNAs) have been suggested to play important regulatory roles in the pathogenesis and progression of many cancers. Examples of LINC00319 sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_194309 (sequence identification number: 45) or NR_026960.1 (sequence identification number: 46).

[0093] Cytochrome P450 Family 3 Subfamily A Member 7 (also known as CYP3A7) is a gene encoding a member of the cytochrome P450 enzyme superfamily involved in drug metabolism and the synthesis of cholesterol, steroids, and other lipids. Such enzymes hydroxylate testosterone and dehydroepiandrosterone 3-sulfate, which are involved in the formation of estriol during pregnancy. Such genes are part of a cluster of related genes on chromosome 7q21.1. An example of the CYP3A7 sequence is available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_000765 (sequence identification number: 47).

[0094] Member 10 of the ATP-binding cassette subfamily A (also known as ABCA10) codes for a membrane-associated protein belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC proteins transport various molecules across extracellular and intracellular membranes. The ABC gene is divided into seven distinct subfamilies (ABC1, MDR / TAP, MRP, ALD, OABP, GCN20, and White). ABCA10 is a member of the ABC1 subfamily. The members of the ABC1 subfamily include the only main ABC subfamily found entirely in multicellular eukaryotes. Such genes are clustered with four other ABC1 family members on 17q24. An example of the ABCA10 sequence is available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_080282.3 (sequence identification number: 48).

[0095] Nogin (also known as NOG) codes for a secreted polypeptide that binds to and inactivates members of the transforming growth factor-beta (TGF-beta) superfamily signaling proteins, such as bone morphogenetic protein-4 (BMP4). Without being bound by theory, it is believed that such proteins may play a major role in creating morphogenetic gradients due to their more efficient diffusion through the extracellular matrix compared to members of the TGF-beta superfamily. NOG appears to have polymorphic effects in both early and late developmental stages. An example of a NOG sequence is available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_005450.4 (sequence identification number: 49).

[0096] The repulsion-inducing molecule BMP co-receptor A (also known as RGMA) is a gene encoding a member of the repulsion-inducing molecule family. The coded protein is a glycosylphosphatidylinositol-fixed glycoprotein that functions as an axon-inducing protein in the developmental and adult central nervous systems. Such proteins may also function as tumor suppressors in some cancers. Examples of RGMA sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_020211.2 (sequence identification number: 50) or NM_001166283.1 (sequence identification number: 51).

[0097] SPANX (a sperm protein associated with the nucleus on the X chromosome) family member N3 (also known as SPANXN3) is a protein-coding gene. An example of the SPANXN3 sequence is available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_001009609 (sequence identification number: 52).

[0098] Pepsinogen-5, Group I (also known as PGA5 or pepsinogen A) codes for the protein precursor of pepsin, a digestive enzyme and member of the peptidase A1 family of endopeptidases. The coded precursor is secreted by gastric principal cells and undergoes autocatalytic cleavage under acidic conditions to form an active enzyme that functions in the digestion of dietary proteins. Such genes are found in a chromosome 11-associated gene cluster, each encoding one of the multiple types of pepsinogen. An example of a PGA5 sequence is available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_014224.4 (sequence identification number: 53).

[0099] Myosin VIIA and Rab interaction proteins (also known as MYRIP) code for Rab effector proteins involved in melanosomal transport, acting as a link between melanosomal-binding RAB27A and motor proteins MYO5A and MYO7A. These Rab effector proteins function as protein kinase A-fixation proteins (AKAPs) and can facilitate exocytosis, including insulin release, by acting as scaffolding proteins that link PKAs to components of the exocytosis mechanism. Examples of MYRIP sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_015460 (sequence identification number: 54) or NM_001284423.1 (sequence identification number: 55).

[0100] The potassium calcium-activated channel subfamily N member 2 (also known as KCNN2) gene is a member of the KCNN family among potassium channel genes. The coding protein is an integrated membrane protein that forms a voltage-independent calcium-activated channel with three different calmodulin-binding subunits. Alternative splicing of such a gene leads to a number of transcript variants. Examples of KCNN2 sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NM_170775.2 (sequence identification number: 56) or NM_001278204.1 (sequence identification number: 57).

[0101] NALCN antisense RNA 1 (also known as NALCN-AS1) is an RNA gene and is related to the non-coding RNA class. Examples of NALCN-AS1 sequences are available in the NCBI nucleotide database (nucleotide sequences) under reference sequence NW_011332700.1 (sequence identification number: 58) or NR_047687.1 (sequence identification number: 59).

[0102] In certain embodiments, the present disclosure provides an rAAV package and / or producer 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 parent cells.

[0103] In certain embodiments, the present disclosure provides an rAAV package and / or producer cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parent cells.

[0104] In certain embodiments, the present disclosure provides a list of genes that, when individually tuned in rAAV packaging and / or producer cell lines, enhance the production of rAAV compared to a control parent cell line. In some aspects, tuning of different combinations of genes in rAAV packaging and / or producer cell lines increases the production of rAAV. In some aspects, tuning 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 rAAV packaging and / or producer cell lines leads to increased rAAV production compared to a control parent cell line.

[0105] Method for modulating one or more genes and / or proteins

[0106] Modifying (e.g., reducing) the expression or activity of a gene (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, or NALCN-AS1) may be achieved by different mechanisms including, but not limited to, altering one or more of the following: 1) the gene copy number, 2) the transcription or translation of the gene, 3) the transcript stability or lifetime, 4) the copy number of mRNA or miRNA, 5) the availability of non-coding RNA or non-coding RNA target sites, 6) the location or extent of post-translational modification on the protein, or 7) the activity of the protein. Tools that can be used to regulate gene expression include, but are not limited to, nucleases, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), antisense RNA oligonucleotides (ASO), gene disruption, or partial or complete gene deletion.

[0107] Nuclease

[0108] In certain embodiments, gene modification is achieved using zinc finger nucleases (ZFNs). Synthetic ZFNs consist of, for example, a zinc finger binding domain fused with a FokI DNA cutting domain. ZFNs can be designed / manipulated to edit the genomes of cells, including knocking out or knocking in gene expression in a wide range of organisms without limitation. Meganucleases, transcriptional activator-like effector nucleases (TALENs), or CRISPR-associated proteins (e.g., Cas nucleases) with short palindromic repeats of regular intervals clustering, and triplexes can also be used for genome manipulation in a wide range of cell types. The described reactants can be used to target promoters, protein-coding regions (exons), introns, 5' and 3' UTRs, etc.

[0109] Coordinating double-stranded RNA ( dsRNA ) molecule

[0110] In certain embodiments, double-stranded RNA (dsRNA) molecules may be used to regulate the expression of one or more genes in the cell lines described herein (e.g., rAAV packaging and / or producer cell lines). The dsRNA molecules may be designed to antagonize one or more genes by sequence homology-based targeting of the corresponding RNA sequences. Such dsRNA may be small interfering RNA (siRNA), small hairpin RNA (shRNA), or microRNA (miRNA). The sequence of such dsRNA comprises a portion of the mRNA that is complementary to the one or more genes to be regulated. Such a portion may be 100% homology to the target portion within the mRNA, but a lower level of complementarity (e.g., 90% or more or 95% or more) may also be used. Typically, percentage complementarity is determined over a certain length of consecutive nucleic acid residues. The dsRNA molecule of the present disclosure may have at least 80% complementarity with respect to a target site in mRNA when 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 respect to a target site in mRNA over the entire length of the dsRNA molecule.

[0111] Another gene targeting reactant using the RNA interference (RNAi) pathway is small hairpin RNA, also referred to as shRNA. For example, shRNA delivered to a cell via an expression construct (e.g., a plasmid, a lentivirus) has the ability to provide a long-term reduction of gene expression in an anachronous or regulated manner, depending on the type of promoter used. In one embodiment, the genome of a lentivirus particle is modified to include one or more shRNA expression cassettes that target a gene (or genes) of interest. Such a lentivirus infects a cell, stably integrates its viral genome into the host genome, and then can express shRNA in an anachronous, regulated, or (where multiple shRNAs are expressed) both anachronous and regulated manner. Accordingly, in some embodiments, shRNA may be designed to target a single gene or individual variants of multiple closely related gene family members. Individual shRNAs may coordinate a set of targets having similar or redundant functional or sequence motifs. A person skilled in the art would know that a lentivirus construct may contain cloned DNA or an ORF expression construct.

[0112] In the embodiments described herein, gene-targeting reactants, including small interfering RNAs (siRNAs) as well as microRNAs (miRNAs), may be used to modulate gene function. SiRNAs and miRNAs may extensively include chemical modifications, levels of complementarity to the target transcript of interest, and designs to enhance stability, cell delivery, specificity, and functionality (see U.S. Patent No. 8,188,060). Additionally, such reactants may be designed to target genes in various regions (including 5' UTRs, open reading frames, and 3' UTRs of mRNA) or (in some cases) promoter / enhancer regions of genomic DNA encoding the gene of interest. Gene modulation (e.g., reduction of gene expression, knockdown) may be achieved by introducing a single siRNA or miRNA or a pool of multiple siRNAs or miRNAs targeting different regions of a single mRNA transcript (into the cell). Synthetic siRNA / miRNA delivery can be achieved by any number of methods, including, but not limited to, 1) self-delivery, 2) lipid-mediated delivery, 3) electroporation, or 4) vector / plasmid-based expression systems. The RNA molecule introduced may be referred to as an exogenous nucleotide sequence or a polynucleotide. In some embodiments, the siRNA may be designed to target individual variants of a single gene or multiple closely related gene family members.

[0113] siRNA may 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, siRNA comprising a nucleotide sequence selected from sequence identification numbers 1 to 11 or a variant thereof is used to reduce the expression of a target gene.

[0114] Table 1 : siRNA sequences used to reduce gene expression

[0115]

[0116]

[0117]

[0118] * siRNA sequences (sense and antisense) used to reduce gene expression. Lowercase nucleotides in the sequence represent the 3' overhang.

[0119] 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 anti-sense strand.

[0120] 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 anti-sense strand.

[0121] 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 anti-sense strand.

[0122] 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 anti-sense strand.

[0123] 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 anti-sense strand.

[0124] 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 anti-sense strand.

[0125] 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 anti-sense strand.

[0126] 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 anti-sense strand.

[0127] 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 anti-sense strand.

[0128] 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 anti-sense strand.

[0129] 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 anti-sense strand.

[0130] antisense RNA oligonucleotide ( ASO )

[0131] Antisense RNA oligonucleotides (ASOs) may be used to modulate the expression of one or more genes in rAAV packaging and / or producer cell lines. Typically, ASOs are used to reduce the expression of one or more genes. Using known techniques and based on knowledge of the sequences of one or more genes to be modulated, ASO molecules may be designed to antagonize one or more genes by sequence homology-based targeting of the corresponding RNA. The ASO sequence may contain a nucleotide sequence that is complementary to the target portion of the mRNA or lncRNA produced from one or more genes. Such a portion may be 100% complementary to the target portion within the mRNA or lncRNA, but a lower level of complementarity (e.g., 90% or more or 95% or more) may also be used.

[0132] In some embodiments, the ASO may be an antisense RNA oligonucleotide in which at least one nucleoside linkage in the sequence is a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylenephosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, 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 in the antisense RNA oligonucleotide sequence is a phosphorothioate linkage. In some embodiments, all internucleoside linkages in the antisense RNA oligonucleotide sequence are phosphorothioate linkages.

[0133] CRISPR genome editing

[0134] In some embodiments, the packaging of rAAV and / or the coordination of gene expression in producer cell lines is performed using CRISPR genome editing. CRISPR genome editing typically comprises the following two distinct components: (1) guide RNA and (2) endonuclease, specifically a CRISPR-associated (Cas) nuclease (e.g., Cas9). The guide RNA is a combination of endogenous bacterial crRNA and tracer RNA into a single chimeric guide RNA (gRNA) transcript. Without being bound by theory, it is believed that when gRNA and Cas are expressed within a cell, the genomic target sequence may be modified or permanently disrupted.

[0135] The gRNA / Cas complex is recruited to the target sequence by base-pairing between the gRNA sequence and the complement to the target DNA sequence among the reduction genes (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, or NALCN-AS1). For successful binding of Cas, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. Binding of the gRNA / Cas complex orients Cas to the genomic target sequence of one or more of the genes of this disclosure, thereby allowing wild-type Cas to cleave both strands of DNA, causing a double-strand break. This can be repaired through one of the following two common repair pathways: (1) a non-homologous end-linking DNA repair pathway or (2) a homology-induced repair pathway. Non-homologous repair pathways can result in insertions or deletions at double-strand breaks, which can lead to frameshifts and / or immature stop codons that effectively disrupt the open reading frame of the target gene. Homologous-induced repair pathways require the presence of a repair template used to repair double-strand breaks.

[0136] Any suitable gRNA pair may be used for CRISPR genome editing. Typically, a 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, a gRNA pair is used to modulate (e.g., reduce or remove / knockout) the expression of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and / or NALCN-AS1.

[0137] gRNA pairs can be designed using known techniques and based on knowledge of the sequences of one or more genes to be tuned, typically using any suitable computer program available in the public. Knockout packaging and / or producer cells can be generated using any suitable technique using standard techniques known in the relevant technical field and suitable kits available in the market.

[0138] gRNA pairs can be delivered to the producer cell line of the present disclosure by any suitable means. Suitable techniques are known in the art and include the use of plasmids, viruses, and bacterial vectors to deliver gRNA pairs to the producer cell line. Typically, gRNA pairs are delivered using plasmid DNA.

[0139] gRNA pairs may 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 may also be used to modulate the expression of genes. In some embodiments described herein, 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 may be used to modulate the expression of KCNN2, LINC00319, RGMA, and SPANXN3. In some embodiments, the gRNA may be modified to improve editing efficiency by increasing binding to the target site and inhibiting nuclease degradation. In certain embodiments, this modification may be a linkage between a 2' O-methyl analog and a 3' phosphorothioate nucleotide at the terminal three nucleotides on both the 5' and 3' ends of the gRNA. Representative target DNA sequences targeted by a pair of gRNAs used to regulate the gene expression of one or more genes may include any one of nucleotide sequences selected from sequence identification numbers 16-31 listed in Table 2 below or variants thereof.

[0140] Table 2 : Representative target region sequences of gRNA pairs (sequence identification numbers: 12-15) and target DNA sequences (sequence identification numbers: 16-31)

[0141]

[0142]

[0143] For example, a gRNA pair used to target KCNN2 may include a sequence selected from the nucleotide sequences of Sequence Identification Nos. 12-15 (presented in Table 2). In some embodiments, a gRNA pair used to target KCNN2 includes a first gRNA molecule containing the sequence of Sequence Identification No. 12 and a second gRNA molecule containing the sequence of Sequence Identification No. 13. In some embodiments, a gRNA pair used to target KCNN2 includes a first gRNA molecule containing or having the sequence of Sequence Identification No. 14 and a second gRNA molecule containing or having the sequence of Sequence Identification No. 15.

[0144] In some embodiments, a gRNA molecule targeting KCNN2 is a 2' O-methyl analog comprising a 3' phosphorothioate nucleotide linkage at the terminal three nucleotides on either or both of its 5' and 3' ends, comprising the sequence of sequence identification number: 12, 13, 14 or 15.

[0145] A variant gRNA sequence may have at least 80% sequence identity with respect to the sequence of the present disclosure when measured over any suitable sequence length. Typically, percent sequence identity is determined over a certain length of consecutive nucleic acids. A variant gRNA sequence of the present disclosure may have at least 80% sequence identity with respect to the sequence of the present disclosure when measured over, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or more nucleic acid residues. In some embodiments, the variant gRNA molecule has at least 80% sequence identity with respect to the gRNA molecule of the present disclosure over the entire length of the variant gRNA molecule. In some embodiments, a variant gRNA molecule of the present disclosure may be one or more variants of gRNA molecules whose target region is complementary to one of the target sequences of SEQ ID NOs: 16 to 30. A pair of gRNAs of the present disclosure may comprise variants of one or both of a pair of two gRNA sequences targeting a gene selected from, for example, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2, and NALCN-AS1. For example, a variant of a gRNA pair comprising a first gRNA molecule comprising the sequence of sequence identification number: 12 and a second gRNA molecule comprising the sequence of sequence identification number: 13 may comprise 1) the first gRNA molecule comprising the variant of the sequence of sequence identification number: 12, 2) the second gRNA molecule comprising the variant of the sequence of sequence identification number: 13, or 3) both of these.

[0146] Regulation at the protein level

[0147] In another embodiment, the modulation of gene expression and / or activity is performed at the protein (e.g., polypeptide) level. For example, reduction of gene function at the protein level may be achieved by methods including, for example, targeting the protein using small molecules, peptides, aptamers, or destabilizing domains, or other methods capable of down-regulating the activity of the gene product or enhancing its degradation rate. Alternatively, the expressed protein may be modified so that its biological activity is reduced or eliminated through localized mutagenesis and / or the introduction of missense or nonsense mutations. In some embodiments, for example, a small molecule that binds to an active site and inhibits the function of the target protein may be introduced into the packaging and / or producer cells by adding, for example, to the cell culture medium. Alternatively, the function of the target protein may be modulated by introducing, for example, a peptide that interferes with protein-protein interactions into the cell (e.g., the packaging and / or producer cell) (Reference [Shangary et al., (2009) Annual Review of Pharmacology and Toxicology[Refer to 49:223]). Such peptides may be introduced into cells (e.g., packaging and / or producer cells) by, for example, transfection or electroporation, or through an expression construct. Alternatively, peptides may be introduced into cells (e.g., packaging and / or producer cells) by adding one or more residues to facilitate cell delivery (e.g., through conjugation) or by supercharging the molecule to enhance self-delivery. Techniques for expressing peptides include, but are not limited to, the fusion of the peptide into a scaffold or the binding of a signal sequence to stabilize or direct the peptide to a site or compartment of interest. In certain embodiments, the rAAV packaging and / or producer cell line comprises 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 using any of the methods mentioned above.

[0148] of one or more genes and / or proteins Effect of adjustment on expression

[0149] In certain embodiments, the modification method described in this disclosure may be used to produce rAAV packaging and / or producer cell lines that produce high rAAV titers. In certain embodiments, the modification method described in this disclosure may result in a significant reduction 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 reduction in the protein activity expressed by one or more genes (e.g., at least 5%, at least 10%, at least 20%, or a greater reduction). In certain embodiments, the expression of the target gene is 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%) is reduced.

[0150] In certain embodiments, the modulation method described herein may result in a significant reduction in the activity of a protein or RNA expressed by a target gene (e.g., ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1). For example, the method described herein may result in a reduction in the activity of a protein or RNA expressed by a target gene of at least 5%, at least 10%, at least 20%, or more. In certain embodiments, the target gene protein or RNA activity is 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%) is reduced. In addition, the modification of one or more genes may result in the modification of multiple genes (e.g., by miRNA).

[0151] In certain embodiments, the adjustment method described in this disclosure may result in a significant reduction (e.g., at least 5%, at least 10%, at least 20% or more) in the expression of gene products (e.g., gene products of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1). In certain embodiments, the expression of the gene product is 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%) is reduced.

[0152] In certain embodiments, the adjustment method described in this disclosure may result in a significant reduction (e.g., at least 5%, at least 10%, at least 20% or more) of the expression of a polypeptide or polyribonucleotide expressed 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 polyribonucleotide is 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% It is reduced 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%).

[0153] In certain embodiments, the adjustment method described in this disclosure may result in a significant reduction (e.g., at least 5%, at least 10%, at least 20% or more) of the activity of a polypeptide or polyribonucleotide expressed 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 polyribonucleotide is 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%) is reduced.

[0154] In certain embodiments, the expression and / or reduction in activity of one or more genes, proteins, or RNAs in the rAAV packaging and / or producer 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 more).

[0155] In certain embodiments, the reduction in expression and / or activity of one or more genes, proteins, or RNAs in rAAV packaging and / or producer cell lines is intended to be maintained indefinitely or permanently, for example, through the use of gene disruption or partial or complete gene deletion.

[0156] In certain embodiments, the expression and / or reduced activity of one or more genes, proteins, or RNAs in the rAAV packaging and / or producer cell line is maintained for 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 in the culture of the rAAV packaging and / or producer cell line.

[0157] rAAV production Effect of adjustment on

[0158] Modification of one or more genes and / or proteins in rAAV packaging and / or producer cell lines may result in an increase in rAAV titer. In some embodiments, the adjustment results in an increase in rAAV titers produced from rAAV packaging and / or producer cell lines, 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 It is increased 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 rAAV packaging and / or producer cell lines is increased by at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times or more. Any increase in rAAV titer attributable to the adjustment of one or more genes and / or proteins may be compared to the rAAV titer produced from a control parent cell line.

[0159] In some embodiments, the modification of one or more genes and / or proteins in rAAV packaging and / or producer cell lines 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 more.

[0160] rAAV Method of production

[0161] In certain embodiments, the present disclosure describes a method for producing rAAV from an rAAV package and / or producer cell line in which the expression of one or more genes, proteins, or non-coding RNAs is tuned. In certain embodiments, rAAV is produced by infecting cells of an rAAV producer cell line generated by delivering an rAAV vector to a tuned rAAV package cell line. In certain embodiments, rAAV is produced by infecting cells of an rAAV producer cell line in which the expression of one or more genes, proteins, or non-coding RNAs is tuned. In certain embodiments, the production of rAAV from the tuned rAAV package and / or producer cell line is enhanced compared to a control parent cell line.

[0162] 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 enables rAAV replication. In some embodiments, cells of the engineered producer cell line are infected with a helper virus (e.g., adenovirus (AV) or herpes simplex virus).

[0163] rAAV How to harvest

[0164] rAAV particles can be obtained by lysing cells from engineered rAAV packaging and / or producer cells. Lysation of engineered rAAV packaging and / or producer cells can be achieved by methods of chemically or enzymatically treating cells to release infectious virus particles. Such methods include the use of nucleases such as benzonase or DNAse, proteases such as trypsin, or detergents or surfactants. Physical disruption, such as homogenization or grinding, or the application of pressure through a microfluidizer pressure cell, or freeze-thaw cycles may also be used. In certain embodiments, lysates from engineered rAAV packaging and / or producer cells may be used to harvest rAAV particles.

[0165] In certain embodiments, cell culture supernatant can be collected from engineered rAAV packaging and / or producer cells without the need for cell lysis. In certain embodiments of the present disclosure, engineered rAAV packaging and / or producer cells secrete rAAV particles that can be collected from cell culture supernatant without the need for cell lysis. In certain embodiments, engineered rAAV packaging and / or producer cell lines have higher rAAV titers compared to control parent cell lines, and accordingly, more rAAV is harvested from engineered rAAV packaging and / or producer cell lines compared to control parent cell lines.

[0166] After harvesting rAAV particles, it may be necessary to purify the sample containing rAAV, for example, to remove cellular residues resulting from cell lysis. Minimal purification methods for AAV particles are known in the relevant art. Two representative purification methods are cesium chloride (CsCl)- and iodixanol-based density gradient purification. For both of the above methods, the literature [Strobel et al.It is described in [ , Human Gene Therapy Methods., 26(4): 147-157 (2015)]. Minimal purification may also be achieved, for example, using affinity chromatography with an AVB cepharose affinity resin (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Regarding AAV purification methods using AVB cepharose affinity resin, for example, see the literature [Wang et al. [This is described in Mol Ther Methods Clin Dev., 2:15040 (2015)]. After purification, rAAV particles are filtered and can be stored at ≤-60℃.

[0167] In certain embodiments, the present disclosure provides a method for harvesting rAAV particles produced from a engineered rAAV packaging cell line after the cells have been co-infected with two different adenoviruses.

[0168] In a specific embodiment, the present disclosure provides a method for harvesting rAAV particles produced after infection of an rAAV producer cell line generated from a engineered rAAV packaging cell line.

[0169] In a specific embodiment, the present disclosure provides a method for harvesting rAAV particles produced after infection of a engineered rAAV producer cell line with a helper virus.

[0170] rAAV Quantification of particles

[0171] Quantification of rAAV particles is complex due to the fact that AAV infection does not cause cytotoxic effects in vitro and, consequently, plaque assays cannot be used to determine infection titers. However, rAAV particles can be quantified using numerous methods, including quantitative polymerase chain reaction (qPCR) (reference [Clark et al., Hum. Gene Ther. 10, 1031-1039 (1999)]), dot-blot hybridization (reference [Samulski et al., J. Virol. 63, 3822-3828 (1989)]), and optical density of highly purified vector preparations (reference [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, California)). Samples containing rAAV particles can be incubated in the presence of DNase I (100 U / ml; Promega, Madison, Wisconsin) for 60 minutes at 37°C, followed by digestion of proteinase K (Invitrogen, Carlsbad, California) (10 U / ml) for 60 minutes at 50°C, and then denatured at 95°C for 30 minutes. The primer-probe set used must be specific to the non-natural portion of the rAAV vector genome, for example, the poly(A) sequence of the protein of interest. The PCR product can be amplified using any appropriate set of period parameters based on the length and composition of the primers, probes, and the sequence being amplified. Alternative protocols are, for example, [Lock et al. It is disclosed in Human Gene Therapy Methods 25(2): 115-125 (2014).

[0172] Viral genome amplification can also be measured using qPCR technology in a manner similar to that described above. However, to quantify total genome amplification within producer cells, only intracellular samples are collected, and the samples are not treated with DNase I to measure both packaged and unpackaged viral genomes. Viral genome amplification can be calculated on a per-host cell basis by simultaneously measuring host cell housekeeping genes, e.g., RNase P.

[0173] The infectivity of rAAV particles can be determined using the TCID50 assay (tissue culture infection dose at 50%), as described in the literature [Zhen et al., Human Gene Therapy 15:709-715 (2004)]. In this assay, rAAV vector particles are serially diluted and then used to co-infect Rep / Cap-expressing cell lines with AV particles in a 96-well plate. 48 hours after infection, total cellular DNA is extracted from the infected and control wells. Next, rAAV vector replication is measured using qPCR with transgene-specific probes and primers. TCID50 infectivity per milliliter (TCID50 / ml) is calculated by the Karber equation using the ratio of wells positive for AAV at 10-fold serial dilutions.

[0174] Treatment Applications

[0175] The engineered rAAV packaging and / or rAAV produced from producer cell lines described herein may be used, for example, in gene therapy in mammals. The engineered cells described herein may be used in in vitro and / or in vivo gene therapy applications. The engineered cells described herein may be used to deliver, for example, small molecules (e.g., siRNA or sgRNA), peptides, and / or proteins.

[0176] In some embodiments, rAAV produced from the engineered cell lines described herein may be used to treat a disease or disorder in human subjects in need. In certain embodiments, rAAV produced from the engineered cell lines described herein may be administered in conjunction with a pharmaceutically acceptable carrier.

[0177] Any suitable method or route may be used to administer rAAV or rAAV-containing compositions produced from the engineered packaging and / or producer cell lines described herein. Routes of administration include, for example, systemic, oral, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. In some embodiments, rAAV or compositions containing rAAV produced from the engineered packaging and / or producer cell lines are administered intravenously.

[0178] The practice of the present disclosure will be more fully understood from the electric examples provided herein solely for illustrative purposes, and should not be construed as limiting the present disclosure in any way.

[0179] Examples

[0180] Examples 1: Development of the Knockdown Protocol

[0181] By knocking down the housekeeping gene HPRT1, siRNA knockdown experiments were optimized and developed for 6-well and 24-well formats. Experiments performed in the 24-well format were evaluated based on numerous factors, such as seeding density, cell culture conditions (e.g., percentage of carbon dioxide (CO2), percentage of fetal bovine serum (FBS)), the ratio between the transfection product (Lipofectamine® RNAiMax) and siRNA ("ratio"), incubation time, and siRNA concentration. Commercially available siRNA designed for HPRT1 gene knockdown was used to optimize experimental conditions. HeLa producer cells were transfected with Lipofectamine® RNAiMax using variable concentrations of siRNA according to the manufacturer's instructions. The percentage reduction in HPRT1 expression was determined by real-time PCR. The optimized 24-well siRNA knockdown method was able to knock down the highly expressed HPRT1 gene by more than 80% compared to the baseline control. do 2A-D As presented in, 1 x 10 per well 5 Canine cells, a 1:5 ratio between transfection reactant and siRNA, and cells seeded with 8 nM siRNA showed the highest knockdown efficiency. do 2A This presents the effect of variable siRNA concentrations / ratios used on the percentage knockdown of HPRT1. do 2B This presents the effect of variable siRNA concentration / ratio on the percentage expression of HPRT1. For the optimization of the 6-well protocol, two different siRNA concentrations were tested. For the data plotted in Figures 2A and 2B, 5 x 10 4 Pieces, 8 x 10 4 Dogs and 1 x 10 5 The seeding density of dogs was tested. do 2CIt presents the effect of variable siRNA concentrations on the percentage knockdown of HPRT1. do 2D This presents the effect of variable siRNA concentrations on the percentage expression of HPRT1. All experiments were performed in triplicate.

[0182] Examples 2: RNA Sequencing Analysis

[0183] Eight 3-liter bioreactors were operated across two different HeLaS3 producer cell lines under supplemental and non-supplemental production conditions. As non-infectious controls, two additional bioreactors were operated without adenovirus 5 (Ad5). Details regarding bioreactor conditions and production levels are listed in Table 3 below.

[0184] Table 3 : Bioreactor conditions and production levels

[0185]

[0186] Abbreviations used in Table 3: Addition of one or more supplements is indicated by (+); absence of one or more supplements is indicated by (-); MOI - Infection multiplicity.

[0187] Thirty hours after infection with Ad5, samples were retrieved for RNA-Seq. After washing the samples once with PBS, the cell pellets were stored at -80°C until ready for transport. RNA extraction and cDNA synthesis of the extracted RNA were performed using 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, the Ad5 genome, and the AAV2 genome using mapping methods well known in the art. All reads mapped to the Ad5 genome were discarded. Another round of sequencing was performed to augment the reads mapped to the human genome. Differential analysis was performed using the data generated by RNA sequencing (see Table 4).

[0188] Table 4 : Differential analysis

[0189]

[0190] * PCL1- Producer cell line 1; PCL2- Producer cell line 2; No production- Non-infected control cells; Production (NS)- Ad5 infected cells cultured under non-supplementary conditions; Production (S)- Ad5 infected cells cultured under supplementary conditions.

[0191] In this embodiment, differential expression analysis was calculated as the log-fold change (LogFC) in mRNA concentration of the experimental condition compared to the control condition. Upregulated genes were represented by positive LogFC, and downregulated genes were represented by negative LogFC. Differentially expressed genes with a p-value ≤ 0.05 were considered statistically significant. Within each differential analysis, hundreds to thousands of genes were significantly upregulated or downregulated. Filter criteria were established (e.g., Fig. 6 By applying the reference, the dataset was reduced to a manageable number of genes for evaluation. The gene set was aligned and transferred based on the filter criteria as described in Example 6.

[0192] Examples 3: RT- to qPCR From RNA sequence analysis by obtained Evaluation of results

[0193] A small gene set was selected for evaluating RNA sequencing data. RNA-Seq results were verified using RT-qPCR, following methods widely known in the relevant technical field. The ΔΔCt method was used to analyze the data. Trends observed in the RNA-Seq data were independently confirmed using RT-qPCR. do 3A-B is PGA5 ( Fig. 3A ) and SPANXN3 ( Fig. 3B Presents the log-fold change in gene expression obtained from bioinformatics analysis of RNA-Seq data for ). The x-axis represents the conditions under which the producer cell line was grown (Supplementary (Differential Analysis #5 as described in Table 4) vs. Non-Supplementary (Differential Analysis #1 as described in Table 4)), and the y-axis represents the log-fold change in gene expression (LogFC). PGA5 in cells cultured in non-supplemented cell culture medium ( Fig. 3A ) and SPANXN3 ( Fig. 3B The log-fold change in ) expression was plotted against the corresponding gene expression in non-infected cells (cells not infected with helper virus). PGA5 in cells cultured in supplemented cell culture medium ( Fig. 3A ) and SPANXN3 ( Fig. 3B The log-fold change in expression was plotted against the corresponding gene expression in cells cultured in unsupplemented cell culture medium.

[0194] PGA5 and SPANXN3 gene expression in producer cell lines grown under supplemented and non-supplemented conditions was also evaluated by RT-qPCR using methods widely known in the relevant technical field. Fig. 3C-D PGA5 in cells cultured in unsupplemented and supplemented cell culture media compared to non-infected cells (cells not infected with helper viruses) Fig. 3C ) and SPANXN3 ( Figure 3D Presents the RT-qPCR multiple change values ​​of ) expression. do 3A-D It suggests that data obtained from qPCR and RNA sequencing follow the same trend.

[0195] Examples 4: RT- in different producer cell line clones to qPCR From RNA sequence analysis by obtained Evaluation of results

[0196] The results of RNA sequencing analysis were further evaluated by conducting RT-qPCR experiments on RNA extracted from different clones of the HeLa S3 producer cell line. do 4A-B PGA5 in producer cell line clones cultured in unsupplemented and supplemented cell culture media relative to non-infected cells (cells not infected with helper virus) when determined by RT-qPCR ( Fig. 4A ) and SPANXN3 ( Fig. 4B ) presents the multiple change values ​​of expression. 21C5, 3C6, and 2B6 represent different clones of the HeLa producer cell line. do 4C-D PGA5 in producer cell line clones 21C5, 3C6, and 2B6 cultured in supplemental cell culture medium compared to clones cultured in non-supplemental cell culture medium ( Fig. 4C ) and SPANXN3 ( Fig. 4D) suggests a relative multiple increase in expression. These results are further confirmed by the bioinformatics RNA sequencing and RT-qPCR data described in Example 3.

[0197] Examples 5: rAAV At the station The effect of gene knockdown on Korea

[0198] Knockdown experiments were performed by individually knocking down genes in HeLa producer cell lines based on the optimized protocol discussed in Example 1. SiRNA nucleotide sequences were designed for each gene (see Table 1).

[0199] 1 x 10⁶ accompanied by 8 nM siRNA and a siRNA:RNAiMAX ratio of 1:5 5 Conditions were established with a seeding density of [number]. AAV production was induced 24 hours after a decrease in gene expression, and rAAV was harvested 72 hours after infection. Titrations were determined for each sample and compared to a non-targeted missense siRNA control. This experiment was performed independently three times, and after averaging the results, statistical analysis was conducted. do 5A-5C This presents the siRNA results for individual genes in producer cell lines 1-3, respectively, for absolute rAAV titer (GC / mL; GC = genome copy). do 5D-5F It presents a multiple increase in rAAV titers by siRNA of individual genes in different producer cell lines 1-3.

[0200] Figs. 5A-5FAs shown in [the text], a decrease in the expression of KCNN2, LINC00319, RGMA, or SPANXN3 in producer cell lines resulted in statistically significant 2-4-fold higher rAAV titers compared to missense controls. Across all three producer cell lines, these four genes demonstrate a statistically significant positive effect on titers when knocked down. These results indicate that these genes serve as excellent targets for more permanent modifications, such as CRISPR / Cas9 knockout.

[0201] Examples 6: Gene Filtering Methodology

[0202] For Filter 1, genes from Differential Analyses 1 and 7 (as listed in Table 3) were aligned. Differential Analyses 1 and 7 defined genes that were upregulated or downregulated upon the addition of adenovirus 5 under non-supplementary conditions. Analysis 1 examined cells from the 21C5 producer cell line (producer cell line 1, PCL1). Analysis 7 examined cells from 2B6 (producer cell line 2, PCL2). The gene list following this Filter 1 identified genes that were not cell line-specific, and this alignment provided a total of 9,149 genes common to the two producer cell lines.

[0203] For Filter 2, genes from Filter 1 were aligned with the genes presented in Differential Analysis 5. Analysis 5 investigated genes that were upregulated and downregulated in cells from the 21C5 producer cell line (PCL1) under supplementary conditions compared to non-supplementary conditions. The purpose of this differential analysis was to limit the effect of production under supplementary conditions compared to production under non-supplementary conditions. The purpose of aligning the set of genes from Filter 1 with Differential Analysis 5 was to identify genes under improved productivity conditions that 1) are not byproducts of the improved production conditions and 2) are potentially relevant to two different cell lines. After alignment, 374 genes were forward-transferred.

[0204] For Filter 3, only genes with large LogFc thresholds of >2 LogFC ± were forward transposed. This was done to confirm high levels of up / down regulation in the genes being forward transposed and to provide confidence that the selected genes were not RNA-Seq products. After filtering, 77 genes were forward transposed.

[0205] For Filter 4, only genes exhibiting either up-regulation in Differential Analysis 1 and Differential Analysis 5, or down-regulation in both Differential Analysis 1 and Differential Analysis 5, were retained. For example, among the 77 genes, one had to exhibit up-regulation from Differential Analysis 1 and additional up-regulation in Differential Analysis 5, or down-regulation from Differential Analysis 1 and additional down-regulation in Differential Analysis 5. The purpose of this filter was to confirm that for the genes being evaluated, high titer conditions did not have an antagonistic effect on the regulation of that specific gene compared to low titer conditions. After Filter 11, the genes were retained for evaluation. An exemplary flowchart presenting a representative gene filtering methodology is provided. Fig. 6 It is presented in (abbreviation used: LogFC = Log multiple change).

[0206] Table 5 below provides Log2FC data from each comparison during the process of filtering genes important for productivity.

[0207] Table 5 : Log2FC data

[0208]

[0209] Examples 7: KCNN2's Gene Knockout

[0210] In this embodiment, two existing highly optimized monoclonal HeLa producer cell lines (PCL) - 2H5 and 7B12 - were genetically modified so that the KCNN2 gene (previously identified in the RNA-seq screen described herein), which codes for the calcium-activated potassium channel protein SK2, is knocked out.

[0211] KCNN2 was knocked out in 2H5 or 7B12 HeLa cells using an eGFP selectable marker. The putative KCNN2 knockout was enriched with eGFP expression and seeded into 96-well plates. After allowing cell colonies to form and harvesting genomic DNA, 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 high knockout potential were scaled up for further testing.

[0212] The best clones were transferred to serum-free suspension cultures. Clonal productivity was evaluated relative to the parent cell line through 24-well deep rAAV production. Clones were 2 x 10 5Canine cells were seeded in 3 mL of culture at a rate of 1 / mL and infected with Ad5 at an infection multiplicity (MOI) of 50. Four days after infection, rAAV was harvested and evaluated for titer. The multiple increase in titer was normalized relative to the maternal control. The highest clones showed a 1.5–2.7-fold increase in titer compared to the control samples. The 2H5 titer was 2.46 x 10⁶ 9 - 4.98 x 10 10 It was in the GC / mL range ( do 7A When titers were normalized relative to the maternal control group, the multiple increase was observed to be in the range of 1.2–2.7 times ( do 7B The titer of 7B12 is 4.33 x 10 8 - 1.88 x 10 10 It was in the GC / mL range ( do 7C When titers were normalized relative to the maternal control group, the multiple increase was observed to be in the range of 1.5–2.6 times ( do 7D ). Next, clones showing at least a 1.5-fold increase were scaled into shaking flask cultures and seeded into ambr® 15 for the production of supplemented rAAV at high seeding densities. Cells were 1.5 x 10⁶ 6 Cells were seeded at cell / mL and infected with Ad5 at an MOI of 50. rAAV was harvested 4 days after infection and titers were evaluated. The multiple increase in titer was normalized relative to the maternal control. The highest clones showed a 1.5–2.3-fold increase in titer compared to the control samples. The 2H5 titer was 1.5 x 10⁶ 11 - 3.82 x 10 11 It was in the GC / mL range ( do 8A When the titer was normalized relative to the maternal control, the multiple increase was observed to be in the range of 1.3–2.3 times ( do 8B The titer of 7B12 is 2.62 x 10 10 - 1.35 x 10 11It was in the GC / mL range ( do 8C When the titer was normalized relative to the maternal control, the fold increase was in the range of 1.2–1.5 times ( Fig. 8D ).

[0213] These data demonstrate that reducing or removing the expression of one or more of the genes described herein (e.g., through gene knockout) in AAV-producing cells can be used to increase the production of rAAV from engineered cells.

[0214] Examples 8: Multi-combination siRNA Knockdown

[0215] In this embodiment, by performing multiple-combination knockdown of previously identified genes in the RNA-seq screen described herein using siRNA, it was determined whether simultaneously targeting multiple genes produces an additional effect on potency.

[0216] Multiple-combination knockdown was performed using a modification of the method described in Example 5. Briefly, cells were transfected using 8 nM of each siRNA and maintaining an siRNA:RNAiMAX ratio of 1:5. AAV production was induced 24 hours after a decrease in gene expression, and rAAV was harvested 72 hours after infection. Titrates were determined for each sample and compared to a non-targeted missense siRNA control.

[0217] In this example, KCNN2 was knocked down in combination with other previously described siRNA panels. Additionally, RGMA and SPANXN3 were knocked down in combination with each other. In 2H5, the combination knockdown showed a titer increase ranging from 4.6 to 11.4-fold compared to the missense control ( do 9A). In 7B12, combination knockdown showed a potency increase in the range of 3.4–9.7 times compared to the missense control group ( do 9B Although all combinations showed an increase in potency; not all combinations were an improvement compared to knocking down KCNN2 alone. KCNN2 knockdown increased 5.3-fold in 2H5 ( Fig. 9A As ), and in 7B12 increased 5.1 times ( Fig. 9B It led to ).

[0218] These data demonstrate that an additional increase in rAAV production can be obtained through the direct targeting of multiple genomic regions in known high-rAAV titer producing monoclonal PCLs.

[0219] Numbered embodiments

[0220] 1. Recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines 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 parent cells.

[0221] 2. The packaging and / or producer cell line comprising cells in which the expression of KCNN2, LINC00319, RGMA, and SPANXN3 is reduced compared to control parent cells in Embodiment 1.

[0222] 3. A packaging and / or producer cell line in which expression is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), or antisense RNA oligonucleotide (ASO) in Embodiment 1 or 2.

[0223] 4. A packaging and / or producer cell line in which, in any one of embodiments 1-3, expression is reduced using siRNA comprising a nucleotide sequence selected from any one of sequence identification numbers: 1-11.

[0224] 5. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of ATP5EP2 is reduced, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 1 in the sense strand and the nucleotide sequence of Sequence Identification No. 32 in the anti-sense strand.

[0225] 6. A packaging and / or producer cell line in which 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 anti-sense strand.

[0226] 7. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of CYP3A7 is reduced, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 3 in the sense strand and the nucleotide sequence of Sequence Identification No. 34 in the anti-sense strand.

[0227] 8. A packaging and / or producer cell line in which the expression of NOG is reduced in Embodiment 4, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 4 in the sense strand and the nucleotide sequence of Sequence Identification No. 35 in the anti-sense strand.

[0228] 9. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of SPANXN3 is reduced, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 5 in the sense strand and the nucleotide sequence of Sequence Identification No. 36 in the anti-sense strand.

[0229] 10. A packaging and / or producer cell line in which the expression of MYRIP is reduced in the embodiment 4, and the siRNA comprises the nucleotide sequence of sequence identification number: 6 in the sense strand and the nucleotide sequence of sequence identification number: 37 in the anti-sense strand.

[0230] 11. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of KCNN2 is reduced, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 7 in the sense strand and the nucleotide sequence of Sequence Identification No. 38 in the anti-sense strand.

[0231] 12. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of NALCN-AS1 is reduced, and the siRNA comprises the nucleotide sequence of Sequence Identification No. 8 in the sense strand and the nucleotide sequence of Sequence Identification No. 39 in the anti-sense strand.

[0232] 13. A packaging and / or producer cell line according to Embodiment 4, wherein the expression of RGMA is reduced and the siRNA comprises the nucleotide sequence of Sequence Identification No. 9 in the sense strand and the nucleotide sequence of Sequence Identification No. 40 in the anti-sense strand.

[0233] 14. In embodiment 4, the expression of PGA5 is reduced, and the siRNA comprises the sequence of sequence identification number: 10 in the sense strand and the sequence of sequence identification number: 41 in the anti-sense strand, packaging and / or producer cell line.

[0234] 15. In embodiment 4, the expression of ABCA10 is reduced, and the siRNA comprises the sequence of sequence identification number: 11 in the sense strand and the sequence of sequence identification number: 42 in the anti-sense strand, packaging and / or producer cell line.

[0235] 16. A packaging and / or producer cell line in which the nuclease of Embodiment 3 is selected from the group consisting of zinc finger nuclease (ZFN), meganuclease, transcription activator-like effector nuclease (TALEN), or short palindromic repeat of clustering regular intervals (CRISPR) associated protein.

[0236] 17. A packaging and / or producer cell line in which expression is reduced using CRISPR genome editing in any one of embodiments 1-16.

[0237] 18. In Embodiment 17, expression is reduced using a guide RNA pair, wherein each guide RNA is

[0238] (a) Sequence identification number: Includes a sequence selected from the nucleotide sequence of 12-15, and / or,

[0239] (b) Targeting a target DNA sequence selected from any one of the nucleotide sequences of sequence identification number: 16-31

[0240] Packaging and / or producer cell lines.

[0241] 19. A packaging and / or producer cell line according to embodiment 18, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule comprising the sequence of sequence identification number: 12 and a second gRNA molecule comprising the sequence of sequence identification number: 13.

[0242] 20. A packaging and / or producer cell line according to embodiment 18, wherein the gRNA pair is used to target KCNN2 and comprises a first gRNA molecule comprising the sequence of sequence identification number: 14 and a second gRNA molecule comprising the sequence of sequence identification number: 15.

[0243] 21. Packaging and / or producer cell line in embodiment 19 or 20, wherein each gRNA molecule is a 2' O-methyl analog comprising a 3' phosphorothioate nucleotide linkage at the terminal three nucleotides on either or both of its 5' and 3' ends.

[0244] 22. A packaging and / or producer cell line in which gene expression is eliminated compared to a control parent cell in any one of embodiments 1-21.

[0245] 23. In any one of embodiments 1-22, the packaging and / or producer cell line is a human cell line.

[0246] 24. In embodiment 23, the packaging and / or producer cell line is a human cell line that is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

[0247] 25. In any one of embodiments 1-24, the cell line is an rAAV packaging cell line.

[0248] 26. In any one of embodiments 1-24, the cell line is an rAAV producer cell line.

[0249] 27. A cell line according to Embodiment 26 in which the rAAV titer is increased by about 1.5 to about 7 times compared to the rAAV titer produced from a cell line containing control parent cells.

[0250] 28. Lysate of a cell line according to any one of embodiments 1-27.

[0251] 29. Cell culture supernatant from a cell line according to any one of embodiments 1-27.

[0252] 30. A method for producing a producer cell line comprising delivering a recombinant adeno-associated virus (rAAV) vector to cells of a packaging cell line according to embodiment 25.

[0253] 31. A method for producing rAAV, comprising infecting cells of a producer cell line produced by the method according to embodiment 30 with a helper virus.

[0254] 32. A method for producing rAAV, comprising infecting cells of a producer cell line according to embodiment 26 with a helper virus.

[0255] 33. A method according to embodiment 31 or 32, wherein rAAV is harvested from a producer cell line.

[0256] 34. A method in which, in any one of embodiments 31-33, the production of rAAV is enhanced compared to a control parent cell line.

[0257] 35. A method for identifying one or more genes associated with the production of rAAV, comprising the following:

[0258] Adding one or more supplements that increase rAAV titer in cell lines;

[0259] Measuring total gene expression for the transcriptome in supplemented and non-supplemented cell lines;

[0260] Obtaining a list of genes that are differentially expressed between supplemental and non-supplemental cell lines; and

[0261] Identifying one or more genes associated with the production of rAAV.

[0262] 36. A method according to embodiment 35, wherein one or more supplements added to the cell line comprise dexamethasone, hydrocortisone, prednisolone, methylprednisolone, betamethasone, cortisone, prednisone, budesonide, or triamcinolone.

[0263] 37. A method for producing rAAV packaging and / or producer cell lines that promote increased production of rAAV, comprising adjusting the expression of one or more genes identified using the method according to Embodiment 35.

[0264] 38. A method in any one of embodiments 35-37 in which the cell line is an rAAV packaging cell line.

[0265] 39. A method in any one of embodiments 35-37 in which the cell line is an rAAV producer cell line.

[0266] 40. A method according to Embodiment 39 in which the rAAV producer cell line increases the rAAV titer by at least 1.5 times compared to the rAAV titer produced by an rAAV producer cell line without the adjustment of one or more corresponding gene expressions.

[0267] 41. A method in any one of embodiments 37-40, wherein adjusting expression includes reducing the expression of one or more genes.

[0268] 42. A method in any one of embodiments 37-40, wherein adjusting expression includes removing one or more gene expressions.

[0269] 43. A method in any one of embodiments 30-42 in which the cell line is a human cell line.

[0270] 44. The method of embodiment 43, wherein the human cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

[0271] 45. Recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines 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 compared to corresponding non-modified parent cells.

[0272] 46. ​​rAAV packaging and / or producer cell line in which 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.

[0273] 47. rAAV packaging and / or producer cell line according to embodiment 46, wherein the cell line is engineered to include a genetic disruption or partial or complete genetic deletion in at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1.

[0274] 48. rAAV packaging and / or producer cell line according to embodiment 47, wherein the cell line is engineered to include genetic disruption of at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and / or NALCN-AS1.

[0275] 49. rAAV packaging and / or producer cell line according to embodiment 47, wherein the cell line is engineered to include genetic disruption in at least two genes selected from ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1.

[0276] 50. rAAV packaging and / or producer cell line according to embodiment 47, wherein the cell line is engineered to include 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.

[0277] 51. rAAV packaging and / or producer cell line according to embodiment 47, wherein the cell line is engineered to include 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.

[0278] 52. Recombinant adeno-associated virus (rAAV) packaging and / or producer cell lines exhibiting reduced expression and / or activity of polypeptides or polyribonucleotides expressed from at least one of ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, RGMA, SPANXN3, PGA5, MYRIP, KCNN2 and NALCN-AS1 compared to the corresponding parent cell line.

[0279] Include as a reference

[0280] The full disclosures of each patent document and scientific paper referred to herein are incorporated by reference for all purposes.

[0281] Equivalents

[0282] The present invention may be embodied in other specific forms without departing from its spirit or essential features. Accordingly, the above embodiments should be regarded as illustrative rather than limiting the invention as described herein in all respects. Accordingly, the scope of the invention is defined by the appended claims rather than by the above description, and all variations falling within the meaning and scope of the claim equivalents are to be encompassed therein.

Claims

Claim 1 a) reduced expression of potassium calcium-activated channel subfamily N member 2 (KCNN2) compared to the corresponding non-transformed maternal cells, or reduced expression of KCNN2 and one or more genes selected from the group consisting of repulsion-inducing molecule BMP co-receptor A (RGMA), ATP synthase F1 subunit epsilon pseudogene 2 (ATP5EP2), long intergene non-protein coding RNA 319 (LINC00319), cytochrome P450 family 3 subfamily A member 7 (CYP3A7), ATP binding cassette subfamily A member 10 (ABCA10), Noggin (NOG), SPANX family member N3 (SPANXN3), pepsinogen A5 (PGA5), myosin VIIA and Rab interacting protein (MYRIP) and NALCN antisense RNA 1 (NALCN-AS1); and / or b) a recombinant adeno-associated virus (rAAV) package and / or producer cell line comprising a plurality of engineered cells having reduced activity of a gene product expressed from KCNN2 compared to a corresponding non-modified parent cell, or one or more genes selected from the group consisting of RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and NALCN-AS1 and reduced activity of a gene product expressed from KCNN2. Claim 2 rAAV packaging and / or producer cell lines in which: a) the expression of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and / or NALCN-AS1 is reduced indefinitely or permanently and / or; or b) the activity of gene products expressed from KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and / or NALCN-AS1 is reduced indefinitely or permanently. Claim 3 rAAV packaging and / or producer cell lines according to paragraph 2, wherein a plurality of engineered cells comprise a genetic disruption or genetic deletion in at least one of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and / or NALCN-AS1. Claim 4 In paragraph 3, an rAAV packaging and / or producer cell line wherein a plurality of engineered cells comprises genetic disruption of at least one of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and / or NALCN-AS1. Claim 5 In paragraph 3, an rAAV packaging and / or producer cell line in which a plurality of engineered cells comprise genetic disruption of at least two genes selected from KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1. Claim 6 In paragraph 3, an rAAV packaging and / or producer cell line wherein a plurality of engineered cells comprise a gene deletion in at least one of KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP and / or NALCN-AS1. Claim 7 rAAV packaging and / or producer cell lines, wherein a plurality of engineered cells comprise gene deletions in at least two genes selected from KCNN2, RGMA, ATP5EP2, LINC00319, CYP3A7, ABCA10, NOG, SPANXN3, PGA5, MYRIP, and NALCN-AS1. Claim 8 rAAV packaging and / or producer cell lines in which expression and / or activity is reduced using clustered regularly spaced short palindromic repeat (CRISPR) genome editing. Claim 9 In claim 8, the CRISPR genome editing uses a guide RNA (gRNA) pair, wherein the gRNA pair comprises: (a) a sequence selected from the nucleotide sequence of sequence identification numbers: 12-15 and / or (b) a target DNA sequence selected from any one of the nucleotide sequences of sequence identification numbers: 16-31, rAAV packaging and / or producer cell line. Claim 10 In claim 9: a) a gRNA pair used to target KCNN2 in a plurality of cells, 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; or b) a gRNA pair used to target KCNN2 in a plurality of cells, comprising 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, wherein the rAAV packaging and / or producer cell line. Claim 11 In claim 10, rAAV packaging and / or producer cell lines in which each gRNA molecule is a 2' O-methyl analog comprising a 3' phosphorothioate internucleotide linkage on the terminal three nucleotides on either or both of its 5' and 3' ends. Claim 12 Reduced expression and / or activity of polypeptides or polyribonucleotides expressed from potassium calcium-activated channel subfamily N member 2 (KCNN2) compared to the corresponding parental cell line, or one or more genes selected from the group consisting of the repulsion-inducing molecule BMP co-receptor A (RGMA), ATP synthase F1 subunit epsilon pseudogene 2 (ATP5EP2), long intergene non-protein coding RNA 319 (LINC00319), cytochrome P450 family 3 subfamily A member 7 (CYP3A7), ATP binding cassette subfamily A member 10 (ABCA10), Noggin (NOG), SPANX family member N3 (SPANXN3), pepsinogen A5 (PGA5), myosin VIIA and Rab interacting protein (MYRIP), and NALCN antisense RNA 1 (NALCN-AS1), and polypeptides expressed from KCNN2 or a recombinant adeno-associated virus (rAAV) package and / or producer cell line comprising a plurality of engineered cells exhibiting reduced expression and / or activity of polyribonucleotides. Claim 13 In paragraph 12, rAAV packaging and / or producer cell lines in which expression and / or activity is reduced using a nuclease, double-stranded RNA (dsRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA) or antisense RNA oligonucleotide (ASO). Claim 14 In paragraph 13: (a) the expression and / or activity of ATP5EP2 in multiple cells is reduced using siRNA, wherein 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 anti-sense strand; and (b) the expression and / or activity of LINC00319 in multiple cells is reduced using siRNA, wherein 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 anti-sense strand; and (c) the expression and / or activity of CYP3A7 in multiple cells is reduced using siRNA, wherein 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 anti-sense strand; (d) the expression and / or activity of NOG in multiple cells is reduced using siRNA, wherein 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 anti-sense strand and / or; (e) the expression and / or activity of SPANXN3 in multiple cells is reduced using siRNA, wherein 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 anti-sense strand and / or; (f) the expression and / or activity of MYRIP in multiple cells is reduced using siRNA, wherein 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 anti-sense strand and / or; (g) Expression and / or activity of KCNN2 in multiple cells is reduced using siRNA, wherein 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 anti-sense strand;(h) the expression and / or activity of NALCN-AS1 in multiple cells is reduced using siRNA, wherein the siRNA contains the nucleotide sequence of SEQ ID No. 8 in the sense strand and the nucleotide sequence of SEQ ID No. 39 in the anti-sense strand and / or; (i) the expression and / or activity of RGMA in multiple cells is reduced using siRNA, wherein the siRNA contains the nucleotide sequence of SEQ ID No. 9 in the sense strand and the nucleotide sequence of SEQ ID No. 40 in the anti-sense strand and / or; (j) the expression and / or activity of PGA5 in multiple cells is reduced using siRNA, wherein the siRNA contains the sequence of SEQ ID No. 10 in the sense strand and the sequence of SEQ ID No. 41 in the anti-sense strand and / or; (k) rAAV packaging and / or producer cell lines in which the expression and / or activity of ABCA10 in multiple cells is reduced using siRNA, wherein the siRNA contains the sequence of SEQ ID NO: 11 in the sense strand and the sequence of SEQ ID NO: 42 in the anti-sense strand.; Claim 15 In paragraph 1, the cell line is a human cell line rAAV packaging and / or producer cell line. Claim 16 In paragraph 15, the human cell line is a rAAV packaging and / or producer cell line that is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line. Claim 17 rAAV packaging and / or lysate of a producer cell line according to any one of claims 1 to 16. Claim 18 rAAV packaging and / or cell culture supernatant of a producer cell line according to any one of claims 1 to 16. Claim 19 A method for producing a cell lysate, comprising growing a plurality of cells of the rAAV packaging and / or producer cell line of any one of claims 1 to 16 in a culture, lysing the cells to obtain a cell lysate, and then harvesting the cell lysate. Claim 20 A method for obtaining a cell culture supernatant, comprising growing a plurality of cells of the rAAV packaging and / or producer cell line of any one of claims 1 to 16 in a culture, and harvesting the supernatant therefrom. 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