Modified mammalian cells for improved production

Abstract

Provided herein are engineered or modified cell lines in which the expression of at least one of ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, HOXA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, PARD6G-AS1, ACTA1, ACTC1, ADCY10, ADCY4, ADORA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLD1, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, and YY1 is altered as compared to a control cell line. Also provided are methods of producing recombinant AAV particles in said engineered or modified cell lines.

Description

[0001] MODIFIED MAMMALIAN CELLS FOR IMPROVED PRODUCTION CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial No.

[0002] 63 / 711,825, filed October 25, 2024, 63 / 879,099, filed September 10, 2025, and 63 / 711,808, filed October 25, 2024, the contents of which are hereby incorporated by reference in their entireties.

[0003] BACKGROUND

[0004] Adeno-associated virus (AAV) is a DNA parvovirus that infects humans and various other animal species. AAV contains a single-stranded DNA genome encoding sets of replication (Rep) and capsid (Cap) proteins. Flanking the Rep and Cap open reading frames at the 5’ and 3’ ends are inverted terminal repeat sequences (ITRs), which act as origins of nucleic acid replication and as packaging signals for the virus. Recombinant AAV (rAAV)-based vectors are one of the most promising vehicles for human gene therapy. rAAV vectors are under consideration for a wide variety of gene therapy applications. In particular, rAAV vectors can deliver therapeutic genes to dividing and nondividing cells, and these genes can persist for extended periods without integrating into the genome of the target cells.

[0005] Although rAAV manufacturing has improved over the years, several issues remain. Development of rAAV-based therapies at the preclinical stage require large amounts of rAAV vectors to complete studies such as toxicology, biodistribution, and expression studies prior to clinical studies in human patients. However, current rAAV production results in lower than desirable titer yields. Therefore, there remains a need for more efficient rAAV production with higher viral titer for preclinical and clinical applications.

[0006] SUMMARY OF THE INVENTION

[0007] In one aspect, provided herein is a engineered cell line in which the expression and / or activity of at least one of ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, PARD6G-AS1, RP11-268J15.6, andACTAl, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP IB, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2 is altered as compared to a control cell line.

[0008] In certain embodiments, the expression of at least one of the transcription factor genes listed in Tables 1-4 is altered as compared to a control cell line. In certain embodiments, the expression of at least one of the transcription factor target genes listed in Tables 1-4 is altered as compared to a control cell line.

[0009] In certain embodiments, the expression of at least one gene is reduced and / or expression of at least one gene is increased, as compared to the control cell line.

[0010] In certain embodiments, the cell line is modified by a modulator that increases or decreases the expression and / or activity of at least one gene.

[0011] In certain embodiments, the cell line comprises a genetic modification.

[0012] In certain embodiments, the modulator is a nuclease. In certain embodiments, the nuclease is selected from the group consisting of zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0013] In certain embodiments, the modulator is a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

[0014] In certain embodiments, the modulator comprises a template for homologous arm mediated recombination.

[0015] In certain embodiments, the modulator comprises an inhibitor of at least one gene selected from the group consisting of: KAT5, DBH, ITGAM, HHEX, HDAC5, SSI 8, FGFR3, RIGI / DDX58, H0XA5, FOXL2, ITGA2B, TNFAIP3, THEM6, HSP90B 1, EHHADH, POLN, CHD1, NFKBIA, RELB, RXRA, CDKN1A, NFKBland ISYNA1. In certain embodiments, the inhibitor comprises a synthetic small molecule inhibitor.

[0016] In certain embodiments, the modulator comprises an agonist of at least one gene selected from the group consisting of: GATA1, LIF, ASNS, RPA1, TCF7, ADM2, NFKB2, BCL2, NME1, NR3C1, NGEF, GJB3, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1. In certain embodiments, the agonist comprises a synthetic small molecule agonist.

[0017] In certain embodiments, the cell line is a mammalian cell line or an insect cell line. In certain embodiments, the mammalian cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or HEK293 cell line. In certain embodiments, the mammalian cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line. In certain embodiments, the insect cell line is an Sf9 cell line or a Hi5 cell line.

[0018] In certain embodiments, the cell line is a modified or engineered host cell line for production of viral particles. In certain embodiments, the cell line is a producer cell line. In certain embodiments, the producer cell line further comprises stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest.

[0019] In certain embodiments, the producer cell line further comprises AAV helper function encoding sequences of an AAV helper virus. In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the AAV helper virus is a herpes simplex virus (HSV).

[0020] In certain embodiments, the AAV helper function encoding sequences comprise at least one of Ela, Elb, E2A, L4, E4, and VA.

[0021] In certain embodiments, the titer of AAV produced from the engineered or modified cell line is at least about 1.5-fold higher compared to the titer of AAV produced from a cell line in which the expression of the gene is not altered.

[0022] In another aspect, provided herein is a method for identifying a gene associated with increasing the production of recombinant AAV (rAAV) particles from a cell, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, F0XL2, GATA1, HDAC5, HOXA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, andPARD6G-ASl, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADC Y 10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

[0023] In certain embodiments, the method further comprises determining the titer of rAAV particles expressed from the cell and comparing the titer to a reference rAAV titer, wherein the reference rAAV titer is that of rAAV particles expressed from a cell that has not been cultured in the presence of the modulator but is otherwise identical.

[0024] In certain embodiments, if the titer of rAAV is increased compared to the reference rAAV titer, the gene is identified as a target for knock-down or knock-out in the cell. In certain embodiments, if the titer of rAAV particles is decreased compared to the reference rAAV titer, the gene is identified a target for over-expression in the cell.

[0025] In an aspect, provided herein is a method of modulating gene expression in a cell line for increasing the production of rAAV particles from a cell, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of: ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS IL, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, and PARD6G-AS1, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

[0026] In certain embodiments, the modulator is DMSO, DMF, NMP, or dihydrolevoglucosenone (Cyrene). In certain embodiments, the concentration of DMSO is at least about 0.5%.

[0027] In another aspect, provided herein is a method for increasing the production of recombinant AAV particles, wherein the method comprises culturing a cell in the presence of a modulator, wherein the expression or activity of at least one of the following genes : DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SS18, THEM6, and TNFAIP3 is reduced as compared to a control cell line, and / or in which the expression of at least one of ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1 is increased as compared to a control cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

[0028] In certain embodiments, the method increases recombinant viral titer by at least 1.5-fold compared to a method comprising a control cell. In certain embodiments, the modulator comprises a synthetic small molecule inhibitor of at least one of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SS18, THEM6, and TNFAIP3.

[0029] In certain embodiments, the modulator comprises a synthetic small molecule agonist of at least one gene selected from the group consisting of: ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YYL

[0030] In certain embodiments, the modulator is DMSO, DMF, NMP, or dihydrolevoglucosenone (Cyrene). In certain embodiments, the concentration of DMSO is at least about 0.5%. In certain embodiments, the modulator is an inhibitor or agonist of a pathway regulated by any one of the genes as disclosed in the present application.

[0031] In certain embodiments, the modulator is a nuclease. In certain embodiments, the nuclease is selected from the group consisting of zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), a clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0032] In certain embodiments, the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

[0033] In certain embodiments, the modulator comprises a template for homologous arm-mediated recombination.

[0034] In certain embodiments, the cell line is a mammalian cell line or an insect cell line. In certain embodiments, the mammalian cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or human embryonic kidney (HEK) 293 cell line. In certain embodiments, the mammalian cell line is a HeLa cell line or a HEK293 cell line.

[0035] In certain embodiments, the insect cell line is an Sf9 cell line or a Hi5 cell line.

[0036] In an aspect, provided herein is an engineered or modified cell line produced by a method comprising knocking down the expression or activity of at least one of the genes selected from the group consisting of DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SSI 8, THEM6, and TNFAIP3.

[0037] In certain embodiments, the knockdown is carried out by using at least one of homology directed recombination, RNA based knock-down, a CRISPR-Cas enzymatic method.

[0038] In yet another aspect, provided herein is an engineered or modified cell produced by a method comprising increasing the expression or activity of at least one of the genes selected from the group consisting of ASNS, BCL2, CDKN1A, COL20A1, FAM46C, GATA1, GJB3, LAS IL, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

[0039] In certain embodiments, increasing the expression or activity is carried out by transient delivery or stable integration of a target expression modulation construct into the host cell line using at least one of random integration, targeted integration (e.g., targeted integration at a safe harbor locus), homology directed recombination, a transposase-based recombination, a lentiviral based integration, an integrase mediated integration or a CRISPR-Cas activation method, optionally wherein the Cas comprises a dCas, such as dCas9.

[0040] In certain embodiments, the cell line further comprises stably integrated nucleic acid sequences encoding AAV Rep and / or cap, AAV ITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus. In certain embodiments, the cell line further comprises transiently transfected nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus.

[0041] In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the AAV helper virus is a herpes simplex virus (HSV).

[0042] In certain embodiments, the AAV helper genes comprise at least one of: El a, Elb, E2A, L4, E4, and VA.

[0043] In certain embodiments, provided herein is a recombinant viral particle obtained from the engineered or modified cell line as provided herein. In certain embodiments, the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle.

[0044] In an aspect, provided herein is a method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: transiently transfecting the host cell line with one or more plasmids comprising: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid encoding a gene of interest.

[0045] In certain embodiments, the method further comprises transfecting the host cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus. In certain embodiments, the AAV helper virus is an adenovirus. In some embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the AAV helper virus is a herpes simplex virus (HSV).

[0046] In another aspect, provided herein is a method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: stably transfecting the producer cell line as provided herein with: (i) nucleic acids encoding AAV Rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, and (iii) a nucleic acid encoding a gene of interest.

[0047] In certain embodiments, the method further comprises transfecting the producer cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus. In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the AAV helper virus is a herpes simplex virus (HSV).

[0048] In certain embodiments, the rAAV comprises an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV 10, AAVrh 10, AAV 11, AAV 12, AAV 13, AAV D J, a goat A AV, abovine AAV, or a mouse AAV, AAV2 / 207m8, or AAV LK03, or hybrids, or variants or chimeras thereof. In one aspect, provided herein is an engineered cell line in which the expression of glyceradehyde-3 -phosphate dehydrogenase (GAPDH) is downregulated compared to a control cell line, wherein the cell line is a host cell line for the production of viral particles. In certain embodiments, the engineered cell line is an engineered human embryonic kidney (HEK) 293 cell line and the control cell line is a HEK293 cell line.

[0049] In one aspect, provided herein is an engineered human embryonic kidney (HEK) 293 cell line in which the expression of glyceradehyde-3 -phosphate dehydrogenase (GAPDH) is downregulated compared to a control HEK293 cell line, wherein the HEK293 cell line is a host HEK293 cell line for the production of viral particles.

[0050] In certain embodiments, the GAPDH is downregulated by a modulator that decreases the expression and / or activity of the GAPDH. In certain embodiments, the cell line comprises a genetic modification. In certain embodiments, the modulator is a nuclease. In certain embodiments, the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system. In certain embodiments, the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO). In certain embodiments, the modulator comprises a template for homologous arm-mediated recombination. In certain embodiments, the modulator comprises an inhibitor of GAPDH. In certain embodiments, the inhibitor comprises a synthetic small molecule inhibitor.

[0051] In one aspect, the HEK293 cell line is a producer HEK293 cell line.

[0052] In certain embodiments, the producer cell line further comprises stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest. In certain embodiments, the producer cell line further comprises AAV helper function encoding sequences of an AAV helper virus. In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the AAV helper function encoding sequences comprise at least one of: El a, Elb, E2A, L4, E4, and VA. In certain embodiments, the titer of rAAV produced from the engineered cell line is at least about 1.5-fold higher compared to the titer of rAAV produced from a cell line in which the expression of the gene is not altered.

[0053] In certain aspects, the disclosure here provides a method of downregulating gene expression of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) for increasing the production of rAAV particles from a cell, wherein the method comprises culturing the cell in the presence of a modulator that downregulates the expression or activity of GAPDH.

[0054] In certain embodiments, the modulator is dimethylsulfoxide (DMSO), dimethylformamide (DMF), (N-methylpyrrolidone (NMP), or dihydrolevoglucosenone (Cyrene). In certain embodiments, the concentration of DMSO is at least about 0.5%.

[0055] In certain aspects, the disclosure herein provides a method for increasing the production of rAAV particles, wherein the method comprises culturing a HEK293 cell in the presence of a modulator, wherein the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) is decreased as compared to a control HEK293 cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

[0056] In certain embodiments, the method increases recombinant viral titer by at least 1.5-fold compared to a method comprising a control HEK293 cell. In certain embodiments, the modulator comprises a synthetic small molecule inhibitor of GAPDH. In some embodiments, the modulator is dimethylsulfoxide (DMSO), dimethylformamide (DMF), (N-methylpyrrolidone (NMP), dihydrolevoglucosenone (Cyrene), DC-5163, 1,2,3,4,6-penta-O-galloyl-P-D-glucopyranose (PGG), or 4-octyl itaconate (4-OI). In certain embodiments, the concentration of DMSO is at least about 0.5%. In certain embodiments, the modulator is an inhibitor of a pathway regulated by GAPDH. In some embodiments, the modulator is a nuclease. In certain embodiments, the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

[0057] In certain embodiments, the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO). In certain embodiments, the modulator comprises a template for homologous arm-mediated recombination.

[0058] In certain aspects, the disclosure herein provides an engineered HEK293 host cell line produced by a method comprising knocking down the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), wherein the engineered HEK293 cell line is for the production of viral particles.

[0059] In certain embodiments, the engineered HEK293 host cell line produced by the method of described herein, wherein the knockdown is carried out using at least one of: homology-directed recombination, RNA-based knock-down, and a CRISPR-Cas enzymatic method.

[0060] In certain embodiments, the engineered HEK293 host cell line produced by the method disclosed herein, further comprising stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus.

[0061] In certain embodiments, the engineered HEK293 host cell line produced by the method disclosed herein, wherein the AAV helper virus is an adenovirus.

[0062] In certain embodiments, the engineered HEK293 host cell line produced by the method disclosed herein, wherein the adenovirus is Ad5 or Ad2.

[0063] In certain embodiments, the engineered HEK293 host cell line produced by the method of claim 100, wherein the AAV helper function encoding sequences comprise at least one of: Ela, Elb, E2A, L4, E4, and VA.

[0064] In certain embodiments, a recombinant viral particle obtained from the engineered HEK293 host cell line produced by the method described herein.

[0065] In certain embodiments, the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle.

[0066] In one aspect, disclosed herein a method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: transiently transfecting the host HEK293 cell line disclosed herein with one or more plasmids comprising: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, and (iii) nucleic acid encoding a gene of interest.

[0067] In certain embodiments, the method further comprising transfecting the host HEK293 cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus.

[0068] In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2.

[0069] In one aspect, as disclosed herein a method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: stably transfecting the producer HEK293 cell line of claim 78 with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, and (iii) nucleic acid encoding a gene of interest.

[0070] In certain embodiments, the method further comprising transfecting the producer HEK293 cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus. In some embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the rAAV comprises an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVDJ, agoatAAV, abovine AAV, oramouseAAV, AAV2 / 207m8, or AAVLK03, or hybrids, or variants or chimeras thereof.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS FIGs 1A-1F demonstrate that dimethyl sulfoxide (DMSO) enhances recombinant AAV (rAAV) production from a model producer cell line. FIG. 1A shows a schematic diagram of AAV producer cell line (PCL) and production. Triple play plasmids (TPP) harboring Rep, Cap and a Gene of Interest (GOI) were stably transfected into parental cells to generate the producer cell line. rAAV production is induced upon wtAd5 infection of PCL. FIG. IB shows the effect of DMSO which increased AAV production from PCL1 in a dose-dependent manner up to 1%. DMSO concentrations higher than 1% (e.g., 1.5%, 2%, and 3%) resulted in no increase or lower levels of A AV product! on. AAV (FIG. IB) and Ad5 (FIG. ID) replication and production were analyzed from whole cell culture collected at 3 days post infection. AAV (FIG. IB) and Ad5 (FIG. ID) viral production was represented by DNase-resistant genome (DRG) copies. AAV (FIG. 1C) and Ad5 (FIG. IE) viral replication was represented by total genome (TVG) copies. Relative rAAV volumetric productivity is shown as a bar graph and normalized to a non-DMSO control. 1% DMSO impact on AAV production was also assessed at typical infection density (X) as well as a 4-fold higher density (Y). 1% DMSO increased PCL1 production at both infection cell densities (FIG. IF). P values were calculated using Prism 2-way ANOVA with n=3. Unless noted otherwise, X was the infection cell density for all the PCL culture infected at suspension condition for all studies, results shown in the subsequent figures.

[0072] FIGs. 2A-2G demonstrate that DMSO enhanced AAV cap protein expression and capsid production in PCL1. FIG. 2A is a schematic diagram of the experimental set up and sample collection. Two PCLs, PCL1 and PCL2 were infected in 250 mL shake flask with wtAd5 for rAAV production with or without 1% DMSO supplementation. DMSO was either added 3 hours prior infection (-3h) or at the same time of infection (Oh), as shown in FIG.2A.

[0073] Samples were collected at Ohpi, 24hpi, 48hpi and 72hpi for cell viability% and Rep / Cap protein expression (western blot) detection. RNA-Seq samples were collected at Ohpi, 24hpi and 48hpi from non-treated control flask and -3h DMSO treated flask of both PCLs. rAAV genome and capsid titer were determined using qPCR and ELISA respectively using 72hpi collected samples. FIG.2B shows relative rAAV productivity. FIG.2C shows AAV replication assessed by total vector genome copies via qPCR. FIG. 2D shows AAV capsid production (VP) evaluated by ELISA. FIG. 2E shows AAV genome packaging percentage (DRG / TVG). FIG. 2F shows full capsid percentage. The full capsid percentages were estimated using DRG / VP. AAV Cap and Rep expression pattern of PCL1(AAV2-SEAP) and PCL2 were determined using western blotting, and tubulin expression was used as loading control, as shown in FIG. 2G.

[0074] All qPCR results were presented as bar graph in relative fold change normalized to non-DMSO control condition. Three independent experiments were conducted and average values were presented. P values were calculated using Prism 2-way ANOVA with n=3.

[0075] FIGs. 3A-3E demonstrate that DMSO did not significantly affect Ad5 production nor infection efficiency. FIG. 3A shows PCL1 and FIG. 3B shows PCL2 cell viability at Ohpi, 24hpi, 48hpi and 72hpi, assessed using the same experimental set up as described in FIG. 2A.

[0076] In FIG. 3C relative Ad5 productivity is shown, as determined by qPCR, and presented as bar graph normalized to control condition of each PCL. FIG.3D shows AdEl A protein expression of PCL1 and PCL2 at different time points as determined by western blotting. Tubulin expression was used as loading control. FIG.3E shows the impact of DMSO on Ad5 infection. This was determined by assaying the infection efficiency of Ad5-GFP in PCL1 and PCL2. GFP positive cell percentages were captured using Celigo at 24hpi. P values were calculated using Prism 2-way ANOVA with n=3.

[0077] FIGs. 4A-4D are provided to illustrate that DMSO treatment improved AAV productivity in Ambr®250 bioreactors with no adverse impact on vector quality. PCL1 cells were seeded into six Ambr®250 bioreactors pre-equilibrated with production medium at set pH, temperature and gas control. 1% DMSO was added to the three reactors at the time of cell seeding. wtAd5 infection was performed by adding wtAd5 at 3h post cell inoculation. Cultures were harvested at 72 hpi for AAV productivity analysis and vector purification for quality understanding. FIG. 4A shows the relative AAV productivity from Ambr®250 bioreactors harvest. 1%DMSO treated samples were normalized to non-DMSO control. FIG. IB shows VP ratios of purified vectors, using SDS-PAGE Coomassie blue staining. Equal numbers of purified vector genome copies of each sample were loaded onto SDS-PAGE based on purified vector titer. FIG. 4C shows capsid full percentage, as determined by analytical ultracentrifugation (AUC). FIG. 4D shows vector potency, which was evaluated with an enzyme activity assay specific for the transgene of PCL1. Cells were transduced with purified vectors from control or 1% DMSO treated condition at lE3vg / cell and lE4vg / cell. Culture supernatants were collected 6 days post transduction and assayed for the enzyme activity of the transgene. Relative activity from le3vg / cell and le4vg / cell transduction was shown as bar graph normalize to control condition. P values were calculated using Prism 2-way ANOVA with n=3. FIG. 5A-5B show that DMSO enhanced rAAV productivity in a HEK293 transient transfection platform. HEK293 cells were transiently transfected to produce two rAAV vectors of different serotypes and transgenes along with mock or transfection only controls. 1% DMSO or culture medium was added at time of infection. Whole cells culture was collected at 3 days post-transfection (dpt) for AAV productivity analysis (FIG. 5A) and cell pellets were collected for KAT5 mRNA analysis (FIG. 5B). Relative rAAV productivity from 1% DMSO treatment is shown in FIG. 5A as bar graph normalized to each serotype non treated condition. Relative KAT5 mRNA expression from HEK293 cells treated with 1% DMSO is shown as a bar graph in FIG. 5B normalized to HEK293 non-transfection control without DMSO treatment. P values were calculated using Prism 2-way ANOVA with n=3.

[0078] FIGs. 6A-6C provide results of a screening study using siRNA-based knockdown, to elucidate possible mechanism of action of DMSO. To validate differentially expressed genes identified by RNA-Seq analysis, siRNA knockdown was performed as illustrated in FIG. 6A.

[0079] AAV productivity upon target knockdown was assessed. Two siRNAs were used for each of the target selected. 20pM of each siRNA were transfected into PCL1 cells using Lonza 4D nucleofector strip format. Cells were cultured for 2 days after resuspending and seeding into 24 well plates before infection with wtAd5 in 96 well plates with or without 1% DMSO. Whole infected culture was then collected at 48 hours post infection (4 days post siRNA transfection) for AAV productivity analysis via TaqMan qPCR. AAV productivity results are shown in FIG.

[0080] 6B. Open circle represents AAV titers from control conditions and filled circle represents titers from 1% DMSO treated conditions. The siRNA results summary is shown in FIG. 6C to highlight one target: KAT5, which showed increased AAV productivity after siRNA knockdown but 1% DMSO did not lead to further increase of the productivity. siRNA RNA-Seq sample collection was carried out as described in Example 1 FIG. 2A. RNA-Seq analysis performed was described in Example 2.

[0081] FIGS. 7A-7E illustrate that the KAT5 knock down recapitulated the impact of DMSO on AAV Rep / Cap and Adenovirus E1A expression. siRNA transfection and wtAd5 infection were performed as described in FIG. 6 A but using nucleocuvette for siRNA transfection instead of strip format. AAV productivity analysis results are shown in FIG. 7A. Consistent with siRNA screening results shown in Fig. 6B, siKAT5 increased AAV production to the similar level as seen in the 1% DMSO treatment. Combination of siKAT5 and DMSO did not show further AAV production improvement. Further analysis was carried out to determine the effect of siKAT5 treatment on AAV replication (FIG. 7B), Rep and Cap expression (FIG. 7C) and Ad5 El A protein expression by western blot (FIG.7D) and El A mRNA expression (FIG. 7E). AAV replication and production were analyzed by qPCR, Rep / Cap and Ad5 El A protein expression by Western blot, E1A mRNA analysis by RT-qPCR. All quantifications are presented as bar graphs using values from siN.C transfection and infection without DMSO as reference for normalization. Tubulin expression was used as loading control for western blot. P values were calculated using Prism 2-way ANOVA n=3.

[0082] FIGs. 8A-8C show the effect of DMSO treatment in additional PCLs. It was observed that DMSO increased AAV production partly via KAT5 in multiple PCLs. The impact of DMSO on additional PCL3 and PCL4 were tested along with PCL1 and PCL2, in deep well plates (FIG. 8A). 1% DMSO was added into the production medium at time of wtAd5 infection and AAV productivity was analyzed at 72 hpi. KAT5 knockdown using siKAT5 was evaluated in PCL1 to PCL6 (FIG.8B), using experimental conditions similar to FIG. 6 A. FIG.

[0083] 8C shows KAT5 expression down-regulation upon DMSO treatment, as analyzed in PCL1 to PCL5 and parental cells, with or without wtAd5 infection. Producer cells were seeded into spin tubes and wtAd5 were added to one half of the total spin tubes at time of cell seeding for KAT5 expression analysis upon infection. 1% DMSO was added into half of the infected culture and half of the non-infection culture for evaluation of KAT5 expression following DMSO treatment with or without wtAd5 infection. Cell pellets were collected at 72 hpi and KAT5 expression was determined using RT-qPCR.

[0084] FIGs. 9A-9C demonstrate that DMSO supplementation affected cytokine production. In FIG. 9A differentially expressed cytokine gene transcripts from RNA-Seq analysis described in Example 2 are shown as a heatmap. FIG. 9B shows cytokine gene mRNA expression of PCL1 as determined by qPCR using a human cytokine array panel. Results were presented as relative log2 fold change normalized to non-treated condition. Samples used for this analysis were collected 24hpi. FIG. 9C shows the expression levels of secreted cytokine proteins determined using Luminex assay of cell culture supernatants. Results are shown as relative log2 foldchange normalized to non-treated condition. Samples used for the analysis were collected at 24hpi.

[0085] FIGs. 10A-10D provide the results of a study that shows DMSO and siKAT5 down-regulated CCND1 expression. To further investigate KAT5 targets identified in RNA-Seq analysis as described in Example 2, siKAT5 knockdown from PCL1 cells was performed as described in FIG. 7. These were analyzed for CCND1 and IL-6 mRNA expression using RT-qPCR. FIG. 10A shows CCND1 level was down-regulated upon DMSO treatment or siKAT5 knockdown alone. siKAT5 knockdown and DMSO together resulted in further reduction in CCND1 expression levels. As shown in FIG. 10B, IL-6 mRNA was only reduced in the DMSO treated condition. siKAT5 treatment did not lead to IL-6 mRNA expression down-regulation. CCND1 protein expression as determined by western blot (FIG. IOC) showed consistent pattern to that of CCND1 mRNA. Both siKAT5 or DMSO treatments, separately, reduced CCND1 protein expression and the two together had the lowest CCDN1 expression. CCND1 siRNA and IL-6 siRNA knockdown had no impact on AAV production under control or DMSO condition (FIG. 10D). siRNA transfection and AAV productivity assessment were performed as previously described in Fig. 6A. AAV productivities were normalized to that of siN.C no DMSO control and relative productivities were presented.

[0086] FIG. 11A-11C shows the acquisition and analysis pipeline of the RNA-Seq data to identify candidate regulators to enhance rAAV production. FIG. 11A shows PCL1 and PCL2 were cultured in medium with (treatment) and without (control) the 1% DMSO added. FIG.

[0087] 11B shows that RNA-Seq data were collected and analyzed at 0, 24 and 48 hours from 36 samples. FIG. 11C shows the analysis workflow using a number of techniques, and identification of candidate target genes that could be associated with an increase in AAV titer.

[0088] FIG. 12A-12E shows the higher-level analysis of the RNA-Seq data. FIG. 12A illustrates significantly enriched biological processes (BP) and molecular functions (MF) Gene Ontology (GO) terms. The vertical dotted line represents a false discovery rate (FDR) corrected p-value cutoff of 0.05. GO terms with p-values < 0.05 are considered significant. FIG. 12B -FIG. 12E is a series of heatmaps of genes that were differentially expressed for PCL1 and PCL2 cells cultured in control vs. DMSO-supplemented media over time for select GO terms.

[0089] FIG. 13A-13B illustrate the detailed immediate gene regulatory network of (A) KAT5 and (B) GATA1 and transcriptional support of their participation in the response to DMSO supplementation and accompanying increase in rAAV titer. FIG. 13A shows the immediate downstream regulatory network of KAT5, which is predicted to show inhibition upon DMSO treatment, based on the activation of downstream regulatory genes it is known to suppress or activate. Gene expression data supporting this and shown here comes from RNA-Seq at 0 hours post Ad5 infection. FIG. 13B shows the regulatory network immediately downstream and immediately upstream of GATA1, based on the expression of downstream targets at 0 hours post Ad5 infection.

[0090] FIGs. 14A-14B are heatmaps illustrating KAT5 and GATA1 downstream target gene expression as influenced by Ad5 infection over time. Log2(Fold Change) expression was calculated at 0, 24, and 48 hours post Ad5 transfection for PCL1 and PCL2. Expression is ordered by time point for each clone and transcription factor targets are hierarchically clustered. FIG. 15 illustrates an upregulation of CREB3L3 in PCLs cultured in media supplemented with DMSO when compared to cells cultured in control media. Fold-change comparison of DESeq2 RLE normalized counts between PCL1 in supplemented and control media (x-axis) and PCL1 and PCL2 in control media (y-axis) at time of infection (0 hpi). CREB3L3 is specifically indicated. Fold-Change > 2 is indicated by grey dashed line in both comparisons. FDR p-adjusted values in either comparison are dark grey circles if < 0.05, and dark grey diamonds if < 0.05 with a fold-change > 2 in both comparisons. Genes with adjusted p-value > 0.05 not shown.

[0091] FIG. 16 depicts relative AAV titers following siRNA knockdown of select genes. FIG. 17 depicts clustering of up-regulated genes in the AAV biosynthesis framework at 0 hours post infection (hpi). For each circle node, the radius represents the log2 fold change.

[0092] FIG. 18 depicts clustering of up-regulated genes in the AAV biosynthesis framework at 3 hours post infection (hpi). For each circle node, the radius represents the log2 fold change.

[0093] FIG. 19 is a graph illustrating rAAV productivity of HEK293 cells transfected with siRNA to GAPDH (siGAPDH) or control siRNA (siN.C). The cells were transfected with 100 pM, 200 pM, or 300 pM siRNA.

[0094] FIG. 20 is a graph illustrating rAAV productivity of HEK293 cells transfected with 100 pM siGAPDH or siN.C and AAV production transfection constructs with different Cap genes and different transgenes.

[0095] DETAILED DESCRIPTION

[0096] Before the subject matter of the present disclosure is described, it is to be understood that the subject matter is not limited to particular methods and experimental conditions described as such methods and conditions can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting because the scope of the disclosure will be limited only by the appended claims.

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0098] The techniques and procedures described or referenced herein are described in Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (R. I. Freshney, 6th ed., J. Wiley and Sons, 2010); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: ALaboratory Notebook (J. E. Cellis, ed., Academic Press, 1998); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A. Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: APractical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J.B. Lippincott Company, 2011). All publications mentioned herein are incorporated herein by reference in their entirety.

[0099] Definitions

[0100] As used herein, the term “vector” refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and non-viral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo, such as but not limited to a viral particle (e.g., an adeno-associated virus (AAV)). A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

[0101] As used herein, a “control” can refer to cells cultured in reference media without DMSO treatment. In addition, “control,” “control cell,” or “control cell line” when referring to the cells, refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a modified or engineered cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration that resulted in the modified or engineered cell; (b) a cell of the same genotype as the modified or engineered cell but that has been transformed with a null construct (i.e., with a construct that has no known effect on a target gene of interest); or, (c) a cell genetically identical to the modified or engineered cell but that is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype. The control cell that is cultured in the reference media without DMSO treatment (untreated media) may be a wild-type (e.g., not engineered or modified) cell. In certain embodiments, the engineered or modified cell line has altered activity and / or expression of a target gene at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100% compared to the control cell.

[0102] The terms “modified” or “engineered” as used herein describe a cell (e.g., a mammalian host cell) wherein expression of a gene is modified from its wild-type level of expression. The expression of a gene may be modified so that it is expressed at lower levels than wild-type levels. The expression of a gene may be modified so that it is expressed at higher levels than wild-type levels. The “modified” or “engineered” cell may be modified or engineered using at least one of a number of known genetic techniques, such as one that transiently or stably introduces an exogenous genetic cassette. The terms may also be used to describe a cell that has been exposed to an exogenous agent, e.g., a nucleic acid or oligonucleotide or a supplement such as DMSO that causes a modification (e.g., an increase or decrease in gene expression) in the cell. In some embodiments, the nucleic acid or oligonucleotide is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

[0103] In some embodiments, the terms “genetically modified” or “genetically engineered” can describe a cell in which the expression of at least one gene is reduced and / or the expression of at least one gene is increased through genetic modification of the cell’s DNA. In some embodiments, the cell’s DNA is modified by a modulator that increases or decreases the expression and / or activity of at least one gene. In some embodiments, the modulator is a nuclease. In some embodiments, the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system. In some embodiments, the modulator comprises a template for homologous arm mediated recombination. In certain embodiments, the term “treatment cell line” as provided herein can be used interchangeably with a cell that is “modified” or “engineered”.

[0104] The term “plasmid” or “plasmid backbone” refers to an extrachromosomal circular DNA capable of autonomous replication in a given cell. A plasmid can include a selection gene in order to select or to identify a cell transfected therewith. Exemplary plasmids include but are not limited to those derived from pBR322, pUC, pUC19, pUC57, pJ241, or pJ247, pBluescript, pREP4, pCEP4, and pCI. Plasmids can also be engineered by standard molecular biology techniques (Sambrook et ah, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), N.Y.).

[0105] As used herein, the term “gene” refers to the segment of a DNA molecule that codes for a polypeptide chain (e.g., the coding region). In some embodiments, a gene can include regions involved in producing the polypeptide chain that are positioned immediately preceding, following, and / or intervening the coding region (e.g., regulatory elements such as a promoter, enhancer, polyadenylation sequence, 5 '-untranslated region, 3 '-untranslated region, or intron).

[0106] As used herein, the term “regulatory element” refers to one or more nucleic acid molecules, such as promoters, enhancers, terminators, polyadenylation sequences, introns, and the like, that provide for the expression of a coding nucleic acid molecule in a cell.

[0107] As used herein, the term “promoter element” refers to a nucleic acid sequence that assists with controlling expression of a coding nucleic acid molecule. A promoter element can be located 5' of the translation start site of a gene, or 3' of the coding nucleic acid molecule. In some embodiments, a promoter useful for gene therapy can be derived from a native gene of a target protein. In some embodiments, a promoter useful for gene therapy can be specific for expression in a particular cell or tissue of the target organism (e.g., a liver-specific promoter or muscle-specific promoter). Non-limiting examples of well-characterized promoter elements include the CMV early promoter, CMV enhancer / chicken (Lactin promoter (CGA), the 3 -actin promoter, chicken (Lactin (CBA) promoters, and the methyl CpG binding protein 2 (MeCP2) promoter. A promoter can be a constitutive promoter or an inducible promoter.

[0108] As used herein, the term the term “polynucleotide” or “nucleic acid” or “oligonucleotide” refers to a polymeric molecule of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double- or multi -stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as can typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be an oligodeoxynucleoside phosphoramidate (P — NH2) or a mixed phosphoramidate-phosphodiester oligomer. In addition, a double-stranded polynucleotide can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.

[0109] Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2 '-amino functionalization) or hybrids thereof.

[0110] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues can contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native nucleic acid molecule, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0111] As used herein, the term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide, or components thereof such as amino acids or nucleotides, that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0112] As used herein, the term “derivative” refers to a nucleic acid, peptide, or protein (e.g., an AAV capsid protein) or a variant or analog thereof comprising one or more mutations and / or chemical modifications as compared to a corresponding full-length wild-type nucleic acid, peptide or protein. Non-limiting examples of chemical modifications involving nucleic acids include, for example, modifications to the base moiety, sugar moiety, phosphate moiety, phosphate-sugar backbone, or a combination thereof. A nucleic acid molecule that encodes mutant gene constructs that can be useful with the plasmid system described herein can be identical to a wild type (i.e., unmutated) nucleic acid molecule or can be a different coding nucleic acid molecule, which nucleic acid molecule, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the wild type coding nucleic acid molecule. One of ordinary skill in the art will recognize that each codon in a nucleic acid molecule (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each variation of a nucleic acid which encodes a same polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual gene therapy constructs.

[0113] One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid molecule that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded nucleic acid molecule is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. Conservative amino acid substitutions providing functionally similar amino acids are well known in the art. Dependent on the functionality of the particular amino acid, e.g., catalytic, structural, or sterically important amino acids, different groupings of amino acid can be considered conservative substitutions for each other.

[0114] As used herein, the term “percent (%) sequence identity” with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987), Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W / Z, where W is the number of nucleotides scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

[0115] As used herein, the term “isolated” when referring to a molecule (e.g., nucleic acid or protein) or cell means the molecule or cell has been identified and separated and / or recovered from a component of its natural environment.

[0116] As used herein, the term “helper virus” for AAV refers to a virus that allows AAV to be replicated and packaged by a host cell. A number of helper viruses are known including adenoviruses, herpesviruses, baculovirus, and poxviruses such as vaccinia. Adenoviruses encompass a number of different subgroups. Numerous adenoviruses of human, non-human mammalian and avian origin are known and are readily available (e.g., from ATCC). Viruses of the herpes family include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV), and pseudorabies viruses (PRV).

[0117] As used herein, the term “recombinant viral vector” refers to a recombinant polynucleotide vector comprising one or more heterologous nucleic acid molecules (i.e., nucleic acid molecule not of viral origin). In the case of recombinant AAV vectors, the recombinant nucleic acid molecule is flanked by at least one inverted terminal repeat sequence (ITR). In some embodiments, the recombinant nucleic acid molecule is flanked by two ITRs.

[0118] As used herein, the term “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector comprising one or more heterologous nucleic acid molecules (i.e., nucleic acid molecules not of AAV origin) that are flanked by at least one AAV inverted terminal repeat sequence (ITR). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV Rep and Cap gene products (i.e., AAV Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then a rAAV vector can be referred to as a “pro-vector” which can be “rescued” by replication. An rAAV vector may be encapsidated in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, e.g., an AAV particle. A rAAV vector can be packaged into an AAV virus capsid to generate a “recombinant adeno-associated viral particle (rAAV particle)”. “Recombinant AAV” (rAAV) and “AAV” are used interchangeably throughout the present disclosure.

[0119] As used herein, the term “rAAV virus” or “rAAV viral particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome.

[0120] As used herein, the term “heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. As it relates to nucleic acid molecules, such as coding sequences and / or control sequences, the term denotes nucleic acid molecules that are not normally j oined together and / or are not normally associated with a particular cell. A cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide acid molecule with respect to the vector. Thus, a “heterologous” nucleic acid molecule can be a nucleic acid molecule from an organism other than AAV or which is synthetically derived. As used herein, the term “operably-linked” refers to the association of two or more elements of a nucleic acid molecule that are physically linked so that the function of one of the elements is affected by another. For example, a regulatory DNA element is said to be “operably linked to” or “associated with” a DNA element that codes for an RNA or a polypeptide if the two elements are situated such that the regulatory DNA element affects expression of the coding DNA element (i.e., that the coding nucleic acid molecule or functional RNA is under the transcriptional control of the promoter). Coding nucleic acid molecules can be operably-linked to regulatory elements in sense or antisense orientation.

[0121] As used herein, the term “transgene” refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and / or expressed under appropriate conditions. A transgene can confer a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or diagnostic outcome. In another aspect, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA, or shRNA. A transgene can be as few as a couple of nucleotides or at least about 50, 100, 150, 200, 250, 300, 350, 400, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 7,500 nucleotides long or longer. A transgene can be for example, a viral genome. A transgene can be coding or non-coding nucleic acid molecule, or a combination thereof. A transgene can include one or more regulatory elements whereby transgene expression can be controlled.

[0122] As used herein, the terms “genome particles (gp)” or “genome copies” as used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures such as described in the Examples herein, or for example, in Clark et al. (1999) Hum. Gene Then, 10: 1031-1039; Veldwijk et al. (2002) Mol. Then, 6:272-278.

[0123] As used herein, the term “vector genome (vg)” can refer to one or more polynucleotides comprising a set of the polynucleotide molecules of a vector, e.g., a viral vector. A vector genome can be encapsidated in a viral particle. Depending on the particular viral vector, a vector genome can comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or double-stranded RNA. A vector genome can include endogenous nucleic acid molecules associated with a particular viral vector and / or any heterologous nucleic acid molecules inserted into a particular viral vector through recombinant techniques. For example, a recombinant AAV vector genome can include at least one ITR sequence flanking a promoter, a stuffer, a nucleic acid molecule of interest (e.g., an RNAi), and a polyadenylation sequence. A complete vector genome can include a complete set of the polynucleotide molecules of a vector. In some embodiments, the nucleic acid titer of a viral vector can be measured in terms of vector genome (vg / mL). Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

[0124] As used herein, the term “inverted terminal repeat” or “ITR” sequence refers to relatively short sequences found at the termini of viral genomes which are in opposite orientation.

[0125] As used herein, the term “AAV inverted terminal repeat (ITR)” sequence refers to an approximately 145-nucleotide sequence that is present at both termini of the native singlestranded AAV genome. The outermost 125 nucleotides of the ITR can be present in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter regions of self-complementarity (designated A, A, B, B', C, C and D regions), allowing intra-strand base-pairing to occur within this portion of the ITR.

[0126] As used herein, the term “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results (e.g., amelioration of symptoms, achievement of clinical endpoints, and the like). An effective amount can be administered in one or more administrations. In terms of a disease state, an effective amount is an amount sufficient to ameliorate, stabilize, or delay development of a disease.

[0127] As used herein, the terms “subject”, “patient”, “individual”, and “animal” are used interchangeably and refer to mammals, including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In certain embodiments, the individual or subject is a human.

[0128] As used herein, the term “treatment” refers to an approach for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, preventing spread (e.g., metastasis) of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. The term “treat” is the verb form of “treatment”. “Treatment” can also refer to addition of a substance to a biological or non-biological sample in an attempt to cause a physical or chemical change in the substance.

[0129] As used herein, a “therapeutic” agent (e.g., a therapeutic polypeptide, nucleic acid, or transgene) is one that provides a beneficial or desired clinical result. As such, a therapeutic agent can be used in a treatment as described above. Ranges can be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±10%, up to ±5%, and up to ±1% of a given value. For example, as used herein, the expression “about 100” includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.) Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, or within 2-fold of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.

[0130] Recombinant AAV Producer Cell Lines and Methods Thereof

[0131] In certain embodiments, the present disclosure provides compositions and methods for generating a producer cell line used for rAAV production. In certain embodiments, the compositions and methods comprise alteration of the expression of at least one of ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, and PARD6G-AS1 in a host cell line to generate an engineered or modified producer cell line. In certain embodiments, the expression of at least one of the transcription factor genes listed in Tables 1-4 is altered as compared to a control cell line. In certain embodiments, the expression of at least one of the transcription factor target genes is listed in Tables 1-4 is altered as compared to a control cell line.

[0132] In certain embodiments, provided herein is an engineered or modified host cell line produced by a method comprising knocking down the expression or activity of at least one of the genes selected from the group consisting of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGVDDX58, SSI 8, THEM6, and TNFAIP3. In certain embodiments, provided herein is an engineered or modified host cell line produced by a method comprising increasing the expression or activity of at least one of the genes selected from the group consisting of ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS IL, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, and PLEKH02.

[0133] Table 1. TFs and all targets associated with increase in viral titer as predicted by IPAfor PCL1.

[0134] Transcription Transcription Target Name

[0135] Factor Factor

[0136] Target

[0137] ALAS2 5 '-aminolevulinate synthase 2

[0138] BCL2L1 BCL2 like 1

[0139] GATA1

[0140] GATA2 GATA binding protein 2

[0141] GP9 glycoprotein IX platelet

[0142] IL13 interleukin 13

[0143] ITGA2B integrin subunit alpha 2b

[0144] ITGAX integrin subunit alpha X

[0145] KIT MBP myelin basic protein

[0146] Pim-2 proto-oncogene, serine / threonine

[0147] PIM2

[0148] kinase

[0149] PTGDR2 prostaglandin D2 receptor 2

[0150] SKI SKI proto-oncogene

[0151] SLC19A1 solute carrier family 19 member 1

[0152] SPTB spectrin beta, erythrocytic

[0153] BGLAP bone gamma-carboxyglutamate protein

[0154] H0XA5 CDKN1A cyclin dependent kinase inhibitor 1A

[0155] EGR1

[0156] growth arrest and DNA damage inducible

[0157] GADD45B

[0158] beta

[0159] LIF LIF interleukin 6 family cytokine

[0160] PTN RUNX2 RUNX family transcription factor 2

[0161] SAT1 spermidine / spermine N1 -acetyltransferase 1 NGEF BCL11B BCL11 transcription factor B

[0162] CCNJL cyclin J like

[0163] C0L2A1 collagen type II alpha 1 chain

[0164] CXCR4 C-X-C motif chemokine receptor 4

[0165] FGFR3 fibroblast growth factor receptor 3

[0166] FLT4

[0167] hes related family bHLH transcription factor

[0168] HEY1

[0169] with YRPW motif 1

[0170]

[0171] IL12RB2 interleukin 12 receptor subunit beta 2 Transcription Transcription Target Name

[0172] Factor Factor

[0173] Target

[0174] long intergenic non-protein coding RNA LINC01140

[0175] 1140

[0176] MAFB MAF bZIP transcription factor B SLC6A9 solute carrier family 6 member 9 TAGLN transgelin

[0177] TERT telomerase reverse transcriptase TNFSF10 TNF superfamily member 10

[0178] ADRB2 adrenoceptor beta 2

[0179] asparagine synthetase (glutamineASNS PELP1 hydrolyzing)

[0180] BCL2L1 BCL2 like 1

[0181] CCNG2 cyclin G2

[0182] CDKN1A cyclin dependent kinase inhibitor 1A CTSH cathepsin H

[0183] CXXC5 CXXC finger protein 5 DGKA diacylglycerol kinase alpha

[0184] FGFR3 fibroblast growth factor receptor 3 F0XQ1 forkhead box Q 1

[0185] LEP leptin

[0186] MAP IB MRAS muscle RAS oncogene homolog MRC1 mannose receptor C-type 1

[0187] PRMT6 protein arginine methyltransferase 6 SESN2 sestrin 2

[0188] TAGLN transgelin

[0189] CABP7 calcium binding protein 7 SS18 calcium voltage-gated channel subunit CACNA1G

[0190] alpha 1 G

[0191] CBX4 chromobox 4

[0192] growth arrest and DNA damage inducible GADD45B

[0193] beta

[0194] GCH1 GTP cyclohydrolase 1

[0195] JAG2 jagged canonical Notch ligand 2 NGFR nerve growth factor receptor

[0196] TENT5C terminal nucleotidyltransferase 5C ADAM metallopeptidase with

[0197] RRAS2 ADAMTS5

[0198] thrombospondin type 1 motif 5

[0199] CAV1 caveolin 1

[0200] COL6A3

[0201] FGB fibrinogen beta chain

[0202] F0SL1 FOS like 1, AP-1 transcription factor subunit NIDI

[0203]

[0204] PGF placental growth factor Transcription Transcription Target Name

[0205] Factor Factor

[0206] Target

[0207] TNFAIP3 TNF alpha induced protein 3

[0208] ABCA1 ATP binding cassette subfamily A member 1 S0RL1 BMP2 bone morphogenetic protein 2

[0209] CACHD1

[0210] CAV1 caveolin 1

[0211] CDH7

[0212] COL5A2

[0213] FGFR3 fibroblast growth factor receptor 3 FGG fibrinogen gamma chain

[0214] potassium voltage-gated channel subfamily KCND1

[0215] D member 1

[0216] NIDI NRP2 neuropilin 2

[0217] PGF placental growth factor BMP2 bone morphogenetic protein 2

[0218] CHD1

[0219] CXCL2

[0220] ICAM1 intercellular adhesion molecule 1

[0221] IER3 immediate early response 3

[0222] LIF LIF interleukin 6 family cytokine CXCL2

[0223] FOXL2

[0224] FAS Fas cell surface death receptor

[0225] ICAM1 intercellular adhesion molecule 1

[0226] IER3 immediate early response 3

[0227] LIF LIF interleukin 6 family cytokine SPRY1 sprouty RTK signaling antagonist 1 ZNF165 zinc finger protein 165 ICAM1 intercellular adhesion molecule 1 JAK1 JunB proto-oncogene, AP-1 transcription JUNB

[0228] factor subunit

[0229] LIF LIF interleukin 6 family cytokine SMAD7

[0230] TNS4

[0231] NPM1 BMP2 bone morphogenetic protein 2

[0232] COL6A2 collagen type VI alpha 2 chain

[0233] CXCL2

[0234] DnaJ heat shock protein family (Hsp40) DNAJB2

[0235] member B2

[0236] FOXC2

[0237] HILPDA hypoxia inducible lipid droplet associated ICAM1 intercellular adhesion molecule 1

[0238]

[0239] JAG1 jagged canonical Notch ligand 1 Transcription Transcription Target Name

[0240] Factor Factor

[0241] Target

[0242] JunB proto-oncogene, AP-1 transcription

[0243] JUNB

[0244] factor subunit

[0245] LIF LIF interleukin 6 family cytokine

[0246] MY016

[0247] NUAK1

[0248] PGF PMEPA1

[0249] TGFBI transforming growth factor beta induced

[0250] TNFRSF25 TNF receptor superfamily member 25

[0251] WNT5A Wnt family member 5A

[0252] ZC3H12A zinc finger CCCH-type containing 12A

[0253] BMP2 bone morphogenetic protein 2

[0254] S0RL1 EFNA1 ephrin Al

[0255] EPHB2 EPH receptor B2

[0256] FGF7 fibroblast growth factor 7

[0257] FZD4 frizzled class receptor 4

[0258] ICAM1 intercellular adhesion molecule 1

[0259] NRP2 neuropilin 2

[0260] PGF RTN4R

[0261] SSTR1 somatostatin receptor 1

[0262]

[0263] WNT5A Wnt family member 5A

[0264] Table 2. TFs and all targets associated with increase in viral titer as predicted by IPA for PCL2.

[0265] IPA IPATF Target Name

[0266] TF target

[0267] ANLN ACER2 alkaline ceramidase 2

[0268] AEN apoptosis enhancing nuclease

[0269] BTG2 BTG anti-proliferation factor 2

[0270] CDH10 cadherin 10

[0271] CDKN1A cyclin dependent kinase inhibitor 1A

[0272] calcium release activated channel regulator

[0273] CRACR2B

[0274] 2B

[0275] DPYSL4 dihydropyrimidinase like 4

[0276] GAL3ST4 galactose-3-O-sulfotransferase 4

[0277] INKA2 inka box actin regulator 2

[0278] MYH16 myosin heavy chain 16 pseudogene

[0279] PDE2A phosphodiesterase 2A

[0280] SESN2 sestrin 2

[0281] SPATAI 8 spermatogenesis associated 18

[0282]

[0283] SULF2 sulfatase 2 IPA IPATF Target Name

[0284] TF target

[0285] TP53 induced glycolysis regulatory TIGAR

[0286] phosphatase

[0287] TNFRSF10A TNF receptor superfamily member 10a VWCE von Willebrand factor C and EGF domains BCL2L1 BCL2 like 1

[0288] BGLAP bone gamma-carboxyglutamate protein CDKN1A cyclin dependent kinase inhibitor 1A C0L1A2

[0289] BMP2

[0290] C0L2A1 collagen type II alpha 1 chain

[0291] CTSK cathepsin K

[0292] DBH dopamine beta-hydroxylase

[0293] DLX2 distal-less homeobox 2 F3 coagulation factor III, tissue factor FGF2 fibroblast growth factor 2

[0294] FST follistatin

[0295] growth arrest and DNA damage inducible GADD45B

[0296] beta

[0297] GREM1 gremlin 1, DAN family BMP antagonist HAS2 hyaluronan synthase 2 ID2 inhibitor of DNA binding 2

[0298] IGFBP3 insulin like growth factor binding protein 3 KLF10 KLF transcription factor 10

[0299] KLF9 KLF transcription factor 9

[0300] MRAS muscle RAS oncogene homolog MSX1 msh homeobox 1

[0301] MSX2 msh homeobox 2

[0302] NOG noggin

[0303] PH0X2A paired like homeobox 2A PLXNA2 plexin A2

[0304] RUNX3 RUNX family transcription factor 3 SMAD1 SMAD family member 1

[0305] SMAD6 SMAD family member 6

[0306] SMAD7 SMAD family member 7

[0307] SOST sclerostin

[0308] SPARC STAR steroidogenic acute regulatory protein TAGLN transgelin

[0309] TIMP3

[0310] TNFSF11 TNF superfamily member 11

[0311] VDR vitamin D receptor

[0312] ZBTB2 zinc finger and BTB domain containing 2 NGEF ART1 ADP-ribosyltransferase 1

[0313]

[0314] C0L2A1 collagen type II alpha 1 chain IPA IPATF Target Name

[0315] TF target

[0316] CXCR4 C-X-C motif chemokine receptor 4 FGFR3 fibroblast growth factor receptor 3 FLT4

[0317] H2BC17 H2B clustered histone 17 IL12RB2 interleukin 12 receptor subunit beta 2 KIF5C

[0318] LILRA6 leukocyte immunoglobulin like receptor A6 MAFB MAF bZIP transcription factor B METTL7A thiol methyltransferase 1A RYR1 ryanodine receptor 1

[0319] SCARF 1 scavenger receptor class F member 1 SLC6A9 solute carrier family 6 member 9 TAGLN transgelin

[0320] TAS2R4 taste 2 receptor member 4 TERT telomerase reverse transcriptase TNFSF10 TNF superfamily member 10

[0321] UPK3B uroplakin 3B

[0322] RASSF6 BMI1 BMI1 proto-oncogene, polycomb ring finger BTG2 BTG anti-proliferation factor 2 CDKN1A cyclin dependent kinase inhibitor 1A TP73 tumor protein p73

[0323] CABP7 calcium binding protein 7

[0324] calcium voltage -gated channel subunit SS18 CACNA1G

[0325] alpha 1 G

[0326] CBX4 chromobox 4

[0327] growth arrest and DNA damage inducible GADD45B

[0328] beta

[0329] GCH1 GTP cyclohydrolase 1 MSX1 msh homeobox 1

[0330] NFATC1 nuclear factor of activated T cells 1 NGFR nerve growth factor receptor

[0331] PDGFB platelet derived growth factor subunit B SLC34A2 solute carrier family 34 member 2 TENT5C terminal nucleotidyltransferase 5C TFRC ANKRD22 ankyrin repeat domain 22

[0332] CDKN1A cyclin dependent kinase inhibitor 1A FAM20C golgi associated secretory pathway FAM20C

[0333] kinase

[0334] ID2 inhibitor of DNA binding 2

[0335] ITGAM integrin subunit alpha M PLD4 phospholipase D family member 4 PRTN3 proteinase 3

[0336] SLC26A11 solute carrier family 26 member 11

[0337]

[0338] SLC2A14 solute carrier family 2 member 14 IPA IPATF Target Name

[0339] TF target

[0340] SULF2 sulfatase 2

[0341] TNFSF10 TNF superfamily member 10

[0342] activity regulated cytoskeleton associated ARC KAT5 protein

[0343] CCND1 cyclin D 1

[0344] IL6

[0345] PTPRZ1

[0346] VGF VGF nerve growth factor inducible ADAM metallopeptidase with BCOR AD AMTS 1

[0347] thrombospondin type 1 motif 1 ADM adrenomedullin

[0348] F0XQ1

[0349] GAS1

[0350] CPA4 carboxypeptidase A4

[0351] cytochrome P450 family 1 subfamily B CBX5 CYP1B1

[0352] member 1

[0353] FGFBP1 fibroblast growth factor binding protein 1 F0XA1 forkhead box Al

[0354] F0XQ1

[0355] IGFL2-AS1 IGFL2 antisense RNA 1

[0356] KIT MUC13 mucin 13, cell surface associated OLR1 oxidized low density lipoprotein receptor 1 RUNX2

[0357] TIMP4 TIMP metallopeptidase inhibitor 4 TM4SF1 transmembrane 4 L six family member 1 TXNIP thioredoxin interacting protein BMP2 bone morphogenetic protein 2 CHD1

[0358] CXCL2 C-X-C motif chemokine ligand 2 CXCL3 C-X-C motif chemokine ligand 3 IER3 immediate early response 3

[0359] IL6

[0360] LIF LIF interleukin 6 family cytokine PTGS2 prostaglandin-endoperoxide synthase 2 CXCL8 C-X-C motif chemokine ligand 8 ECSIT IER3 immediate early response 3

[0361] IL6

[0362] IRF7

[0363] NFKBIA NFKB inhibitor alpha PTGS2 prostaglandin-endoperoxide synthase 2 TNFAIP3 TNF alpha induced protein 3

[0364] SCD CEBPB CCAAT enhancer binding protein beta CXCL3 C-X-C motif chemokine ligand 3

[0365]

[0366] CXCL8 C-X-C motif chemokine ligand 8 IPA IPATF Target Name

[0367] TF target

[0368] homocysteine inducible ER protein with

[0369] HERPUD1

[0370] ubiquitin like domain 1

[0371] heat shock protein family A (Hsp70) member

[0372] HSPA5

[0373] 5

[0374] CYP51A1

[0375] THEM6 homocysteine inducible ER protein with

[0376] HERPUD1

[0377] ubiquitin like domain 1

[0378] HMGCS1 3 -hydroxy-3 -methylglutaryl-CoA synthase 1 HSP90B1 heat shock protein 90 beta family member 1

[0379] heat shock protein family A (Hsp70) member

[0380] HSPA5

[0381] 5

[0382] MSM01 methylsterol monooxygenase 1

[0383] BMP2 bone morphogenetic protein 2

[0384] WWTR1 CXCL2 C-X-C motif chemokine ligand 2

[0385] CXCL3 C-X-C motif chemokine ligand 3

[0386] CXCL8 C-X-C motif chemokine ligand 8

[0387] EFNA3

[0388] FOXA1 forkhead box Al

[0389] IL6

[0390] IRF7

[0391] IRX3 iroquois homeobox 3

[0392] ITGB8 integrin subunit beta 8

[0393] PLIN4

[0394] THY1

[0395]

[0396] TNFSF9 TN F superfamily member 9

[0397] Table 3. Additional TFs and targets associated with increase in viral titer as predicted by IPA for PCL1.

[0398] IPA IPATF Target Name

[0399] TF target

[0400] ADM2 IGFBP3 insulin like growth factor binding protein 3

[0401] LEP leptin

[0402] MTUS1 microtubule associated scaffold protein 1

[0403] TNFRSF9

[0404] BCL2L1 BCL2 like 1

[0405] BCL2

[0406] CCNE1 cyclin E 1

[0407] CDKN1A cyclin dependent kinase inhibitor 1A

[0408] NES nestin

[0409] PRDM1 PR / SET domain 1

[0410] CCND1 AREG amphiregulin

[0411] CCNE1 cyclin E 1

[0412] CDCA2 cell division cycle associated 2

[0413]

[0414] CDK6 cyclin dependent kinase 6 IPA IPATF Target Name

[0415] TF target

[0416] CDKN1A cyclin dependent kinase inhibitor 1A CPED1 cadherin like and PC-esterase domain containing 1 FZD7 frizzled class receptor 7

[0417] GNMT glycine N-methyltransferase

[0418] H2BC21 H2B clustered histone 21

[0419] H4C8 H4 clustered histone 8

[0420] IER3 immediate early response 3

[0421] ITGB3 integrin subunit beta 3

[0422] ITIH2 inter-alpha-trypsin inhibitor heavy chain 2 LAMB2 laminin subunit beta 2

[0423] MAP3K5 mitogen-activated protein kinase kinase kinase 5 MYBL1 MYB proto-oncogene like 1

[0424] MYO7A myosin VIIA

[0425] MYT1 myelin transcription factor 1

[0426] RGS2 regulator of G protein signaling 2 SATB1

[0427] SCG5 secretogranin V

[0428] SI sucrase-isomaltase

[0429] TM7SF2 transmembrane 7 superfamily member 2 TMEM204 transmembrane protein 204

[0430] TP53INP2 tumor protein p53 inducible nuclear protein 2 UAP1 UDP-N-acetylglucosamine pyrophosphorylase 1 BCL2L1

[0431] FGFR3 BCL2 like 1

[0432] CDKN1A cyclin dependent kinase inhibitor 1A CLDN5 claudin 5

[0433] FRMD3 FERM domain containing 3

[0434] ZNF589 zinc finger protein 589

[0435] GATA1 ITGA2B integrin subunit alpha 2b

[0436] ACTA1 actin alpha 1, skeletal muscle

[0437] HDAC5 CDKN1A cyclin dependent kinase inhibitor 1A

[0438] CKM creatine kinase, M-type

[0439] DAPK1 death associated protein kinase 1 NR4A1 nuclear receptor subfamily 4 group A member 1 TAGLN transgelin

[0440] TNFRSF1A TNF receptor superfamily member 1A TUBB3 tubulin beta 3 class III

[0441] HHEX CCN1 cellular communication network factor 1

[0442] CCN2 cellular communication network factor 2 FGF2 fibroblast growth factor 2

[0443] IL12A interleukin 12A

[0444] IL15 interleukin 15

[0445] IL17F interleukin 17F

[0446]

[0447] PPBP pro-platelet basic protein IPA IPATF Target Name

[0448] TF target

[0449] SLC10A1 solute carrier family 10 member 1 TNFSF10 TNF superfamily member 10

[0450] TNFSF14 TNF superfamily member 14

[0451] HNF4A TCF7 transcription factor 7

[0452] H0XA5 LIF LIF interleukin 6 family cytokine

[0453] CCNG2 cyclin G2

[0454] LAS IL CDKN1A cyclin dependent kinase inhibitor 1A CHST6 carbohydrate sulfotransferase 6 CPT1C carnitine palmitoyltransferase 1C CRABP2 cellular retinoic acid binding protein 2 DGKA diacylglycerol kinase alpha

[0455] SESN2 se strin 2

[0456] SUSD2 sushi domain containing 2

[0457] BCL11B BCL11 transcription factor B

[0458] CCNJL cyclin J like

[0459] NGEF

[0460] C0L2A1 collagen type II alpha 1 chain

[0461] CXCR4 C-X-C motif chemokine receptor 4

[0462] FGFR3 fibroblast growth factor receptor 3 FLT4

[0463] hes related family bHLH transcription factor with HEY1

[0464] YRPW motif 1

[0465] IL12RB2 interleukin 12 receptor subunit beta 2 LINC01140 long intergenic non-protein coding RNA 1140 MAFB MAF bZIP transcription factor B SLC6A9 solute carrier family 6 member 9 TAGLN transgelin

[0466] TERT telomerase reverse transcriptase TNFSF10 TNF superfamily member 10

[0467] CCN2 cellular communication network factor 2 NME1

[0468] CDKN1A cyclin dependent kinase inhibitor 1A

[0469] FZD1 frizzled class receptor 1

[0470] GEMIN5 gem nuclear organelle associated protein 5 MMP14 matrix metallopeptidase 14

[0471] PTN NR3C1 LIF LIF interleukin 6 family cytokine

[0472] PELP1 FGFR3 fibroblast growth factor receptor 3

[0473] PTEN AKAP1 A-kinase anchoring protein 1

[0474] AKR1C3 aldo-keto reductase family 1 member C3 ARID2 AT-rich interaction domain 2 BCL3 BCL3 transcription coactivator BPGM bisphosphoglycerate mutase

[0475] CBX4 chromobox 4

[0476]

[0477] CDKN1A cyclin dependent kinase inhibitor 1A IPA IPATF Target Name

[0478] TF target

[0479] CXCL11 C-X-C motif chemokine ligand 11 CXCR4 C-X-C motif chemokine receptor 4 CYP27A1

[0480] DUSP1 dual specificity phosphatase 1

[0481] F3 coagulation factor III, tissue factor

[0482] FGFR3 fibroblast growth factor receptor 3 GATA2 GATA binding protein 2

[0483] GGT1 gamma-glutamyltransferase 1 HCLS1 hematopoietic cell-specific Lyn substrate 1

[0484] hyperpolarization activated cyclic nucleotide gated HCN2

[0485] potassium and sodium channel 2

[0486] HGFAC HGF activator

[0487] IDI1 isopentenyl-diphosphate delta isomerase 1 IFNGR2 interferon gamma receptor 2

[0488] IGFBP3 insulin like growth factor binding protein 3 IL4R interleukin 4 receptor

[0489] JunD proto-oncogene, AP-1 transcription factor JUND

[0490] subunit

[0491] NFIL3 nuclear factor, interleukin 3 regulated NR3C1 nuclear receptor subfamily 3 group C member 1 PLAU plasminogen activator, urokinase PRDM1 PR / SET domain 1

[0492] SAT1 spermidine / spermine N1 -acetyltransferase 1 SH2B2 SH2B adaptor protein 2

[0493] THBS1 thrombospondin 1

[0494] TNFRSF10D TNF receptor superfamily member lOd TNFRSF14 TNF receptor superfamily member 14 TNFRSF9

[0495] TNFSF10 TNF superfamily member 10

[0496] RXRA TCF7 transcription factor 7

[0497] SMAD4 ACVR1C activin A receptor type 1C

[0498] BGLAP bone gamma-carboxyglutamate protein CCN2 cellular communication network factor 2 CCNE1 cyclin E 1

[0499] CCNG2 cyclin G2

[0500] CDK17 cyclin dependent kinase 17

[0501] CDK6 cyclin dependent kinase 6

[0502] CDKN1A cyclin dependent kinase inhibitor 1A C0L1A2

[0503] CSF1 colony stimulating factor 1

[0504] DAPK1 death associated protein kinase 1 EPB41L5 erythrocyte membrane protein band 4.1 like 5

[0505]

[0506] FST follistatin IPA IPATF Target Name

[0507] TF target

[0508] FSTL3 follistatin like 3

[0509] GADD45B growth arrest and DNA damage inducible beta GUCY2F guanylate cyclase 2F, retinal HMCN1 hemicentin 1

[0510] HMGA2 high mobility group AT-hook 2 HMOX1 heme oxygenase 1

[0511] ID2 inhibitor of DNA binding 2 IER3 immediate early response 3 ITGB7 integrin subunit beta 7

[0512] JAG2 jagged canonical Notch ligand 2

[0513] MUC4 mucin 4, cell surface associated PILRA paired immunoglobin like type 2 receptor alpha PLAU plasminogen activator, urokinase SERTAD1 SERTA domain containing 1 SPI1 Spi-1 proto-oncogene

[0514] SSTR2 somatostatin receptor 2

[0515] TERT telomerase reverse transcriptase THBS1 thrombospondin 1

[0516] TIMP3 TIMP metallopeptidase inhibitor 3 TNFRSF10A TNF receptor superfamily member 10a CABP7 calcium binding protein 7

[0517] SS18 CACNA1G calcium voltage-gated channel subunit alphal G CBX4 chromobox 4

[0518] GADD45B growth arrest and DNA damage inducible beta GCH1 GTP cyclohydrolase 1

[0519] JAG2 jagged canonical Notch ligand 2

[0520] NGFR nerve growth factor receptor TENT5C terminal nucleotidyltransferase 5C AXIN2 axin 2

[0521] TCF7 BCL6 BCL6 transcription repressor

[0522] MMP14 matrix metallopeptidase 14

[0523] NES nestin

[0524] PRDM1 PR / SET domain 1

[0525] RORC RAR related orphan receptor C SPI1 Spi-1 proto-oncogene

[0526] TCF7 transcription factor 7

[0527] TGM2 BCL6 BCL6 transcription repressor

[0528] CCRL2 C-C motif chemokine receptor like 2 CDK6 cyclin dependent kinase 6

[0529] CFP complement factor properdin CSRNP1 cysteine and serine rich nuclear protein 1 E2F5 E2F transcription factor 5

[0530]

[0531] ICAM3 intercellular adhesion molecule 3 IPA IPATF Target Name

[0532] TF target

[0533] ITGAM integrin subunit alpha M

[0534] ITGAX integrin subunit alpha X

[0535] ITGB3 integrin subunit beta 3

[0536] LGALS9 galectin 9

[0537] MAFB MAF bZIP transcription factor B NLRC4 NLR family CARD domain containing 4 PLEKH02 pleckstrin homology domain containing 02 RFLNB refilin B

[0538] SEMA7A semaphorin 7A (John Milton Hagen blood group) SERPINB2 serpin family B member 2

[0539] SIGLEC12 sialic acid binding Ig like lectin 12 SLFN5 schlafen family member 5

[0540] THBS1 thrombospondin 1

[0541] UBA7 ubiquitin like modifier activating enzyme 7 TP53 LIF LIF interleukin 6 family cytokine

[0542] APAF1 apoptotic peptidase activating factor 1 TRPC1

[0543] CDK6 cyclin dependent kinase 6

[0544] CDKN1A cyclin dependent kinase inhibitor 1A

[0545] transient receptor potential cation channel subfamily TRPC1

[0546] C member 1

[0547] ABCA1 ATP binding cassette subfamily A member 1

[0548] ADAM metallopeptidase with thrombospondin type ADAMTS5

[0549] 1 motif 5

[0550] IL6

[0551] CCND1 cyclin D 1

[0552] CYBB FGA fibrinogen alpha chain

[0553] FGB fibrinogen beta chain

[0554] FGG fibrinogen gamma chain

[0555] FLU GP1BA

[0556] interferon induced protein with tetratricopeptide IFIT1

[0557] repeats 1

[0558] IL1RL1

[0559] ITGB3

[0560] KIT MAP2

[0561] NES nestin

[0562] PCSK1

[0563] PGF placental growth factor

[0564] PLEK PPBP pro-platelet basic protein

[0565] RUNX2 RUNX family transcription factor 2 TERT

[0566]

[0567] THBS1 thrombospondin 1 IPA IPATF Target Name

[0568] TF target

[0569] TLR3

[0570] MAP2K1 CCND1 cyclin D 1

[0571] ITGA2B

[0572] PRKCA CCND1 cyclin D 1

[0573] TRPC1

[0574] SALL4 CCND1 cyclin D 1

[0575] S0RL1 FGFR3 fibroblast growth factor receptor 3 SP1 CCND1 cyclin D 1

[0576] STAT3 CCND1 cyclin D 1

[0577] SYVN1 CCND1 cyclin D 1

[0578] BCL2L1 FAS Fas cell surface death receptor BRD4 CCND1 cyclin D 1

[0579] CHD1 LIF LIF interleukin 6 family cytokine EGFR CCND1 cyclin D 1

[0580] ESR2 CCND1 cyclin D 1

[0581] F0XL2 FAS Fas cell surface death receptor LIF LIF interleukin 6 family cytokine HGF CCND1 cyclin D 1

[0582] JAK1 LIF LIF interleukin 6 family cytokine JAK2 CCND1 cyclin D 1

[0583] NPM1 LIF LIF interleukin 6 family cytokine RAFI CCND1 cyclin D 1

[0584] FAS Fas cell surface death receptor LIF LIF interleukin 6 family cytokine SIX2 FAS Fas cell surface death receptor STAT3 CCND1 cyclin D 1

[0585] FAS Fas cell surface death receptor LIF LIF interleukin 6 family cytokine SYVN1 CCND1 cyclin D 1

[0586] TGFA CCND1 cyclin D 1

[0587] C5AR1 complement C5a receptor 1 TGM2 CCND1 cyclin D 1

[0588] CCRL2

[0589] DAPK2 death associated protein kinase 2 E2F5 E2F transcription factor 5

[0590] GCA IFI35 interferon induced protein 35 ITGB3

[0591] MAFB MDN1 midasin AAA ATPase 1 SMPD1 sphingomyelin phosphodiesterase 1

[0592]

[0593] THBS1 Table 4. Additional TFs and targets associated with increase in viral titer as predicted by IPAfor PCL2

[0594] IPA IPATF Target Name

[0595] TF target

[0596] APP NFKB1 nuclear factor kappa B subunit 1

[0597] BCL2L1

[0598] BCL2 BCL2 like 1

[0599] CCNE1 cyclin El

[0600] CDKN1A cyclin dependent kinase inhibitor 1A

[0601] NES nestin

[0602] PRDM1 PR / SET domain 1

[0603] AR androgen receptor

[0604] AREG amphiregulin

[0605] CCNE1 cyclin El

[0606] CCND1 CDK6 cyclin dependent kinase 6

[0607] CDKN1A cyclin dependent kinase inhibitor 1A

[0608] CPED1 cadherin like and PC-esterase domain containing 1 E2F8 E2F transcription factor 8

[0609] EPHA2 EPH receptor A2

[0610] ERRFI1 ERBB receptor feedback inhibitor 1

[0611] FZD7 frizzled class receptor 7

[0612] H2BC21 H2B clustered histone 21

[0613] H4C8 H4 clustered histone 8

[0614] H0MER2 homer scaffold protein 2

[0615] IER3 immediate early response 3

[0616] ITGB3

[0617] ITIH2 inter-alpha-trypsin inhibitor heavy chain 2

[0618] KDM6B lysine demethylase 6B

[0619] KRT4 keratin 4

[0620] LAMB2 laminin subunit beta 2

[0621] membrane associated guanylate kinase, WW and

[0622] MAGE PDZ domain containing 2

[0623] MARCKS MTFR2 mitochondrial fission regulator 2

[0624] MYBL1 MYB proto-oncogene like 1

[0625] MY07A myosin VIIA

[0626] MYT1 myelin transcription factor 1

[0627] RGS2 regulator of G protein signaling 2

[0628] SCG5 secretogranin V

[0629] SI sucrase-isomaltase

[0630] TMEM204 transmembrane protein 204

[0631] TP53INP2 tumor protein p53 inducible nuclear protein 2

[0632] UAP1 UDP-N-acetylglucosamine pyrophosphorylase 1

[0633]

[0634] CTNNB1 LIF LIF interleukin 6 family cytokine IPA IPATF Target Name

[0635] TF target

[0636] TCF7 transcription factor 7

[0637] transient receptor potential cation channel subfamily F2 TRPC1

[0638] C member 1

[0639] ACTA1 actin alpha 1, skeletal muscle

[0640] HDAC5 CD27 CD27 molecule

[0641] CDKN1A cyclin dependent kinase inhibitor 1A CKM creatine kinase, M-type

[0642] DAPK1 death associated protein kinase 1 LM0D2 leiomodin 2

[0643] MEG3

[0644] NR4A1 nuclear receptor subfamily 4 group A member 1 SMAD7 SMAD family member 7

[0645] TAGLN transgelin

[0646] TNFRSF1A TNF receptor superfamily member 1A TUBB3 tubulin beta 3 class III

[0647] CCN1 cellular communication network factor 1 HHEX CCN2 cellular communication network factor 2

[0648] FGF2 fibroblast growth factor 2

[0649] IL17F interleukin 17F

[0650] PPBP SLC10A1 solute carrier family 10 member 1 TNFSF10 TNF superfamily member 10

[0651] TNFSF14 TNF superfamily member 14

[0652] HIF1A ITGA2B integrin subunit alpha 2b

[0653] HIVEP1 NFKB1 nuclear factor kappa B subunit 1

[0654] IL13 FGFR3 fibroblast growth factor receptor 3

[0655] BTG2 BTG anti-proliferation factor 2 LAS IL CCNG2 cyclin G2

[0656] CDKN1A cyclin dependent kinase inhibitor 1A CEMIP cell migration inducing hyaluronidase 1 CHST6 carbohydrate sulfotransferase 6 CPT1C carnitine palmitoyltransferase 1C SESN2 sestrin 2

[0657] SPACA6 sperm acrosome associated 6

[0658] SUSD2 sushi domain containing 2

[0659] NGEF ART1 ADP-ribosyltransferase 1

[0660] C0L2A1 collagen type II alpha 1 chain

[0661] CXCR4 C-X-C motif chemokine receptor 4 FGFR3 fibroblast growth factor receptor 3 FLT4

[0662] H2BC17 H2B clustered histone 17

[0663] IL12RB2 interleukin 12 receptor subunit beta 2

[0664]

[0665] KIF5C IPA IPATF Target Name

[0666] TF target

[0667] LILRA6 leukocyte immunoglobulin like receptor A6 MAFB MAF bZIP transcription factor B METTL7A thiol methyltransferase 1A

[0668] RYR1 ryanodine receptor 1

[0669] SCARF 1 scavenger receptor class F member 1 SLC6A9 solute carrier family 6 member 9 TAGLN transgelin

[0670] TAS2R4 taste 2 receptor member 4

[0671] TERT telomerase reverse transcriptase TNFSF10 TNF superfamily member 10

[0672] UPK3B uroplakin 3B

[0673] CCN2 cellular communication network factor 2 NME1 CDKN1A cyclin dependent kinase inhibitor 1A

[0674] FZD1 frizzled class receptor 1

[0675] GEMIN5 gem nuclear organelle associated protein 5 METTL1 methyltransferase 1, tRNA methylguanosine MMP14 matrix metallopeptidase 14

[0676] PTN SMAD7 SMAD family member 7

[0677] NR3C1 LIF LIF interleukin 6 family cytokine

[0678] NFKB1 nuclear factor kappa B subunit 1 PAF1 DDX58 RNA sensor RIG-I

[0679] LIF LIF interleukin 6 family cytokine PRKCD

[0680] transient receptor potential cation channel subfamily TRPC1

[0681] C member 1

[0682] PTEN ADM adrenomedullin

[0683] AKAP1 A-kinase anchoring protein 1

[0684] AKR1C3 aldo-keto reductase family 1 member C3

[0685] AR androgen receptor

[0686] BCL3 BCL3 transcription coactivator BMI1 BMI 1 proto-oncogene, polycomb ring finger CBX4 chromobox 4

[0687] CDKN1A cyclin dependent kinase inhibitor 1A CXCL11 C-X-C motif chemokine ligand 11 CXCR4 C-X-C motif chemokine receptor 4 CYP27A1

[0688] DTX1 deltex E3 ubiquitin ligase 1

[0689] DUSP1 dual specificity phosphatase 1 ERRFI1 ERBB receptor feedback inhibitor 1

[0690] F3 coagulation factor III, tissue factor

[0691] FGFR3 fibroblast growth factor receptor 3 GATA2 GATA binding protein 2

[0692]

[0693] GGT1 gamma-glutamyltransferase 1 IPA IPATF Target Name

[0694] TF target

[0695] HAVCR1 hepatitis A virus cellular receptor 1 HCLS1 hematopoietic cell-specific Lyn substrate 1 HGFAC HGF activator

[0696] IDI1 isopentenyl-diphosphate delta isomerase 1 IGFBP3 insulin like growth factor binding protein 3 INPPL1 inositol polyphosphate phosphatase like 1 JunD proto-oncogene, AP-1 transcription factor JUND

[0697] subunit

[0698] MAFK MAF bZIP transcription factor K

[0699] NEU1 neuraminidase 1

[0700] NFIL3 nuclear factor, interleukin 3 regulated NFKB1 nuclear factor kappa B subunit 1

[0701] NKX3-1 NK3 homeobox 1

[0702] NR3C1 nuclear receptor subfamily 3 group C member 1 PLEC plectin

[0703] PRDM1 PR / SET domain 1

[0704] SAT1 spermidine / spermine N1 -acetyltransferase 1 SMAD6 SMAD family member 6

[0705] THBS1

[0706] TNFRSF14 TNF receptor superfamily member 14 TNFSF10 TNF superfamily member 10 VEGFC vascular endothelial growth factor C RELA NFKB1 nuclear factor kappa B subunit 1 RNASEH2B DDX58 RNA sensor RIG-I

[0707] RXRA TCF7 transcription factor 7

[0708] SMAD4 ACVR1C activin A receptor type 1C

[0709] BGLAP bone gamma-carboxyglutamate protein BMI1 BMI 1 proto-oncogene, polycomb ring finger CCN2 cellular communication network factor 2 CCNE1 cyclin El

[0710] CCNG2 cyclin G2

[0711] CDK6 cyclin dependent kinase 6 CDKN1A cyclin dependent kinase inhibitor 1A C0L1A2

[0712] CSF1 colony stimulating factor 1 CXCL3 C-X-C motif chemokine ligand 3 DAPK1 death associated protein kinase 1 EPB41L5 erythrocyte membrane protein band 4.1 like 5 F0SL1 FOS like 1, AP-1 transcription factor subunit FST follistatin

[0713] FSTL3 follistatin like 3

[0714] GADD45B growth arrest and DNA damage inducible beta

[0715]

[0716] GAST gastrin IPA IPATF Target Name

[0717] TF target

[0718] GUCY2F guanylate cyclase 2F, retinal HMCN1 hemicentin 1

[0719] HMOX1 heme oxygenase 1

[0720] ID2 inhibitor of DNA binding 2 IER3 immediate early response 3 IL31 interleukin 31

[0721] ITGB7 integrin subunit beta 7

[0722] KITLG KIT ligand

[0723] MUC4 mucin 4, cell surface associated NUP153 nucleoporin 153

[0724] PDGFB platelet derived growth factor subunit B PILRA paired immunoglobin like type 2 receptor alpha SERTAD1 SERTA domain containing 1 SMAD3 SMAD family member 3

[0725] SMAD7 SMAD family member 7

[0726] SPI1 Spi-1 proto-oncogene

[0727] SSTR2 somatostatin receptor 2

[0728] STAR steroidogenic acute regulatory protein TERT telomerase reverse transcriptase

[0729] THBS1

[0730] TIMP3

[0731] TNFRSF10A TNF receptor superfamily member 10a ZFP36 ZFP36 ring finger protein CABP7 calcium binding protein 7 SS18 CACNA1G calcium voltage-gated channel subunit alphal G CBX4 chromobox 4

[0732] GADD45B growth arrest and DNA damage inducible beta GCH1 GTP cyclohydrolase 1

[0733] MSX1 msh homeobox 1

[0734] NFATC1 nuclear factor of activated T cells 1 NGFR nerve growth factor receptor PDGFB platelet derived growth factor subunit B SLC34A2 solute carrier family 34 member 2 TENT5C terminal nucleotidyltransferase 5C TGFB1 LIF LIF interleukin 6 family cytokine

[0735] TGM2 BCL6 BCL6 transcription repressor

[0736] BTG2 BTG anti-proliferation factor 2

[0737] CCRL2 C-C motif chemokine receptor like 2 CDK6 cyclin dependent kinase 6 CEACAM1 CEA cell adhesion molecule 1

[0738] CFP complement factor properdin CSRNP1 cysteine and serine rich nuclear protein 1

[0739]

[0740] DAPK2 death associated protein kinase 2 IPA IPATF Target Name

[0741] TF target

[0742] DNAAF2 dynein axonemal assembly factor 2 E2F5 E2F transcription factor 5

[0743] HK3 hexokinase 3

[0744] ITGAM integrin subunit alpha M

[0745] ITGAX integrin subunit alpha X

[0746] ITGB3

[0747] LGALS9 galectin 9

[0748] MAFB MAF bZIP transcription factor B MDN1 midasin AAA ATPase 1

[0749] M0RC2 MORC family CW-type zinc finger 2 NLRC4 NLR family CARD domain containing 4 0AS2 2'-5'-oligoadenylate synthetase 2 PLEKH02 pleckstrin homology domain containing 02 RFLNB refilin B

[0750] SEMA6B

[0751] SEMA7A semaphorin 7A (John Milton Hagen blood group) SERPINB2 serpin family B member 2

[0752] SIGLEC12 sialic acid binding Ig like lectin 12 SLFN5 schlafen family member 5

[0753] THBS1

[0754] TNFAIP2 TNF alpha induced protein 2

[0755] TP63 tumor protein p63

[0756] UBA7 ubiquitin like modifier activating enzyme 7 WDR3 WD repeat domain 3

[0757] TP53 LIF LIF interleukin 6 family cytokine

[0758] NFKB1 nuclear factor kappa B subunit 1 TP73 FGFR3 fibroblast growth factor receptor 3

[0759] APAF1 apoptotic peptidase activating factor 1 TRPC1 CDK6 cyclin dependent kinase 6

[0760] CDKN1A cyclin dependent kinase inhibitor 1A

[0761] transient receptor potential cation channel subfamily TRPC1

[0762] C member 1

[0763] IKBKB CCND1 cyclin D 1

[0764] KAT5 CCND1 cyclin D 1

[0765] SYVN1 CCND1 cyclin D 1

[0766] ATF4 HSP90B1 heat shock protein 90 beta family member 1 C5 NFKBIA NFKB inhibitor alpha

[0767] CD36 LIF LIF interleukin 6 family cytokine

[0768] CDK9 NFKBIA NFKB inhibitor alpha

[0769] CHD1 LIF LIF interleukin 6 family cytokine

[0770] CSF1 HSP90B1 heat shock protein 90 beta family member 1

[0771]

[0772] ECSIT NFKBIA NFKB inhibitor alpha IPA IPATF Target Name

[0773] TF target

[0774] FN1 NFKBIA NFKB inhibitor alpha

[0775] F0XL2 LIF LIF interleukin 6 family cytokine

[0776] GPER1 NFKBIA NFKB inhibitor alpha

[0777] IL18 LIF LIF interleukin 6 family cytokine

[0778] ADAM metallopeptidase with thrombospondin type AD AMTS 1

[0779] 1 motif 1

[0780] ADAM metallopeptidase with thrombospondin type ADAMTS5

[0781] 1 motif 5

[0782] IL6 CEBPB CCAAT enhancer binding protein beta CEBPD CISH CXCL8 C-X-C motif chemokine ligand 8 CYP1B1 cytochrome P450 family 1 subfamily B member 1 FGB fibrinogen beta chain

[0783] FLU GLI1

[0784] HGF HPGD HSPA5 heat shock protein family A (Hsp70) member 5 IL1RL1

[0785] IL6

[0786] ITGB3

[0787] KIT LAG3

[0788] LDLR low density lipoprotein receptor MAP2

[0789] NFKBIA NFKB inhibitor alpha

[0790] PGF PLEK PPBP PTGS2 prostaglandin-endoperoxide synthase 2 RORC RUNX2

[0791] S1PR3

[0792] STAP2 signal transducing adaptor family member 2 TALI TBC1D9 TBC 1 domain family member 9 THBS1

[0793] TNFRSF25 TNF receptor superfamily member 25

[0794] JUN NFKBIA NFKB inhibitor alpha

[0795] KLF2 NFKBIA NFKB inhibitor alpha

[0796] KLF6 NFKBIA NFKB inhibitor alpha

[0797]

[0798] NPM1 LIF LIF interleukin 6 family cytokine IPA IPATF Target Name

[0799] TF target

[0800] PRKCD LIF LIF interleukin 6 family cytokine

[0801] NFKBIA NFKB inhibitor alpha

[0802] TRPC1

[0803] RAFI LIF LIF interleukin 6 family cytokine

[0804] RELA HSP90B1 heat shock protein 90 beta family member 1

[0805] NFKBIA NFKB inhibitor alpha

[0806] RIPK2 NFKBIA NFKB inhibitor alpha

[0807] SP110 TCF7

[0808] STAT3 LIF LIF interleukin 6 family cytokine

[0809] STAT6 LIF LIF interleukin 6 family cytokine

[0810] TCF7

[0811] TGFB1 HSP90B1 heat shock protein 90 beta family member 1

[0812] LIF LIF interleukin 6 family cytokine

[0813] NFKBIA NFKB inhibitor alpha

[0814] THEM6 HSP90B1 heat shock protein 90 beta family member 1

[0815] TP53 LIF LIF interleukin 6 family cytokine

[0816] NFKBIA NFKB inhibitor alpha

[0817]

[0818] XBP1 HSP90B1 heat shock protein 90 beta family member 1

[0819] In certain embodiments, the expression of at least one gene is reduced and / or expression of at least one gene is increased in the treatment cell line or treatment culture condition, as compared to the control cell line or control culture condition, to generate an engineered or modified cell line as disclosed herein.

[0820] In certain embodiments, the engineered or modified cell line or the treatment cell line is a mammalian cell line or an insect cell line. In certain embodiments, the mammalian cell line is a CHO cell line. In certain embodiments, the mammalian cell line is a Vero cell line. In certain embodiments, the mammalian cell line is a HeLa cell line. In certain embodiments, the mammalian cell line is a MDCK cell line. In certain embodiments, the mammalian cell line is a BHK cell line. In certain embodiments, the mammalian cell line is a A549 cell line. In certain embodiments, the mammalian cell line is an amniocyte cell line. In certain embodiments, the mammalian cell line is a HEK293 cell line. In certain embodiments, the insect cell line is an Sf9 cell line or a Hi5 cell line.

[0821] In certain embodiments, the cell line is an engineered or modified host cell line for the production of viral particles. In certain embodiments, the cell line is a producer cell line.

[0822] In certain embodiments, the engineered or modified cell line is stably transfected with nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences, to generate an engineered or modified producer cell line. As such, in certain embodiments, the engineered or modified producer cell line comprises stably integrated nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences. In certain embodiments, the engineered or modified cell line is transiently transfected with nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences, to generate an engineered or modified producer cell line. As such, in certain embodiments, the engineered or modified cell line comprises transient (e.g., not stably integrated) nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences. In certain embodiments, the sequences are encoded on one plasmid. In certain embodiments, the sequences are on more than one plasmid.

[0823] Recombinant HEK293 AAV Producer Cell Lines and Methods Thereof

[0824] In certain embodiments, the present disclosure provides compositions and methods for generating a HEK293 producer cell line used for rAAV production. In certain embodiments, the compositions and methods comprise alteration of the expression of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) in a host cell line to generate an engineered or modified producer HEK293 cell line.

[0825] As used herein, the term “glyceraldehyde-3 -phosphate dehydrogenase” or “GAPDH” refers to the gene encoding the protein glyceraldehyde-3-phosphate dehydrogenase. The human GAPDH gene corresponds toNCBI gene accession number NG 007073.2. Glyceraldehyde-3 -phosphate dehydrogenase catalyzes the reaction of glyceraldehyde-3 -phosphate to D-gly cerate 1,3-bisphosphate.

[0826] In certain embodiments, provided herein is an engineered or modified HEK293 producer cell line produced by a method comprising knocking down the expression or activity of GAPDH.

[0827] In certain embodiments, the expression of GAPDH is reduced in the treatment HEK293 cell line or treatment culture condition, as compared to the control HEK293 cell line or control culture condition.

[0828] In certain embodiments, the HEK293 cell line is a host cell line for the production of viral particles. In certain embodiments, the cell line is a HEK293 producer cell line.

[0829] In certain embodiments, the engineered or modified HEK293 host cell line is stably transfected with nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences, to generate an engineered or modified HEK293 producer cell line. As such, in certain embodiments, the engineered or modified HEK293 producer cell line comprises stably integrated nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR sequences. In certain embodiments, the engineered or modified HEK293 host cell line is transiently transfected with nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences, to generate an engineered or modified HEK293 host cell line comprises transient (e.g., not stably integrated) nucleic acid sequences encoding AAV Rep and / or Cap and a gene of interest (e.g., a transgene) flanked by inverted terminal repeat (ITR) sequences. In certain embodiments, the sequences are encoded on one plasmid. In certain embodiments, the sequences are on more than one plasmid.

[0830] Helper Nucleic Acid Molecules

[0831] In certain embodiments, the producer cell line further comprises AAV helper function encoding sequences of an AAV helper virus. These sequences include helper virus sequences necessary for AAV replication, such as an adenovirus (Ad) or herpesvirus. In certain embodiments, the AAV helper virus is an adenovirus. In certain embodiments, the adenovirus is Ad5 or Ad2. In certain embodiments, the adenovirus is wild type Ad5 or wild type Ad2. In certain embodiments, the AAV helper virus is a herpes simplex virus (HSV).

[0832] Adenovirus (Ad) helper viruses are small, non-enveloped viruses. The Ad genome encodes about 39 genes, which are classified as either early or late depending on whether they are expressed before or after DNA replication. Helper functions for AAV expression are provided by early Ad transcription units encoding proteins Ela, Elb, E2a, E4, and the transcription unit VARNA. Major late gene products are encoded in the transcription units LI to L5. The L4 region encodes: L4-100K, a translation enhancer protein that targets specific late mRNAs, protein pVIII, a structural protein of the viral capsid, L4-22K, required for viral DNA packaging into the empty capsid, and splicing factor L4-33K.

[0833] In certain embodiments, the helper genes comprise at least one of Ela, Elb, E2a, L4, E4, and VA. Helper genes can be present in a vector, in an adenovirus, or integrated into a cell genome.

[0834] In certain embodiments, Ad nucleic acid molecules encoding helper genes can be present in an Ad genome and / or encapsidated in an Ad vector. In certain embodiments, the helper genes can be an isolated molecule or inserted into one or more expression cassettes, vectors or plasmids.

[0835] Nucleic Acid Molecules Encoding AAV Rep / Cap Proteins

[0836] A Rep coding nucleic acid molecule can encode one or more AAV Rep proteins. In certain embodiments, a Rep coding nucleic acid molecule encodes Rep proteins that are necessary for viral genome replication and packaging into new virions. In certain embodiments, a Rep coding nucleic acid molecule can encode one or more of AAV Rep78, Rep68, Rep52, and Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode at least Rep78 or Rep68 and Rep52 or Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode Rep78 and Rep52 and / or Rep40 protein or Rep68 and Rep52 and / or Rep40. In certain embodiments, a Rep coding nucleic acid molecule can encode Rep78, Rep68, Rep52 and Rep40 proteins.

[0837] A Rep coding nucleic acid molecule can include a wild type or synthetic nucleic acid sequence encoding one or more AAV Rep proteins. A Rep coding nucleic acid molecule can be derived from any AAV, including but not limited to serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV13, or any other AAV, such as a chimeric AAV or a variant AAV. A Rep molecule can include a wild-type sequence modified by insertion, deletion, truncation and / or missense mutations.

[0838] In certain embodiments, a Rep coding nucleic acid molecule can be inserted into one or more expression cassettes, vectors or plasmids as described above. In certain embodiments, Ad nucleic acid molecules and a Rep coding nucleic acid molecule can be present on a single construct (e.g., plasmid backbone). In certain embodiments, a Rep coding nucleic acid molecule can be stably integrated into a genome of a cell line for AAV production.

[0839] A Cap coding nucleic acid molecule can encode one or more AAV Cap proteins. A Cap coding nucleic acid molecule can encode one or more AAV capsid subunits. In certain embodiments, a Cap coding nucleic acid molecule encodes a sufficient number of capsid subunits for producing a functional AAV capsid. AAV capsid structure is described in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0840] A Cap coding nucleic acid molecule can include a wild-type or synthetic nucleic acid molecule encoding one or more AAV Cap proteins. In certain embodiments, a Cap coding nucleic acid molecule can include one or more of VP1, VP2, and VP3. A Cap coding nucleic acid molecule can be derived from any AAV, including but not limited to serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV13, or any other AAV, such as a chimeric AAV or a variant AAV. A Cap coding nucleic acid molecule can include a wild type sequence modified by insertion, deletion, truncation and / or missense mutations. A Cap coding nucleic acid molecule can be modified to provide an altered tropism. In certain embodiments, a Cap coding nucleic acid molecule encodes a capsid protein for transducing a specific cell, e.g., CNS, heart, liver, lung, pancreas, photoreceptor cells, retinal pigment epithelium, and / or skeletal muscle.

[0841] In certain embodiments, a Cap coding nucleic acid molecule can be present in a helper Ad genome and / or an Ad vector. In certain embodiments, a Cap coding nucleic acid molecule can be encapsidated in an Ad vector. In certain embodiments, a Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be present on a single construct (e.g., plasmid backbone). In certain embodiments, a Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be encapsidated in an Ad vector. A Cap coding nucleic acid molecule and a Rep coding nucleic acid molecule can encode proteins from the same AAV type, or from different AAV types. In certain embodiments, a Rep coding molecule, a Cap coding nucleic acid molecule and Ad nucleic acid molecules can be present on a single vector or a single expression cassette. In certain embodiments, a Cap coding nucleic acid molecule can be stably integrated into a genome of a cell line for AAV production.

[0842] In certain embodiments, a Rep coding nucleic acid molecule and a Cap coding nucleic acid molecule can be present on a single Rep-Cap plasmid. A Rep-Cap plasmid can include an AAV promoter to control expression of AAV Rep and Cap proteins described above. A promoter can be any desired promoter, selected based on known considerations, such as the level of expression of a nucleic acid molecule functionally linked to the promoter and the cell type in which the vector is to be used. A promoter can be tissue / cell-specific. A promoter can be a prokaryotic, eukaryotic, fungal, nuclear, mitochondrial, viral, or plant promoter. A promoter can be exogenous or endogenous to the cell type being transduced by a vector. Promoters can be selected from, for example, bacterial promoters, known strong promoters such as SV40 or the inducible metallothionein promoter. A promoter can be a promoter of any AAV serotypes (e.g., an AAVP5 promoter). A promoter can be a chimeric regulatory promoter for targeted gene expression or promoter derived from actin genes, immunoglobulin genes, cytomegalovirus (CMV), adenovirus, bovine papilloma virus, adenoviral promoters, such as the adenoviral major late promoter, an inducible heat shock promoter, respiratory syncytial virus, Rous sarcomas virus (RSV), and the like. Non-limiting examples of suitable plasmid backbones for a Rep-Cap plasmid can include pHLP19, pETC18, pETC19, and pAAV-RC2. In certain embodiments, the engineered or modified cell line produces a titer of rAAV higher than a wild-type parental cell line. In certain embodiments, the titer of rAAV is aboutl.l-, about 1.2-, about 1.3-, about 1.4-, about 1.5-, about 1.6-, about 1.7-, about 1.8-, about 1.9-, about 2-, about 2.5-, about 3-, about 3.5-, about 4-, about 4.5-, about 5-, about 5.5-, about 6-, about 6.5-, about 7-, about 7.5-, about 8-, about 8.5-, about 9-, about 9.5-, about 10-, about 10.5-, about 11-, about 11.5-, about 12-, about 12.5-, about 13-, about 13.5-, about 14-, about 14.5-, about 15-, about 20-, about 30-, about 40-, about 50-, about 60-, about 70-, about 80-, about 90-, about 100-, about 200-, about 500-, about 1000-fold higher compared to the tier of rAAV produced from a cell line in which the expression of the gene is not altered. In certain embodiments, the engineered or modified cell line (e.g., producer cell line) produces a titer of rAAV at least about 1.5-fold higher compared to the titer of rAAV produced from a cell line in which the expression of the gene is not altered (e.g., a wild-type parental cell line).

[0843] In certain embodiments, as provided herein, is a method for identifying a gene associated with increasing the production of rAAV particles from a cell as described herein, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of : ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, and PARD6G-AS1, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP IB, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2. In certain embodiments, as provided herein, is a method of modulating gene expression in a cell line as provided herein, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of: ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS IL, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, and PARD6G-AS1, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB 1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP IB, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, YY1 , EGFR, IGF 1R, INSR, and GRB2.

[0844] In certain embodiments, as provided herein, is a method for increasing the production of rAAV particles, wherein the method comprises culturing the cells as provided herein, in the presence of a modulator, wherein the expression or activity of at least one of the following genes: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SS18, THEM6, TNFAIP3, EGFR, IGF1R, INSR, and GRB2 is reduced as compared to a control cell line, and / or in which the expression of at least one of ASNS, BCL2, CDKN1 A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1 is increased as compared to a control cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

[0845] In certain embodiments, as provided herein, is a method of modulating gene expression in a cell line as provided herein for increasing the production of rAAV particles from the cell, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of: ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGVDDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, FAM46C, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL2A1, GJB3, ISYNA1, PARD6G-AS1, RP11-268J15.6, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

[0846] In certain embodiments, as provided herein, is a method for increasing the production of rAAV particles, wherein the method comprises culturing a cell as provided herein, in the presence of a modulator, wherein the expression or activity of at least one of the following genes: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SS18, THEM6, and TNFAIP3 is reduced as compared to a control cell line, and / or in which the expression of at least one of ASNS, BCL2, CDKN1A, COL20A1, FAM46C, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1 is increased as compared to a control cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

[0847] In certain embodiments, as provided herein, is a method for identifying a gene associated with increasing the production of rAAV particles from a HEK293 cell as described herein, wherein the method comprises culturing the HEK293 cell in the presence of a modulator that alters the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).

[0848] In certain embodiments, as provided herein, is a method of modulating gene expression in the HEK293 cell line as provided herein, wherein method comprises culturing the HEK293 cell in the presence of a modulator that alters the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).

[0849] In certain embodiments, as provided herein, is a method of modulating gene expression in a HEK293 cell line as provided herein for increasing the production of rAAV particles from the HEK293 cell, wherein the method comprises culturing the HEK293 cell in the presence of a modulator that alters the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).

[0850] In certain embodiments, as provided herein, is a method for increasing the production of rAAV particles, wherein the method comprises culturing a HEK293 cell as provided herein, in the presence of a modulator, wherein the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), under conditions that allow for production and / or secretion of the recombinant viral particles.

[0851] AAV Constructs

[0852] Adeno-associated viruses (AAVs) are small, non-pathogenic viruses of the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV is distinct from other members of this family by its dependence upon a helper virus for replication.

[0853] AAV genomes possess a broad host range, transduce both dividing and non-dividing cells in vitro and in vivo, and maintain high levels of expression of the transduced genes. AAV particles are generally heat stable. AAV particles are also resistant to solvents, detergents, changes in pH, and temperature. They may also be column purified and / or concentrated on gradients (e.g., CsCl gradients) or by other means. The AAV genome comprises a singlestranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The approximately 4.7 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. The ends of the genome are short, inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as the origin of viral DNA replication.

[0854] The AAV virion or particle is a non-enveloped, icosahedral particle approximately 25 nm in diameter that comprises an AAV capsid. The AAV particle comprises an icosahedral symmetry comprised of three related capsid proteins - VP1, VP2, and VP3 - which together form the capsid. The genome of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. The VP1, VP2, and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. The left ORF often encodes the non- structural Rep proteins which are involved in regulation of replication and transcription in addition to the often encodes the non- structural Rep proteins, Rep 40, Rep 52, Rep 68, and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68 / 78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities. Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. The genome of an AAV (e.g., an rAAV) may encode some or all of the Rep proteins. The genome of an AAV (e.g., an rAAV) may not encode the Rep proteins. One or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a recombinant polypeptide.

[0855] AAV Constructs

[0856] In some embodiments, the AAV vectors or expression cassettes of the present disclosure are obtained by cloning at least one heterologous nucleic acid molecule of interest (i.e., a transgene) into an AAV construct. For example, an AAV vector or expression cassette can include recombinant DNA containing a heterologous nucleic acid molecule, such as a transgene, flanked by ITR sequences for packaging into an AAV capsid. An AAV vector or expression cassette can be introduced into a eukaryotic cell line for producing AAV.

[0857] An AAV vector or expression cassette can be constructed using techniques for providing operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest, and a transcriptional termination region. The control elements can be selected to be functional in a mammalian cell. Termination signals, such as polyadenylation sites, can be included in the plasmid.

[0858] In certain embodiments, the nucleic acid sequences as disclosed herein, comprise AAV ITR sequences. These ITR sequences are located 5’ and 3’ to a polynucleotide sequence. The AAV ITR sequences can include any cis elements required for packaging allowing for production of recombinant AAV.

[0859] The nucleic acid can include ITR sequences from any AAV serotype. Nucleic acid molecules of AAV ITR regions are known. An ITR can have a wild type nucleic acid sequence, or a modified sequence, e.g., by the insertion, deletion or substitution of nucleotides. An AAV ITR can be derived from any AAV serotype, including without limitation, AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV13, and 5' and 3' ITRs in an AAV vector can be independently selected therefrom.

[0860] The nucleic acid can include a polynucleotide of any length positioned within two AAV ITR sequences (i.e., a polynucleotide can be flanked by ITR sequences). A polynucleotide can be a transgene of 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, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000 or at least 10000 nucleotides. In certain embodiments, an ITR flanked polynucleotide sequence does not exhibit biological activity. For example, enhancers, promoters, splicing regulators, noncoding RNAs, antisense sequences, and / or coding sequences can be absent from a polynucleotide sequence positioned within two AAV ITR sequences. In certain embodiments, each of enhancers, promoters, splicing regulators, noncoding RNAs, antisense sequences, and coding sequences are absent. In certain embodiments, a polynucleotide sequence positioned within two AAV ITR sequences does not include an open reading frame.

[0861] The AAV vector or expression cassettes can include recombinant DNA containing a heterologous nucleic acid molecule, such as a transgene, flanked by ITR sequences for packaging into an AAV capsid. An AAV vector or expression cassette can be introduced into a eukaryotic cell line for producing AAV.

[0862] An AAV vector or expression cassette can be constructed using techniques for providing operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest, and a transcriptional termination region. The control elements can be selected to be functional in a mammalian cell. Termination signals, such as polyadenylation sites, can be included in the plasmid.

[0863] A heterologous nucleic acid molecule of an AAV vector or expression cassette can include a polynucleotide with biological activity of an anti-sense RNA molecule, shRNA, miRNA, a ribozyme, or a gene encoding a polypeptide of interest, and optionally one or more a nucleic acid sequences capable of directing expression of a particular heterologous nucleic acid sequence in an appropriate host cell (e.g., mammal). Non-limiting of nucleic acid sequences capable of directing expression include a promoter and termination signal. In certain embodiments, a heterologous nucleic acid molecule can be included in a chimeric expression cassette. In certain embodiments, an expression cassette can be naturally occurring, optionally obtained in a recombinant form for heterologous expression.

[0864] In certain embodiments, a heterologous nucleic acid molecule can be 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, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 2000, at least 3000, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 7000 nucleotides, at least 7500 nucleotides, or at least 8000 nucleotides.

[0865] In certain embodiments, a heterologous nucleic acid molecule (e.g., a transgene) can encode a polypeptide such as, but not limited to, a clotting factor, an enzyme, an antibody or other polypeptide of interest. In certain embodiments, a heterologous nucleic acid molecule can encode an RNA having a structural or therapeutic function such as, but not limited to, an antisense, siRNA, shRNA, miRNA, EGSs, gRNA, sgRNA, ribozyme, or aptamer.

[0866] In certain embodiments, a heterologous nucleic acid molecule can encode a peptide, polypeptide, or protein that binds to a specific target of interest, which can be useful for the treatment or prevention of disease in a subject. Examples of such heterologous nucleic acid molecules and associated peptides, polypeptides, or proteins include, but are not limited to, a gene encoding antibodies, MHC molecules, T-cell receptors, B-cell receptors, aptamers, avimers, receptor-binding ligands, or targeting peptides. Antibodies useful in the present disclosure can be monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of a immunoglobulin molecule including an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies and specific binding fragments thereof can be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). An antibody can include an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), or an unspecified class.

[0867] A heterologous nucleic acid molecule comprising a gene of interest (e.g., a transgene) can encode a peptide, polypeptide, or protein that can be useful for the treatment or prevention of disease in a subject.

[0868] A heterologous nucleic acid molecule comprising a gene of interest (e.g., a transgene) can be a gene editing molecule used for modifying a genomic locus of interest (i.e., target) in a cell. A modification can include a disruption, deletion, repair, mutation, addition, alteration, or modification of a gene sequence at a target locus in a gene. Examples of gene-editing molecules include, but are not limited to, endonucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, restriction endonucleases, recombinases, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) proteins.

[0869] In certain embodiments, the heterologous nucleic acid molecule comprising a gene of interest can be a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO). In certain embodiments, the heterologous nucleic acid molecule comprising a gene of interest comprises a template for homologous arm mediated recombination.

[0870] In certain embodiments, an AAV vector or expression cassette can be multi-cistronic. A multi -ci str onic vector or expression cassette can simultaneously express two or more (e.g., 2, 3, 4, 5, 6, or more) separate proteins from the same mRNA. In such cases, the multiple heterologous nucleic acid molecules can be separated by an element that allows for separate translation for each gene.

[0871] In certain embodiments, a large heterologous nucleic acid molecule can be split into more than one construct, whereby multiple constructs can express two or more fragments for assembly into a protein of interest in vivo. In certain embodiments, AAV vectors or expression cassettes do not include a reporter gene or a selection marker. In certain embodiments, the transgene-containing vector or expression cassette does not comprise an antibiotic resistance gene. An AAV vector or expression cassette can include a stuffer sequence as described above.

[0872] Genetic Modulators

[0873] In an aspect of the present disclosure, expression of at least one gene is reduced and / or expression of at least one gene is increased in an engineered or modified cell line, as compared to a control cell line. In certain embodiments, the cell line is modified by a modulator that increases or decreases the expression and / or activity of at least one gene. In certain embodiments, the cell line comprises a genetic modification. In certain embodiments, the modulator is a nuclease.

[0874] Zinc Finger Nucleases

[0875] In certain embodiments, the nuclease is a zinc finger nuclease (ZFN). Targeted genetic engineering in many model systems and in human tissue culture cells has historically been challenging. The development of ZFNs dramatically changed the field. ZFNs are artificial endonucleases that consist of a designed zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme.

[0876] The DNA-binding domains of individual ZFNs typically contain between three and six individual zinc finger repeats and can each recognize between 9 and 18 basepairs. If the zinc finger domains perfectly recognize a 3 basepair DNA sequence, a 3-finger array may be generated that can recognize a 9 basepair target site.

[0877] The non-specific cleavage domain from the type II restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. This cleavage domain must dimerize in order to cleave DNA, and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. To let the two cleavage domains dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart. The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5’ edge of each binding site to be separated by 5 to 7 bp.

[0878] A ZFN may be redesigned to cleave new targets by developing ZFPs with new sequence specificities. For genome engineering, a ZFN is targeted to cleave a chosen genomic sequence. The cleavage event induced by the ZFN provokes a cellular repair process that in turn mediates efficient modification of the targeted genomic locus. The modification is dependent on how the cleavage event is repaired. For instance, if the ZFN-induced cleavage event is resolved via non-homologous end joining, this can result in small deletions or insertions, effectively leading to gene knockout. This approach has been used to establish easy and efficient reverse genetics that does not require selection in a number of model organisms. However, if the break is resolved via a homology -based process in the presence of an investigator-provided donor, small changes or entire transgenes can be transferred, often without selection, into a chromosome. This process is often referred to as a “gene correction” and “gene addition”. Using ZFNs in therapeutic applications require the engineering of ZFNs that are highly specific.

[0879] Meganucleases

[0880] In certain embodiments, the nuclease is a meganuclease. Meganucleases are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs). As a result, the recognition site generally occurs only once in any given genome. Among the meganucleases, the LAGLIDADG family of homing endonucleases has become a valuable tool for the study of genomes and genome engineering over the years. Meganucleases are thought of as “molecular DNA scissors” that can be used to replace, eliminate, or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases are used to modify all genome types, whether bacterial, plant or animal.

[0881] Meganucleases are mainly represented by two enzyme families collectively known as homing endonucleases: intron endonucleases and intein endonucleases. Introns propagate by intervening at a precise location in the DNA, where the expression of the meganuclease produces a break in the complementary intron- or intein-free allele.

[0882] The high specificity of meganucleases gives them a high degree of precision and lower cell toxicity than other naturally occurring restriction enzymes. Since being identified in the 1990s, subsequent work has shown that these enzymes are promising tools for genome engineering and gene editing, as they are able to efficiently induce homologous recombination, generate mutations, and alter reading frames.

[0883] TALENS

[0884] In certain embodiments, the nuclease is a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. They are generally made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease (typically FokT), DNA can be cut at specific locations.

[0885] TAL effectors are proteins that are secreted by Xanthomonas bacteria via their type III secretion system when they infect plants. The DNA binding domain contains a repeated highly conserved 33-34 amino acid sequence with divergent 12thand 13thamino acids. These positions, referred to as the Repeat Variable Diresidue (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. This relatively straightforward relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0886] The DNA sequences encoding TALEN constructs can be inserted into plasmids. The cells targeted for genetic engineering are then transfected with the plasmids, and the gene products are expressed and enter the nucleus to access the genome. Alternatively, TALEN constructs can be delivered to the cells as mRNAs, thereby avoiding any potential genomic integration of the TALEN-expressing protein.

[0887] CRISPR / Cas9

[0888] CRISPR RNA-guided genome engineering has revolutionized research into human genetic disease and many other aspects of biology. The most widely used genome editing tool is the type ILA Cas9 from Streptococcus pyogenes strain SF370 (SpCas9). Cas9 forms a ribonucleoprotein (RNP) complex with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) for efficient DNA cleavage both in bacteria and eukaryotes. The crRNA contains a guide sequence that directs the Cas9 RNP to a specific locus via base-pairing with the target DNA to form an R-loop. This process requires the prior recognition of a protospacer adjacent motif (PAM). R-loop formation activates the His-Asn-His (HNH) and RuvC-like endonuclease domains that cleave the target strand and the non-target strand of the DNA, respectively, resulting in a double-strand break (DSB). In some embodiments, the crRNA and tracrRNA are linked to form a single guide RNA (sgRNA). A CRISPR system described herein is composed of the CRISPR nuclease, or a polynucleotide encoding the nuclease, and a guide RNA, such as an sgRNA. In some embodiments, the Cas9 nuclease is a deactivated or dead Cas9 (dCas9). A dCas9 nuclease comprises one or more mutations that reduce or eliminate the nuclease activity of Cas9. For mammalian applications, Cas9 and its guide RNAs can be expressed from DNA (e.g., a viral vector), RNA (e.g., Cas9 mRNA plus guide RNAs in a lipid nanoparticle), or introduced as a ribonucleoprotein (RNP). Viral delivery of Cas9 results and its guides can result in off-target editing, and viral vectors can elicit strong host immune responses (Mingozzi et al. Blood 122, 23-36 (2013)). RNA and RNP delivery platforms of Cas9 are suitable alternatives to viral vectors for many applications and have recently been shown to be effective genome editing tools in vivo (Yin et al. Nature Biotechnology 35, 1179 (2017); Lee et al. eLife 6, e25312 (2017)). RNP delivery of Cas9 also bypasses the requirement for Cas9 expression, leading to faster editing. Furthermore, Cas9 delivered as mRNA or RNP exists only transiently in cells and therefore exhibits reduced off-target editing. For instance, Cas9 RNPs were successfully used to correct hypertrophic cardiomyopathy (HCM) in human embryos without measurable off-target effects (Ma et al. Nature 548, 413 (2017).

[0889] Small Molecules

[0890] In certain embodiments, the engineered or modified cell line or methods as provided herein comprise a modulator that is a small molecule. In certain embodiments, the modulator is DMSO. In certain embodiments, the modulator is DMF. In certain embodiments, the modulator is NMP. In certain embodiments, the modulator is dihydrolevoglucosenone (Cyrene). In certain embodiments, the DMSO used is at a concentration of at least 0.5%. In certain embodiments, the DMSO used is at a concentration of at least 1%. In certain embodiments, the engineered or modified cell line is cultured in the presence of an inhibitor of a gene as disclosed herein. In certain embodiments, the inhibitor inhibits at least one gene selected from the group consisting of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SSI 8, THEM6, and TNFAIP3. In certain embodiments, the inhibitor comprises a synthetic small molecule inhibitor.

[0891] In certain embodiments, the engineered or modified cell line is cultured in the presence of an agonist of a gene as disclosed herein. In certain embodiments, the agonist is an agonist of at least one gene selected from the group consisting of: ASNS, BCL2, CDKN1A, COL20A1, FAM46C, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1 A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP IB, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, and YY1. In certain embodiments, the agonist comprises a synthetic small molecule agonist.

[0892] In certain embodiments, the engineered or modified HEK293 cell line or methods as provided herein comprise a modulator that is a small molecule. In certain embodiments, the modulator is DMSO. In certain embodiments, the modulator is DMF. In certain embodiments, the modulator is NMP. In certain embodiments, the modulator is dihydrolevoglucosenone (Cyrene). In certain embodiments, the DMSO used is at a concentration of at least 0.5%. In certain embodiments, the DMSO used is at a concentration of at least 1%. In certain embodiments, the engineered or modified HEK293 cell line is cultured in the presence of an inhibitor of a gene as disclosed herein. In certain embodiments, the inhibitor inhibits GAPDH. In certain embodiments, the inhibitor comprises a synthetic small molecule inhibitor. In certain embodiments, the inhibitor is DC-5163, 1,2,3,4,6-penta-O-galloyl-P-D-glucopyranose (PGG), or 4-octyl itaconate (4-01). The inhibitor DC-5163 corresponds to CAS number 897771-47-0 and is described in further detail in Li et al. (Biorg Chem. 96: 103620.2020). incorporated herein by reference.

[0893] RNA-Based Modulators

[0894] In certain embodiments, the modulator is an RNA-based modulator. In certain embodiments, the modulator is a double stranded RNA (dsRNA). In certain embodiments, the modulator is a small interfering RNA (siRNA). In certain embodiments, the modulator is a small hairpin RNA (shRNA). In certain embodiments, the modulator is an antisense RNA oligonucleotide. In certain embodiments, the modulator is a micro RNA (miRNA).

[0895] In certain embodiments, the RNA-based modulator may comprise a substitution. In certain embodiments, the RNA-based modulator may comprise chemically modified nucleotides. In certain embodiments, the RNA-based modulator may comprise nonnucleotides (e.g., substitutions or modifications in the backbone, sugar chains, bases or nucleosides). These modifications may allow the RNA-based modulator to have an increased half-life in a subject or biological sample. In certain embodiments, the modifications improve the cellular uptake of the RNA-based modulator.

[0896] DNA Integration of Expression Construct

[0897] In certain embodiments, increasing the expression or activity is carried out by transient delivery or stable integration of a target expression modulation construct into the host cell line.

[0898] In certain embodiments, increasing the expression or activity is carried out by introduction of an exogenous gene or expression cassette into a cell’s DNA via homology-directed repair (HDR), through homologous recombination. HDR is a precise DNA repair process that uses a template to repair double-strand breaks (DSBs) in DNA. Supplementation of homologous DNA templates with site specific nucleases shifts the innate DNA repair pathway to HDR attaining targeted nucleotide changes. All HDR donor templates contain 5’ and 3’ homology sequences flanking the cut site, to exploit the HDR pathway. A wide range of viral derived or synthetically generated double-stranded and single-stranded HDR donor templates are currently tested for single nucleotide changes to large transgene insertion. Viral-derived HDR donors include integrase defective lentiviral vectors (IDLVs), adenovirus 5 / 35 serotype (AdVs), and adeno-associated vector (AAV) and single-stranded oligo deoxynucleotides (ssODNs) is a non-viral option.

[0899] In certain embodiments, the increase in expression or activity comprises a template for homologous arm mediated recombination. In certain embodiments, the modulator affects expression and / or activity of the target gene alone. In certain embodiments, the modulator affects expression and / or activity of the target gene in combination with another modulator as disclosed herein (e.g., CRISPR). In certain embodiments, the homologous arm mediated recombination results in gene knock-in. In certain embodiments, the homologous arm mediated recombination results in gene correction.

[0900] Homologous arm mediated recombination is one of the most commonly used methods to introduce a genetic mutation into the genome (e.g., gene knock-in). It allows desired and controlled genetic modifications in the genome. In certain embodiments, the homologous arm mediated recombination comprises a DNA repair template. In certain embodiments, the DNA repair template is in the form of a plasmid. In certain embodiments, the genetic modification is in an intron. In certain embodiments, the genetic modification is in an exon. In certain embodiments, the genetic modification is found in the promoter. In certain embodiments, the genetic modification is in an untranslated region (e.g., 5’ or 3’ UTR). In certain embodiments, the increase in expression or activity comprises targeted integration, e.g., integration at a safe harbor locus. A number of safe harbor loci are known, and such a locus can be selected from the group consisting of but not limited to: AAVS1, ALB, AngptB, ApoC3, ASGR2, CCR5, Rosa26, FIX, G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpinal, TF, and TTR. Generally, disruption of these loci does not have an adverse effect on the cell, and robust transcription can be used to maintain the expression of an exogenously inserted gene. Insertion of the gene or gene modulation cassettes can be targeted to these safe harbor loci via homology-directed repair and / or CRISPR / Cas9.

[0901] In certain embodiments, the increase in expression or activity is due to random integration of a target expression modulation construct into the DNA of the host cell.

[0902] In certain embodiments, the increase in expression or activity is due to transposase-based recombination. Transposons are genetic elements that generally have modest target site selectivity and can thus insert themselves into many different DNA sites. In transposition, a specific enzyme known as transposase, is usually encoded by the transposon itself, and acts on a specific DNA sequence at each end of the transposon. It first disconnects it from the flanking DNA and then inserts it into a new target DNA site. There is no requirement for homology between the ends of the element and the insertion site.

[0903] In certain embodiments, the increase in expression or activity is due to an integrase mediated integration or a CRISPR-Cas activation method. In certain embodiments, the Cas comprises a dCas, such as dCas9.

[0904] AAV Compositions and Methods of Use

[0905] AAV produced according to one or more embodiments of the present disclosure can be used for the delivery of nucleic acids to cells in vitro, ex vivo, and in vivo. AAV can be advantageously employed to deliver or transfer nucleic acids to animal cells, including mammalian cells.

[0906] In certain embodiments, a heterologous nucleic acid molecule can encode any polypeptide or RNA for production in a cell in vitro, ex vivo, or in vivo. For example, nucleic acids of interest include nucleic acids, such as genes, encoding polypeptides or RNAs. In certain embodiments, an AAV can be used to deliver a reporter, therapeutic (e.g., for medical or veterinary uses), immunogenic (e.g., for vaccines), or diagnostic polypeptide or RNA. In certain embodiments, an AAV can be used to introduce a heterologous nucleic acid sequence into cultured cells and the expressed gene product isolated therefrom. An AAV produced according to the compositions and methods of the present disclosure can be used for delivering heterologous nucleic acids into a broad range of cells, including dividing and non-dividing cells. An AAV can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy. An AAV can be useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA in a subject. A subject can be in need of a beneficial effect provided by expression of an AAV delivered immunogenic or therapeutic polypeptide or a functional RNA. In certain embodiments, AAV can be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject to observe the effects of a polypeptide or functional RNA on a subject, for example, in connection with screening methods.

[0907] EXAMPLES

[0908] The present disclosure is described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and is not limiting.

[0909] Example 1. DMSO supplementation enhances rAAV production in multiple stable producer cells as well as in transiently transfected mammalian host cells

[0910] To assess the effect of DMSO on recombinant AAV (rAAV) vector production, experiments were performed in the context of both stable producer cell lines (PCL) and transiently transfected mammalian host cells. Production of rAAV in the PCLs was initiated by infection with a helper virus, such as wild type adenovirus type 5 (wtAd5) as shown in the schematic (FIG. 1A). For routine PCL infection, cells were seeded at the target cell density in a proprietary production media and infected with wtAd5 at a multiplicity of 100 DNase-resistant genomes (DRG) per cell. For infection in the presence of DMSO, producer cells were treated with 1% DMSO either three hours prior to wtAd5 infection (-3h) or at the time of infection (Oh). Infections were performed in culture formats ranging from static 6-well plates, 96-well plates, suspension 24-deep well plates, suspension spin tubes or shake flasks and cultured at 37°C, 10% CO2 for 48 or 72 hours before harvesting the culture (cells plus media) for analysis of AAV production.

[0911] In an experiment to determine the optimal concentration of DMSO for enhancement of rAAV production in the PCLs, DMSO was supplemented into production media at various concentrations (0, 0.5, 1, 1.5, 2, and 3%) at the time of infection (Oh). AAV vector production (FIG. IB) and rAAV DNA replication (FIG. 1C) in PCL1 were analyzed in whole cell lysates at three days post infection. The impact of DMSO on wtAd5 virus production and adenoviral DNA replication were also assessed following supplementation, as shown in FIGs. ID and IE, respectively. The results of these experiments demonstrate that peak enhancement of rAAV production is achieved at the concentration of 1% DMSO. There is no significant increase in rAAV DNA replication at any of the DMSO concentrations tested. Likewise, DMSO supplementation did not result in an increase of either wtAd5 production or adenoviral DNA replication at any concentration.

[0912] The impact of DMSO on AAV productivity was subsequently assessed for wtAd5 infections performed at a higher cell density. PCL1 was infected with wtAd5 at two different cell densities X and Y (about four-fold higher density than X; X is the standard cell density) and rAAV productivity was assayed by qPCR. Titer values were normalized to the non-treated control for each infection cell density. The results showed that 1% DMSO supplementation at the time of infection increased AAV production for both the standard infection cell density X and four-fold higher cell infection density Y (FIG. IF).

[0913] Supplementation of production medium with 1% DMSO and the impact on rAAV DNA replication and vector production was assessed in two different PCLs (PCL1 and PCL2) as shown in FIG.2A. Addition of 1% DMSO at both 3 hr prior to infection (-3h) and at the time of infection (Oh) increased AAV production from PCL1 but not PCL2 (FIG.2B). To assess the biological factors contributing to the productivity increase, total vector genomes, total capsids, percent full capsids and rAAV genome packaging efficiency were analyzed in samples collected at 72 hr post-infection. rAAV genome packaging efficiency (FIG.2E) represents the ratio of rAAV vector titer (i.e., packaged vector genomes) to total rAAV vector genomes (FIG.

[0914] 2C) (tvg; i.e., total vector DNA copies) as assessed via qPCR. Total capsids were estimated via ELISA (FIG.2D). Capsid packaging efficiency (% full capsids) was estimated as the ratio of rAAV vector titer to total capsids (FIG. 2F). DMSO treatment increased AAV capsid production, AAV vector genome packaging and capsid packaging efficiency in PCL1 but not PCL2, consistent with the effect on rAAV productivity in the former cell line.

[0915] Western blot analysis (FIG. 2G) was performed to evaluate the effect of DMSO on AAV Rep and Cap protein expression. Cell pellets collected at 0, 24, 48 and 72 hours postinfection (hpi) were lysed and subjected to electrophoresis and then transferred to a nitrocellulose membrane. Analysis of AAV Rep and Cap protein expression (FIG.2G) showed that DMSO increased Cap protein and expression of Rep52 / Rep40 in PCL1 but not in PCL2, consistent with AAV capsid production, capsid packaging efficiency and genome packaging results in PCL1. These results suggest that DMSO increases AAV production by improving both capsid production as well as the packaging of rAAV genomes into the capsid.

[0916] Additional parameters were evaluated following treatment of PCL1 and PCL2 with DMSO at both timepoints, 3 hours prior to infection (3h) and at the time of infection (Oh). Cell viability was analyzed at Ohpi, 24hpi, 48hpi, and 72 hpi. The results (FIGs.3A and 3B) show that DMSO had no significant impact on percent viability in either PCL1 or PCL2; one exception is a trend toward an increase in viability at the 72 hpi timepoint in PCL2.

[0917] Because DMSO is known to increase cell permeability, DMSO might exert its effect on rAAV productivity by increasing the efficiency of infection of PCLs with wtAd5. To test this hypothesis, the impact of DMSO treatment on wtAd5 production (FIG. 3C) was assessed following infection of PCL1 and PCL2. Relative Ad5 productivity was determined by qPCR in whole cell lysates and the results were normalized to the control condition (no DMSO) for each PCL. Similar levels of wtAd5 production were observed in PCL1 in DMSO treated and control cultures but a trend towards reduced Ad5 production was observed in PCL2 with DMSO treatment (FIG. 3C). In addition, AdElA protein expression was evaluated as a measure of wtAd5 infectivity. El A protein levels were assessed via Western analysis in PCL1 and PCL2 at 0, 24, 48 and 72 hpi (FIG. 3D). DMSO treatment increased El A expression in both PCL1 and PCL2. Infection of PCL1 and PCL2 with Ad5-GFP was also carried out to assess the impact of DMSO on infection efficiency (FIG. 3E). The percentage of cells expressing GFP was analyzed via Celigo at 24 hpi. The results suggested that DMSO had no impact on Ad5-GFP infection in PCL1 regardless of the time of treatment; however, DMSO appeared to increase Ad5-GFP infection in PCL2 when added at 3 hpi.

[0918] The effect of DMSO on vector quality was analyzed on rAAV produced following wtAd5 infection of PCL1 in ambr250 bioreactors. 1% DMSO was added at time of cell inoculation into the ambr250 modular bioreactor and wtAd5 infection was performed at 3 hours post cell seeding. Analysis of relative AAV productivity showed a two-fold increase in 1% DMSO treated samples (DMSO was added at the time of ambr250 inoculation, which was 3 hours prior to wtAd5 infection) compared to the no-DMSO control. This result is consistent with data generated in smaller scale infections (FIG. 1A and FIG. 2B). FIG. 4B shows SDS PAGE analysis of VP1:VP2:VP3 ratios in purified vector following detection of the Cap proteins by Coomassie blue staining. An equal number of purified vector genome copies of each sample was loaded onto the SDS-PAGE. The results show that the capsid protein ratios in vector produced in the presence and absence of DMSO are similar. Full capsid percentage (FIG. 4C) as determined by analytical ultracentrifugation (AUC) was slightly (1.5-fold) higher for vector produced in the presence of DMSO. Vector potency was evaluated using an enzyme activity assay for the transgene in PCL1 (FIG. 4D). Cells were transduced with vector purified from control or DMSO-treated samples at MOIs of 1E3 vg / cell and 1E4 vg / cell. Culture supernatants were collected 6 days post-infection and assayed for enzyme activity. Relative enzyme activity as shown in the graph was normalized to the control condition (FIG.4D). The results show no significant difference in potency between the two vectors produced in the presence and absence of DMSO.

[0919] Additional experiments were carried out to understand whether the effect of DMSO on rAAV productivity would extend to transiently transfected mammalian cells. For HEK293 cell transfection, Polyplus FectoVIR was used to transfect a serum-free suspension culture of HEK293 (0.5 pg pDNAper 1E6 viable cells). Two or three plasmid DNAs (e.g., pAdhelper as one plasmid and pAAV-Rep / Cap / ITR / transgene as a second plasmid, or pAdhelper, pAAV-RepCap and pITR-transgene as two separate plasmids) were used for the transfection. The transfection complex was generated using a plasmid DNA : FectoVIR ratio of 1:1 at 5% complexing volume. The transfection complex was then added into HEK293 cells within 25 min of initiation of the complex formation. Either 1% DMSO or an equal volume of culture medium was added at the time of transfection. Transfected cultures were collected at 72 hr post-transfection (hpt) for AAV replication and production analysis. In addition, cell pellets were collected for analysis of KAT5 mRNA via qRT-PCR.

[0920] HEK293 cells were transfected to produce two rAAV vectors of different serotypes and transgenes (CapaGenei and CapbGene?) along with transfection reagent only and untransfected controls. Assessment of relative rAAV productivity following DMSO treatment shows about a 2-fold enhancement of rAAV titer with DMSO, regardless of the capsid serotype or transgene. In addition, KAT5 mRNA expression in HEK293 cells treated with 1% DMSO was reduced approximately 2-fold (FIG. 5B). These results suggest that DMSO treatment exerts a positive effect on rAAV production in transiently transfected HEK293 cells as observed in the PCLs. The reduction of KAT5 mRNA expression in DMSO-treated HEK293 cells points toward a possible shared mechanism (see Examples below).

[0921] Example 2. RNA-Seq analysis elucidates regulators of rAAV production induced by DMSO

[0922] To understand the impact of DMSO on host cell gene expression, infected cell pellets were collected for poly-A enriched RNA-Seq analysis. Briefly, AAV producer cell lines were infected with wtAd5 in the presence or absence of DMSO. For control samples (infection in the absence of DMSO), infection was performed as described above. For DMSO supplemented samples, 1% DMSO were added into growth medium 3 hrs prior to wtAd5 infection (-3h) or added at the time of infection (Oh) and kept in the medium throughout the infection. Cell pellets were collected and snap frozen at Oh, 24h and 48h post-infection (FIG. 11A-11B). Whole cultures were collected at 48hpi / 72hpi for AAV titer analysis to confirm the DMSO response. Total RNA extraction, library preparation and Illumina sequencing was performed at Genewiz.

[0923] Following the addition of DMSO, there was several fold-increase in AAV transcriptional expression at 48 hours post wtAd5 infection (as described in previous example). Thus, it was clear that the medium supplementation aided in AAV biosynthesis. However, it was unclear what exact changes in cell state favored this increase in AAV expression. Furthermore, the two producer cell lines responded to DMSO to different degrees, and it was unclear how the compositional differences between cell lines could explain the discrepancy in AAV expression. An initial descriptive summary of these changes as induced by DMSO was pursued by a Gene Ontology (GO) Over Representation Analysis (ORA) on the available RNA-Seq data (FIG. 11C).

[0924] To facilitate this analysis mRNA abundance was quantified in both PCLs to test which GO terms were overrepresented. Specifically, following analysis of differential gene expression in PCL1 in control or supplemented media at each time point, a total of 3327 differentially expressed genes (DEGs) with an absolute log2 fold-change greater than 1 and a Benjamini Hochberg corrected p-value less than 0.05 were retained for the GO ORA. We found that among a set of curated GO terms, the sets of significantly enriched Biological Process (BP; n = 8) and Molecular Function (MF; n = 13) GO terms associated with the change in media are supported by literature on AAV PCLs (FIG. 12A). Significantly enriched pathways associated with differentially expressed genes were measured from the comparison of control and DMSO supplemented media in PCL1 and PCL2 and over representation analysis was conducted using GO terms. For example, the top 3 BPs of cell-cell signaling, translation, and DNA repair are all functions known to be targeted directly or through an intermediary by AAV and Ad5 machinery (FIG. 12A).

[0925] Upon further analysis, these enriched terms were manually categorized in the context of their biological function, indicated that host transport / localization, translation / transcription, signaling / cell communication, and binding machinery were likely to be affected by DMSO supplementation. Of these, the translation / transcription subsystems retained the greatest number of enriched BP (n = 3) and MF (n = 6) GO terms with greatest significance (1.306E-18 < p < 0.043) indicating that supplement-induced changes in upstream transcription factor (TF) expression could be responsible for corresponding increases in rAAV vector titer (Tables 1-4). Correlative support for this observation is drawn from the DE heatmaps of genes as subset by participation in RNA polymerase II cis-regulatory region sequence-specific DNA binding, DNA repair and RNA binding ontologies, where media supplementation of DMSO to PCL1 appears to decrease expression before 24 hours (FIG. 12E). A similar trend is observed for DEGs participating in cell-cell signaling, suggesting an additional role (albeit with unknown impact on viral expression) of media supplementation in signaling and cell communication subsystems (FIG. 12B).

[0926] Differentially expressed genes (DEGs) from the comparison of producer cells infected with wtAd5 in the presence or absence of DMSO were used to generate significantly enriched pathways via GO ORA. The FDR corrected p-value cutoff was 0.05 and is represented by vertical dotted black line. GO terms with a p-value <0.05 were considered as significant (FIG.

[0927] 12A). DEGs were shown as log2 Fold Change in heatmaps for example GO terms (FIG. 12B). FIG. 12C is a table of transcription factors and unique targets associated with an increase in viral titer predicted by Ingenuity Pathway Analysis (IPA).

[0928] To identify potential cell engineering targets, GO ORA results were leveraged, along with an analysis of upstream regulators of differential gene expression induced by DMSO. The rationale for this was the hypothesis that upstream transcription factors (TFs) of differentially expressed genes could be responsible for the corresponding increases in AAV viral expression observed following DMSO treatment. Emulation of the rAAV titer response by knock down (KD), knock out (KO), or overexpression of participating TFs can facilitate both a deeper understanding of the mechanistic networks driving rAAV biosynthesis and can eliminate the costs associated with DMSO supplementation. Several TFs and their targets were identified using IPA through use of the mechanistic and causal network features in the upstream regulator analysis (URA). Multiple candidate TFs and corresponding targets unique to each TF were identified as potential upstream regulators of overall rAAV viral expression (Tables 1-4; FIG.

[0929] 13-14) Further quantitative information on each identified upstream regulator UR and their comprehensive set of downstream targets as identified from IPA URA that pass both mechanistic and causal network filters are listed in Table 3 and Table 4. Additional upstream regulators and targets of interest identified solely from the URA include CCND1, SSI 8, HDAC5, SMAD4, NME1, PTEN, BCL2, laslL, TGM2, NGEF, TRPC1, HHEX, LIF, IL6, FAS, ITGA2B, NFKBIA, HSP90B1, TCF7, RIGI, FGFR3, ADM2, and NFKB1 and can be found in Tables 1-4. Finally, additional genes of interest with known response to viral infection identified solely from differential expression testing analyses were identified. Brief descriptions of example candidate TFs are as follows:

[0930] Chromodomain-Helicase DNA-binding 1 (CHD1) is a chromatin remodeling protein that is highly conserved across species and is named after the functional capacity of their chromo, helicase-like and DNA-binding domains that bind to lysine residues, nucleic acids, and methylated histone H3K4 typically associated with the 5' end of actively transcribed genes.

[0931] Evolutionarily conserved signaling intermediate in toll pathway (ECSIT) is a protein with at least 2 functional isozymes participating in viral infection pathology, alongside typical cellular physiological processes of redox metabolism, innate immunity, and development.

[0932] Forkhead box like 2 (FOXL2) is a member of the functionally diverse FOX family of TFs, all of which are identifiable by a 100-residue forkhead DNA binding domain; FOXL2 functional capacity is pivotal for ovarian development.

[0933] Homeobox A5 (H0XA5) is a member of the HOX family of TFs associated with pivotal roles in embryonic development; H0XA5 is positively regulated by another TF, Yin Yang 1 (YY1) and its expression can upregulate regulatory tumor protein p53 expression.

[0934] Retinoid X receptor alpha (RXRa), in response to retinoic acid (RA) as ligand, participates in either homo- or hetero-dimer complexes with RA receptors to bind the RA response element of target gene promoters in order to regulate their expression.

[0935] Thioesterase superfamily member 6 (THEM6) is a member of the ER-associated THEs participating in the regulation of fatty acid intracellular trafficking, lipogenesis and 0-oxidation; overexpression of THEM6 has been shown to increase de novo sterol synthesis. GATA-binding factor 1 (GATA1) is one of 6 members within the family so called for the recognition and binding of the DNA consensus sequence GATA and maintains essential functions in both normal erythropoiesis and regulation of the Gl / S cell cycle progression. FIG.

[0936] 14B shows GATA1 downstream target gene expression as influenced by Ad5 infection over time.

[0937] Histone acetyltransferase KAT5 is a member of the MYST family of acetyltransferases that can acetylate histones in the nucleosome allowing for the interaction of other TFs. FIG.

[0938] 14A shows KAT5 downstream target gene expression as influenced by Ad5 infection over time.

[0939] Example 3. siRNA knockdown and AAV productivity impact assessment

[0940] To assess the impact of target gene knockdown on AAV production in the PCLs, cells were transfected with siRNA followed by wtAd5 infection in the presence or absence of 1% DMSO treatment at the time of infection (schematic of experimental design provided in FIG. 6A). siRNAs against several target genes of interest identified from the RNA-Seq analysis were resuspended with nuclease-free sterile water to create a 10 pM stock solution (Table 5). 20 pmol siRNA against each target were transfected into 4E5 total viable producer cells in Nucleocuvette Strip format using Lonza 4D nucleofector and SE Cell Line 4D-Nucleofector X kit S (Lonza) following the manufacturer’s protocol. Cells were resuspended and cultured in 24-well plates for 48 hours before infection with wtAd5 and then harvested for assessment of AAV production at 48 hpi (corresponds to 96 hours post-transfection (hpt) with the siRNA). The results of the siRNA screen (bar graph, FIG. 6B; scatterplot, FIG. 6C) identify KAT5 as a promising target. Knockdown of KAT5 increases rAAV productivity about 2-fold while no further increase is observed following addition of DMSO. These results suggest that KAT5 might be involved in the mechanism of action of DMSO.

[0941] PCL1 was transfected with either a negative control siRNA (siN.C) or siKAT5 followed by Ad5 infection as described above using the Lonza 4D nucleocuvette with 4E6 total viable cells. Cell cultures were collected for evaluation of rAAV DNA replication and rAAV production; cell pellets were collected for gene expression analysis via RT-qPCR and assessment of protein expression via Western Blot. rAAV productivity was evaluated following knockdown of the KAT5 gene (FIG. 7A). Values shown in the graph were normalized to the PCL1 transfection with siN.C in the absence of DMSO. In cells transfected with siN.C, supplementation with 1% DMSO increased rAAV productivity approximately 2.5-fold. Cells transfected with siKAT5 showed a similar 2.5-fold increase in rAAV production in both the absence or presence of DMSO. rAAV DNA replication was assessed similarly (FIG. 7B).

[0942] Replication was not significantly increased with DMSO treatment in control cells or with knockdown of KAT5. The results suggest that knockdown of KAT5 simulates the effect of DMSO on rAAV production in PCL1.

[0943] Expression of the AAV Rep and Cap (FIG.7C) and Adenoviral El A (FIG.7D) proteins were evaluated by Western blot. In cells transfected with the control siRNA, an increase in expression of all four AAV Rep proteins (Rep78, 68, 52 and 40) as well as the AAV Cap proteins was observed with DMSO treatment. Knockdown of KAT5 also increases expression of AAV Rep and Cap proteins relative to the control (siN.C, -DMSO) both in the presence and absence of DMSO. Likewise, AdElA protein expression is enhanced by DMSO treatment in control cells and by KAT5 knockdown independent of DMSO treatment (FIG. 7D). Overall, the analysis of AAV Rep, Cap and AdElA expression via Western suggests that KAT5 knockdown recapitulates the effect of DMSO on expression of these viral proteins. To assess whether the increase in AdEl A protein expression results from enhanced El A gene transcription, mRNA levels were analyzed via qRT-PCR (FIG. 7E). Values shown in the graph were normalized to cells transfected with siN.C in the absence of DMSO. Though a trend toward an increase was observed, AdElA mRNA was not significantly increased in control cells treated with DMSO, while in cells transfected with siKAT5, AdElA mRNA was increased approximately 3.5-fold and 6-fold in the absence and presence of DMSO, respectively.

[0944] Table 5. siRNA Target List

[0945] RefSeq

[0946] Gene

[0947] Accession Full Gene Name Gene ID siRNA ID Symbol

[0948] Numbers Number

[0949] NM_02486

[0950] 1 6 ADM2 adrenomedullin 2 79924 s36652

[0951] NM_02486

[0952] 2 6 ADM2 adrenomedullin 2 79924 s36653

[0953] NM_00065

[0954] 3 7 BCL2 B-cell CLL / lymphoma 2 596 S1915

[0955] NM_00065

[0956] 4 7 BCL2 B-cell CLL / lymphoma 2 596 S1916

[0957] NM_05305

[0958] 5 6 CCND1 cyclin D 1 595 s229

[0959] NM_05305

[0960] 6 6 CCND1 cyclin D 1 595 s230

[0961] NM_00127 chromodomain helicase

[0962] 7 0 CHD1 DNA binding protein 1 1105 s2974

[0963] NM_00127 chromodomain helicase

[0964] 8 0 CHD1 DNA binding protein 1 1105 s2975

[0965] NM_01431 DEAD (Asp-Glu-Ala- 9 4 DDX58 Asp) box polypeptide 58 23586 s24143

[0966] NM_01431 DEAD (Asp-Glu-Ala- 10 4 DDX58 Asp) box polypeptide 58 23586 s24144

[0967] NM_01658 ECSIT homolog

[0968] 11 1 ECSIT (Drosophila) 51295 s27873

[0969] NM_01658 ECSIT homolog

[0970] 12 1 ECSIT (Drosophila) 51295 s27874

[0971] enoyl-Coenzyme A,

[0972] hydratase / 3 -hydroxyacyl

[0973] NM_00196 EHHAD Coenzyme A

[0974] 13 6 H dehydrogenase 1962 s4549

[0975] enoyl-Coenzyme A,

[0976] hydratase / 3 -hydroxyacyl

[0977] NM_00196 EHHAD Coenzyme A

[0978] 14 6 H dehydrogenase 1962 s4550

[0979] NM_00004 Fas (TNF receptor

[0980] 15 3 FAS superfamily, member 6) 355 S1506

[0981] NM_00004 Fas (TNF receptor

[0982]

[0983] 16 3 FAS superfamily, member 6) 355 S1507 RefSeq

[0984] Gene

[0985] Accession Full Gene Name Gene ID siRNA ID Symbol

[0986] Numbers Number

[0987] NM_00014 fibroblast growth factor

[0988] 17 2 FGFR3 receptor 3 2261 s5167 NM_00014 fibroblast growth factor

[0989] 18 2 FGFR3 receptor 3 2261 s5168 XM_00113

[0990] 19 1060 FOXL2 forkhead box L2 668 s2068 XM_00113

[0991] 20 1060 FOXL2 forkhead box L2 668 s2069 GATA binding protein 1

[0992] NM_00204 (globin transcription

[0993] 21 9 GATA1 factor 1) 2623 s5593 GATA binding protein 1

[0994] NM_00204 (globin transcription

[0995] 22 9 GATA1 factor 1) 2623 s5594 NM_00547

[0996] 23 4 HDAC5 histone deacetylase 5 10014 si 9462

[0997] NM_00547

[0998] 24 4 HDAC5 histone deacetylase 5 10014 S19463

[0999] NM_01045

[1000] 25 3 Hoxa5 homeobox A5 15402 s67675

[1001] NM_01045

[1002] 26 3 Hoxa5 homeobox A5 15402 s67676 heat shock protein

[1003] NM_00329 90kDa beta (Grp94),

[1004] 27 9 HSP90B1 member 1 7184 S14373 heat shock protein

[1005] NM_00329 90kDa beta (Grp94),

[1006] 28 9 HSP90B1 member 1 7184 S14374

[1007] NM_00060 interleukin 6 (interferon,

[1008] 29 0 IL6 beta 2) 3569 s7311 NM_00060 interleukin 6 (interferon,

[1009] 30 0 IL6 beta 2) 3569 s7312 integrin, alpha 2b

[1010] (platelet glycoprotein lib

[1011] NM_00041 of Ilb / IIIa complex,

[1012] 31 9 ITGA2B antigen CD41) 3674 s7538 integrin, alpha 2b

[1013] (platelet glycoprotein lib

[1014] NM_00041 of Ilb / IIIa complex,

[1015] 32 9 ITGA2B antigen CD41) 3674 s7539 NM_00638 K(lysine)

[1016] 33 8 KAT5 acetyltransferase 5 10524 s20629

[1017] NM_00638 K(lysine)

[1018] 34 8 KAT5 acetyltransferase 5 10524 s20630

[1019] NM_03120 LASl-like (S.

[1020] 35 6 LAS IL cerevisiae) 81887 s37860

[1021] NM_03120 LASl-like (S.

[1022] 36 6 LAS IL cerevisiae) 81887 s37861 leukemia inhibitory

[1023] NM_00230 factor (cholinergic

[1024]

[1025] 37 9 LIF differentiation factor) 3976 s8168 RefSeq

[1026] Gene

[1027] Accession Full Gene Name Gene ID siRNA ID Symbol

[1028] Numbers Number

[1029] leukemia inhibitory

[1030] NM_00230 factor (cholinergic

[1031] 38 9 LIF differentiation factor) 3976 s8169 nuclear factor of kappa

[1032] NM_00399 light polypeptide gene

[1033] 39 8 NFKB1 enhancer in B-cells 1 4790 s9504 nuclear factor of kappa

[1034] NM_00399 light polypeptide gene

[1035] 40 8 NFKB1 enhancer in B-cells 1 4790 s9505 nuclear factor of kappa

[1036] light polypeptide gene

[1037] NM_00107 enhancer in B-cells 2

[1038] 41 7493 NFKB2 (p49 / pl00) 4791 s9507 nuclear factor of kappa

[1039] light polypeptide gene

[1040] NM_00107 enhancer in B-cells 2

[1041] 42 7493 NFKB2 (p49 / pl00) 4791 s9508 nuclear factor of kappa

[1042] light polypeptide gene

[1043] NM_02052 enhancer in B-cells

[1044] 43 9 NFKBIA inhibitor, alpha 4792 s9510 nuclear factor of kappa

[1045] light polypeptide gene

[1046] NM_02052 enhancer in B-cells

[1047] 44 9 NFKBIA inhibitor, alpha 4792 s9511 neuronal guanine

[1048] NM_01985 nucleotide exchange

[1049] 45 0 NGEF factor 25791 s24487 neuronal guanine

[1050] NM_01985 nucleotide exchange

[1051] 46 0 NGEF factor 25791 S230253

[1052] NM_19817 NME / NM23 nucleoside

[1053] 47 5 NME1 diphosphate kinase 1 4830 s9588 NM_19817 NME / NM23 nucleoside

[1054] 48 5 NME1 diphosphate kinase 1 4830 s9590 nuclear receptor

[1055] subfamily 3, group C,

[1056] NM_00102 member 1

[1057] 49 4094 NR3C1 (glucocorticoid receptor) 2908 s6186 nuclear receptor

[1058] subfamily 3, group C,

[1059] NM_00102 member 1

[1060] 50 4094 NR3C1 (glucocorticoid receptor) 2908 s6187 NM_18180 polymerase (DNA

[1061] 51 8 POLN directed) nu 353497 s51479

[1062] NM_18180 polymerase (DNA

[1063] 52 8 POLN directed) nu 353497 s51480

[1064] NM_00031 phosphatase and tensin

[1065] 53 4 PTEN homolog 5728 s325 NM_00031 phosphatase and tensin

[1066]

[1067] 54 4 PTEN homolog 5728 s326 RefSeq

[1068] Gene

[1069] Accession Full Gene Name Gene ID siRNA ID Symbol

[1070] Numbers Number

[1071] v-rel

[1072] reticuloendotheliosis

[1073] NM_00650 viral oncogene homolog

[1074] 55 9 RELB B 5971 S11917 v-rel

[1075] reticuloendotheliosis

[1076] NM_00650 viral oncogene homolog

[1077] 56 9 RELB B 5971 S11918

[1078] NM_00294 replication protein Al,

[1079] 57 5 RPA1 70kDa 6117 S12127

[1080] NM_00294 replication protein Al,

[1081] 58 5 RPA1 70kDa 6117 S12128

[1082] NM_00295 retinoid X receptor,

[1083] 59 7 RXRA alpha 6256 S12384

[1084] NM_00295 retinoid X receptor,

[1085] 60 7 RXRA alpha 6256 S12385

[1086] NM_00535

[1087] 61 9 SMAD4 SMAD family member 4 4089 s8403 NM_00535

[1088] 62 9 SMAD4 SMAD family member 4 4089 s8405 synovial sarcoma

[1089] NM_00563 translocation,

[1090] 63 7 SS18 chromosome 18 6760 S13510 synovial sarcoma

[1091] NM_00563 translocation,

[1092] 64 7 SS18 chromosome 18 6760 S13511

[1093] NM_00320 transcription factor 7 (T- 65 2 TCF7 cell specific, HMG-box) 6932 S13877

[1094] NM_00320 transcription factor 7 (T- 66 2 TCF7 cell specific, HMG-box) 6932 S13878

[1095] NM_19895

[1096] 67 1 TGM2 transglutaminase 2 7052 S14087

[1097] NM_19895

[1098] 68 1 TGM2 transglutaminase 2 7052 S14088

[1099] NM_01664 thioesterase superfamily

[1100] 69 7 THEM6 member 6 51337 s27976

[1101] NM_01664 thioesterase superfamily

[1102] 70 7 THEM6 member 6 51337 s27977

[1103] NM_00629 tumor necrosis factor,

[1104] 71 0 TNFAIP3 alpha-induced protein 3 7128 S14259

[1105] NM_00629 tumor necrosis factor,

[1106] 72 0 TNFAIP3 alpha-induced protein 3 7128 S14260 transient receptor

[1107] NM_00330 potential cation channel,

[1108] 73 4 TRPC1 subfamily C, member 1 7220 S14409 transient receptor

[1109] NM_00330 potential cation channel,

[1110] 74 4 TRPC1 subfamily C, member 1 7220 S14410 76 NM_00078 DBH dopamine beta1621 s3946

[1111] 7 hydroxylase (dopamine

[1112]

[1113] beta-monooxygenase) RefSeq

[1114] Gene

[1115] Accession Full Gene Name Gene ID siRNA ID Symbol

[1116] Numbers Number

[1117] 77 NM_00063 ITGAM integrin, alpha M 3684 s7565

[1118] 2 (complement component

[1119] 3 receptor 3 subunit)

[1120] 78 NM_00063 ITGAM integrin, alpha M 3684 s7566

[1121] 2 (complement component

[1122] 3 receptor 3 subunit)

[1123] 79 NM_01770 FAM46C family with sequence 54855 s29596

[1124] 9 similarity 46, member C

[1125] 80 NM_01770 FAM46C family with sequence 54855 s29597

[1126] 9 similarity 46, member C

[1127] 81 NM_00272 HHEX hematopoietically 3087 s6534

[1128] 9 expressed homeobox

[1129] 82 NM_00272 HHEX hematopoietically 3087 s6535

[1130] 9 expressed homeobox

[1131] 83 NM_00647 TXNIP thioredoxin interacting 10628 s20878

[1132] 2 protein

[1133] 84 NM_00647 TXNIP thioredoxin interacting 10628 s20879

[1134] 2 protein

[1135] 85 NM_13343 ASNS asparagine synthetase 440 S1678

[1136] 6

[1137] 86 NM_13343 ASNS asparagine synthetase 440 S1679

[1138] 6

[1139] 87 NM_07846 CDKN1A cyclin-dependent kinase 1026 s415

[1140] 7 inhibitor 1A (p21, Cipl)

[1141] 88 NM_07846 CDKN1A cyclin-dependent kinase 1026 s416

[1142] 7 inhibitor 1A (p21, Cipl)

[1143] 89 NM_02520 PLEKHO pleckstrin homology 80301 s37165 domain containing,

[1144] 1 2

[1145] family O member 2

[1146] 90 NM_02520 PLEKHO pleckstrin homology 80301 s37166 domain containing,

[1147] 1 2

[1148] family O member 2

[1149] 91 NM_17366 C5ORF64 hypothetical protein 285668 s49998

[1150] FLJ37543

[1151] 7

[1152] 92 NM_17366 C5ORF64 hypothetical protein 285668 s49999

[1153] FLJ37543

[1154] 7

[1155] 93 NM_02088 COL20A collagen, type XX, alpha 57642 s33496

[1156] 1

[1157] 2 1

[1158] 94 NM_02088 COL20A collagen, type XX, alpha 57642 s33497

[1159] 1

[1160] 2 1

[1161] 95 NM_02400 GJB3 gap junction protein, 2707 s5778 beta 3, 3 IkDa

[1162] 9

[1163]

[1164] RefSeq

[1165] Gene

[1166] Accession Full Gene Name Gene ID siRNA ID Symbol

[1167] Numbers Number

[1168] 96 NM_02400 GJB3 gap junction protein, 2707 s5779

[1169] beta 3, 3 IkDa

[1170] 9

[1171] 97 NM_01636 ISYNA1 inositol-3-phosphate 51477 s28195

[1172] synthase 1

[1173] 8

[1174] 98 NM_01636 ISYNA1 inositol-3-phosphate 51477 s28196

[1175] synthase 1

[1176] 8

[1177] 99 NR_028339 PARD6G- PARD6G antisense RNA 100130522 n514828

[1178] 1

[1179] AS1

[1180] 100 NR_028339 PARD6G- PARD6G antisense RNA 100130522 n514829

[1181] 1

[1182] AS1

[1183]

[1184] The universality of the DMSO treatment effect and KAT5 knockdown was assessed in additional cell lines (PCL1-6) which produce five different vectors composed of four different capsids and expressing five transgenes. Relative rAAV productivity was examined in PCL1, PCL2, PCL3, and PCL4, in deep well plates with or without (+ / -) 1 % DMSO treatment at the time of infection (0 h) (FIG. 8A). The results show that DMSO enhances rAAV productivity 2-3 fold in all four PCLs in the deep well culture condition, including PCL2 which showed no AAV productivity response to DMSO in shake flasks in three independent runs shown in FIG.

[1185] 2B

[1186] KAT5 knockdown was evaluated in five different PCLs (FIG. 8B) as previously described in FIG. 6A. Relative rAAV productivity was analyzed following transfection with KAT5 siRNA(s20629) or scramble control siRNA (siN.C) with (+) or without (-) DMSO supplementation and normalized to transfection with the control siRNA in the absence of DMSO treatment. siKAT5 knockdown increased AAV production from PCL4, PCL5, and PCL6 in the absence of DMSO, but not from PCL2 and PCL3, which differs from the observations made upon DMSO treatment in deep well plates as shown in FIG. 8A. However, this was consistent with the results shown in FIG. 2B, showing no effect of DMSO on PCL2 in three independent shake flask tests. In PCL5 and PCL6, siKAT5 or DMSO treatment alone each increased AAV production to a similar level but DMSO treatment in addition to KAT5 knockdown did not lead to a further increase in productivity. In contrast, knockdown of KAT5 in PCL4 in the absence of DMSO increased productivity 4-fold while no significant effect was observed following DMSO treatment. These results suggest DMSO increases AAV production partly via down-regulation of KAT5 at least from PCL1, PCL5 and PCL6.

[1187] To further evaluate if the downregulation of KAT5 mRNA expression is a general cellular response to DMSO treatment, five different PCLs as well as the parental host cell were cultured in spin tubes with or without 1% DMSO and with or without wtAd5 infection. Cell pellet samples were collected at 48 hours post cell seeding for KAT5 mRNA analysis using RT-qPCR (FIG. 8C). Total RNA was extracted using the RNeasy plus kit (Qiagen) according to manufacturer’s instructions, and reverse transcribed to cDNA using Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems,) followed by qPCR with QuantStudio7 flex using primers and probes listed in Table 6. Relative KAT5 mRNA levels shown in the bar graph were calculated following normalization to the uninfected, nontreated control condition for each cell line. The results (FIG. 8C) show that in the absence of DMSO, wtAd5 infection reduces KAT5 mRNA levels in each cell line, with the magnitude ranging from 2.5 to 5-fold. In uninfected cells, KAT5 mRNA is decreased approximately 2-fold by DMSO treatment. In wtAd5 -infected cells, DMSO exerts an additive effect and further suppresses KAT5 mRNA levels in most cell lines (exceptions are PCL4 and the parental cells).

[1188] Table 6. Primers / probe sequences

[1189] Primer, probes Oligo nucleotides Source Identifier BGH-Froward primer TCTAGTTGCCAGCCATCT Integrated DNA

[1190] GTTGT Technologies

[1191] BGH-Reverse primer TGGGAGTGGCACCTTCC Integrated DNA

[1192] A Technologies

[1193] BGH-Probe TCCCCCGTGCCTTCCTTG Integrated DNA

[1194] ACC Technologies

[1195] ElA-Forward primer GTCCTTCTAACACACCTC Integrated DNA

[1196] CTG Technologies

[1197] ElA-Reverse primer CAGGCTGTGGAATGTATC Integrated DNA

[1198] GAG Technologies

[1199] ElA-Probe TGTGCCCCATTAAACCAG Integrated DNA

[1200] TTGCC Technologies

[1201] E2A -Forward primer TTGCGTCGGTGTTGGAGA Integrated DNA

[1202] T Technologies

[1203] E2A -Reverse primer CAAGGCCAAGATCGTGA Integrated DNA

[1204] AGAA Technologies

[1205] E2A-Probe TGCACCACATTTCGGCCC Integrated DNA

[1206] CA Technologies

[1207] HPRT1 primer / probe Invitrogen Hs99999909

[1208]

[1209] 20x mix ml Primer, probes Oligo nucleotides Source Identifier IL-6 primer / probe 20x Invitrogen Hs00174131 mix ml CCND 1 primer / probe Invitrogen Hs00765553 20x mix ml KAT5 primer / probe Invitrogen Hs00197310

[1210]

[1211] 20x mix ml

[1212] Example 4. Cytokine expression analysis

[1213] DMSO has been shown to affect the production of pro-inflammatory cytokines; therefore, cytokine expression was evaluated to determine whether the impact of DMSO on rAAV production might be mediated by cytokine production. Differential expression of cytokine genes (FIG. 9A) in PCL1 and PCL2 were derived via RNA-Seq analysis. RNA-Seq analysis is described in Example 2.

[1214] Cell pellet samples collected at 24 hpi from the control (no DMSO) and DMSO-treated cultures shown in FIGs. 2A-2G were used for cytokine array analysis. Total RNA extraction was performed using the Qiagen RNAeasy kit. Reverse transcription was performed to generate cDNA (Applied Biosystems), and subjected to TaqMan cytokine array analysis (Applied Biosystems) following the manufacturer’s protocol. Array results are shown in FIG. 9B for cytokines with a Ct value less than 30. Values shown are fold change normalized to both a housekeeping gene (HPRT1) and the non-treated control.

[1215] For analysis of cytokine secretion, culture supernatants were collected at 0 and 24 hpi from the control (no DMSO) and DMSO-treated wtAd5 -infected PCL1 cultures. Luminex assays were performed using multiplex human cytokine / chemokine magnetic bead panel (Millipore) following the manufacturer’s protocol by DC3 Therapeutics. Luminex assay results shown in FIG. 9C were for cytokines with values above the limit of quantification (LOQ).

[1216] DMSO treatment led to broad changes in cytokine gene expression as depicted in the RNAseq analysis heatmap (FIG.9A). DMSO treatment reduced mRNA expression of multiple cytokines including IL-6, IL-8, IL-15, IL-12A, IL-1A, IL-1B but expression of IL-18 and TNF were increased. Reductions in IL-6, IL-8 and IL- 15 were confirmed by both the cytokine array (FIG. 9B) and Luminex (FIG. 9C) analyses. Example 5. Knockdown of KAT5 reduced CCND1 expression while not altering IL-6 expression.

[1217] As a histone acetyltransferase, KAT5 may influence the expression of multiple downstream genes. Expression of CCDN1 and IL-6, two genes known to be regulated by KAT5 was assessed in PCL1 following KAT5 gene knockdown in the presence or absence of DMSO. CCND1 and IL-6 mRNA levels were analyzed via qRT-PCR and presented as fold change normalized to the negative control (transfection with siN.C, no DMSO). CCND1 mRNA (FIG.

[1218] 10A) was reduced two-fold (to 50% of the negative control) following DMSO treatment of cells transfected with the control siRNA (siN.C). In cells transfected with siKAT5, a similar two-fold reduction (to 50%) compared to the negative control was observed in the absence of DMSO while treatment with DMSO reduced CCND1 mRNA an additional two-fold (to -25% of the control). CCND1 protein expression as analyzed by Western blot (FIG. IOC) was consistent with the mRNA analysis, showing a reduction with both DMSO and KAT5 knockdown. Overall, these data suggest that both KAT5 knockdown and DMSO reduce the expression of CCND1 and that an additive effect in observed in cells subjected to both treatments.

[1219] Though KAT5 knockdown reduced CCND1 mRNAlevels, IL-6 mRNA abundance was not affected by KAT5 knockdown (FIG.1 OB). Cells transfected with siN.C and siKAT5 showed similar levels of IL-6 mRNA; however, treatment with DMSO reduced IL-6 mRNA in both cases (-8 and ~4-fold, respectively). This result is consistent with the cytokine expression analysis (Example 4). Therefore, expression of IL-6, though reduced by DMSO, is not influenced by KAT5.

[1220] To directly assess the effect of reduced CCND1 and IL-6 expression on rAAV production, the genes were knocked down via transfection of PCL1 with siRNAs targeting each gene as previously described in FIG. 6A. rAAV productivity was not changed compared to the control (transfection with siN.C, no DMSO) following knockdown of CCND1 (FIG. IOC). In contrast, rAAV production was increased -2.5-fold by DMSO treatment in cells transfected with the control siRNA and siCCNDl. A similar result was observed following knockdown of IL-6 (FIG. 10D).

[1221] Overall, the results of these experiments show that DMSO treatment reduces the expression of both CCND1 and IL-6 in PCL1; however, KAT5 influences the expression of CCND1 only. Knockdown of CCND1 or IL-6 did not affect rAAV production, therefore, the effect of KAT5 knockdown on rAAV production is likely not mediated by either CCND1 or IL-6 in the PCLs tested. Notably, KAT5 knockdown increases expression of AdEl A protein (see Example 3). This observation is consistent with a report in the literature that the product of the KAT5 gene, TIP60, represses expression of AdEl A (Gupta et al, Oncogene, 2013). Since AdElA activates transcription of adenoviral genes as well as the AAV p5 promoter which drives transcription of the rep gene, the effect of KAT5 knockdown on AdElA expression may explain in part how DMSO (and knockdown of KAT5) enhances rAAV production.

[1222] Example 6. AAV production from knockdown of select additional genes.

[1223] An siRNA screen in PCL1 was performed for additional target genes identified in the RNA-Seq experiments. Two siRNA candidates for each selected target or negative control were transfected into PCL1 cells using Lonza nucleofector 4D. Transfected cells were cultured for 2 days before infecting with wtAd5 with or without the presence of 1% DMSO. rAAV titer was analyzed to assess the impact of target knockdown with samples collected at 2 days post infection. Productivity from control infection condition without DMSO from each target was normalized to that of siNC. Productivity of each target comparing 1% DMSO to control, no DMSO was also determined. As shown in FIG. 16, knockdown of ISYNA1, led to higher AAV titers. Knockdown of GJB3 which was upregulated in DMSO treated cells showed reduced AAV production.

[1224] Example 7. Reconstruction of the AAV biosynthesis pathway - Network-based analysis.

[1225] To gain a more detailed understanding of the gene expression changes within the AAV biosynthesis pathway, the reconstruction and integrated transcriptomic data was visualized to perform network-based analyses. In this network, each node represents a gene and edges are established between nodes that share the same process, subsystem, or system. Edge weights were computed as a weighted sum of shared annotations, with higher contributions from shared processes, followed by subsystems, and then systems. Node positioning was determined using the Fruchteman-Reingold force-directed algorithm, resulting in a spatial organization that shows a separation of nodes according to system- and sub system -level annotations.

[1226] Differential gene expression data was then incorporated into this network at each timepoint and for each regulation direction (up- and down-regulated genes), allowing the visualization of transcriptional changes in the context of network topology. The log2FoldChange values were used to adjust edge weights based on log2FoldChange similarity between two nodes. The edge weights were multiplied by the similarity of Log2FoldChange values between connected nodes. Using the network with modified edges, initially, the Louvain method for community detection was applied. To ensure robustness and reproducible clustering, consensus clustering was combined with the Louvain clustering method. The community structures were evaluated across a range of resolution parameters and the optimal resolution was selected based on modularity and normalized mutual information (NMI) relative to known system-level annotations.

[1227] The resulting clusters represent groups of genes that shared both similar functional roles, annotated based on the AAV reconstruction, and similar expression changes across different media conditions. Distinct clusters were observed at early time points (0, 3, and 6 hpi) among up-regulated genes in production media compared to growth media (see FIG. 17 for the 0 hpi and FIG. 18 for the 3 hpi clusters). Down-regulated genes or later time points displayed weaker clustering and smaller log2FoldChange magnitudes, suggesting that formation of these clusters may have been primarily driven by topological proximity in the reference network rather than the differential expression. These observations suggest that the transcriptional responses to media conditions are most pronounced during the initial stages of AAV biosynthesis.

[1228] To functionally characterize the resulting clusters, enrichment analysis was performed using Fisher’s exact test to identify significantly overrepresented subsystems and processes. At the early timepoints, clusters with stronger internal connectivity and larger log2FoldChange magnitudes were enriched for ‘ Ad5 binding’ which is a subsystem representing the initial stage of Ad5 infection of producer cells required to initiate AAV biomanufacturing. Majority of the genes within these clusters were also annotated under the process ‘lysosomal exocytosis,’ which is nested within the ‘ Ad5 binding’ subsystem and is known to be indirectly involved in viral entry by facilitating lipid composition via ASMase. Additionally, clusters related to ‘DNA repair’ subsystem were identified. Collectively, these results demonstrate that our AAV reconstruction network provides a resource that enables systemic analysis of transcriptomic data in the context of AAV biosynthesis. By integrating expression dynamics with curated biological relationships, this approach enabled the identification of functionally relevant gene clusters, highlighting key time points of regulatory divergence, and offered mechanistic insights into AAV biosynthesis. The genes identified in this example are recited below in Table 7. Table 7. Genes identified in network clustering analysis at 0 hpi and 3 hpi that are upregulated

[1229] condition cluster gene

[1230] Oh up 0 ACTA1

[1231] Oh up 0 GCGR

[1232] Oh up 0 KCNB1

[1233] Oh up 0 SYT1

[1234] Oh up 0 SYT5

[1235] Oh up 0 MAP IB

[1236] Oh up 0 NPM2

[1237] Oh up 3 MYH11

[1238] Oh up 3 MYH3

[1239] Oh up 3 MYH7B

[1240] Oh up 3 EXOC3L1

[1241] Oh up 3 SCRIB

[1242] Oh up 3 LAT2

[1243] Oh up 3 SYT2

[1244] Oh up 3 BAIAP3

[1245] Oh up 3 SYTL1

[1246] Oh up 3 NR4A3

[1247] Oh up 8 ITGAM

[1248] Oh up 8 MYH15

[1249] Oh up 8 ACTC1

[1250] Oh up 8 HAP1

[1251] Oh up 8 RAB25

[1252] Oh up 8 BTK

[1253] Oh up 8 NKD2

[1254] Oh up 8 TRIM72

[1255] Oh up 8 NLRP5

[1256] Oh up 8 RAB44

[1257] Oh up 8 H4C7

[1258] Oh up 8 ATP1A4

[1259] Oh up 8 ATP1B2

[1260] Oh up 8 ATP2B2

[1261] Oh up 8 GHSR

[1262] Oh up 8 PTGER3

[1263] Oh up 8 HCAR1

[1264] Oh up 8 ADCY4

[1265] 3h up 21 SNRNP70

[1266] 3h up 21 RIF1

[1267] 3h up 21 CXADR

[1268] 3h up 21 GSK3B

[1269] 3h up 21 ITGAM

[1270] 3h up 21 MYH11

[1271] 3h up 21 MYH15

[1272]

[1273] 3h up 21 ACTA1 condition cluster gene

[1274] 3h up 21 ACTC1 3h up 21 EXOC3L1 3h up 21 NAPA

[1275] 3h up 21 LIN7C 3h up 21 STX3

[1276] 3h up 21 HAP1

[1277] 3h up 21 LAT2

[1278] 3h up 21 RAB40C 3h up 21 BTK

[1279] 3h up 21 GCGR

[1280] 3h up 21 TXLNA 3h up 21 NKD2 3h up 21 VPS 18 3h up 21 CBL

[1281] 3h up 21 TRIM72 3h up 21 SYT2

[1282] 3h up 21 NR4A3 3h up 21 CLTC

[1283] 3h up 21 WASL 3h up 21 SYNJ2 3h up 21 GAK

[1284] 3h up 21 DYNLRB1 3h up 21 DCTN5 3h up 21 TNPO2 3h up 21 ADORA2A 3h up 21 ATP1B2 3h up 21 CRHR2 3h up 21 GHSR

[1285] 3h up 21 NFKBIA 3h up 21 VAV3 3h up 21 ADCY10 3h up 21 NPM2 3h up 101 XRN2

[1286] 3h up 101 CCNA1 3h up 101 BAZ1B 3h up 101 PIAS1 3h up 101 UNC5A 3h up 101 DAPK1 3h up 101 TNFSF10 3h up 101 CD14

[1287] 3h up 103 YY1

[1288] 3h up 103 POLE3 3h up 103 POLE

[1289] 3h up 103 POLDI 3h up 103 FEN1

[1290]

[1291] 3h up 103 RPA4 condition cluster gene

[1292] 3h up 103 H2BC5

[1293] 3h up 103 H4C8

[1294] 3h up 103 TERF2

[1295] 3h up 103 RNF168

[1296] 3h up 103 RAD9A

[1297] 3h up 103 TOP3A

[1298] 3h up 103 PIAS4

[1299] 3h up 103 TRIM25

[1300] 3h up 103 UBA7

[1301] 3h up 103 FANCA

[1302] 3h up 103 SPHK1

[1303] 3h up 103 POLH

[1304] 3h up 103 REV1

[1305] 3h up 103 ALKBH5

[1306] 3h up 103 RNF111

[1307] 3h up 103 NFRKB

[1308] 3h up 103 INO80

[1309] 3h up 103 COPS3

[1310] 3h up 103 EME2

[1311] 3h up 103 POLN

[1312] 3h up 103 PAXIP1

[1313] 3h up 103 RNF4

[1314] 3h up 103 PPP4R2

[1315] 3h up 103 RTEL1

[1316] 3h up 103 EYA3

[1317] 3h up 103 EYA4

[1318]

[1319] 3h up 103 MBD4

[1320] Example 8. Decreasing levels of GAPDH results in higher AAV titer produced from HEK293 cells.

[1321] HEK293 cells were transfected with siRNA to GAPDH (siGAPDH) to decrease GAPDH expression or with a control siRNA (siN.C) along with AAV production transfection constructs pAdhelper and a triple-play construct consisting of AAV Rep, Cap, and ITR-transgene. The transfected cells were cultured for three days before harvesting to assess AAV titer. AAV titer was assessed by real-time PCR (qPCR). Results from siGAPDH were normalized to the siN.C control (FIG. 19) Three different concentrations of siRNA in the culture media were tested: 100 pM, 200 pM, and 300 pM per milliliter of HEK293 cell culture. rAAV production increased with GAPDH knockdown and showed trend of siGAPDH dose dependent increase. HEK293 cells were then transfected with 100 pM of either siGAPDH or siN.C along with the AAV production transfection constructs pAdhleper and a triple-play construct. Four different AAV constructs comprising different transgenes (Capagenel, Capbgene?, Capcgene3, and Capagene^ were used to assess rAAV productivity from HEK293 cells. Virus production was increased with all four constructs when GAPDH expression was decreased (FIG.20).

[1322] These results demonstrate increased AAV production with a decrease in GAPDH expression in producer cells (e.g., HEK293 cells).

[1323] Example 9. Growth Factor Receptors & RTK- Associated Proteins modulate AAV titer production

[1324] EGFR (Epidermal Growth Factor Receptor)

[1325] EGFR is a receptor tyrosine kinase that, upon ligand binding and dimerization, activates multiple cascades (RAS / RAF / MEK / ERK MAPK pathway, PI3K / Akt, JAK / STAT, PLCy) promoting proliferation, survival, migration, and inflammatory responses. Noh, S. S. & Shin, H. J. Role of virus-induced EGFR trafficking in proviral functions. Biomolecules 13, 1766 (2023). In adenovirus infection, EGFR plays a multifaceted role. The physical process of adenovirus entry (binding to its primary receptor and integrins) can transiently activate EGFR or EGFR-related signaling as a stress response, even without EGF ligand. Id. This “stress-induced” EGFR activation helps trigger the host innate immune response. For example, adenovirus entry rapidly stimulates the Raf / MEK / ERK pathway and immediate early cytokines like IL-8 in epithelial cells. Bruder, J. T. & Kovesdi, I. Adenovirus infection stimulates the Raf / MAPK signaling pathway and induces interleukin-8 expression. J. Virol. 71, 398-404 (1997). However, adenovirus also downregulates EGFR during infection: the adenoviral E3 region proteins (e.g. RIDa / complex) bind and reroute EGFR to endosomes / lysosomes, causing EGFR degradation. Noh et el. (2023), Biomolecules 13, 1766. This occurs within the early phase and serves to limit NF-KB signaling and inflammatory / apoptotic responses that EGFR might mediate. Id. Additionally, adenovirus El A and E4orfl oncoproteins manipulate EGFR pathways: E1A can repress some growth genes, and E4orfl hijacks EGFR’s tyrosine kinase activity in a ligand-independent manner to activate RasZERK and PI3K signaling. Kong, K., Kumar, M., Taruishi, M. & Javier, R. T. Adenovirus E4-ORF1 dysregulates epidermal growth factor and insulin / insulin-like growth factor receptors to mediate constitutive Myc expression. J. Virol. 89, 10774-10785 (2015). E4orfl forms a complex with EGFR (and the membrane scaffold Dlgl) and even links EGFR with insulin / IGF receptors, resulting in constitutive EGFR phosphorylation and downstream signaling. Id. Biological context: Early on, EGFR activation contributes to pro-viral conditions (enhancing cell survival and suppressing antiviral cytokines) but must be balanced; the virus later dampens EGFR to avoid excessive inflammation. Noh et el. (2023), Biomolecules 13, 1766. In HeLa cells, which natively express EGFR, wild-type adenovirus infection would transiently exploit EGFR signaling to promote an environment favorable for viral DNA replication and to suppress interferon / NF-KB responses, then reduce surface EGFR to prevent the cell from undergoing inflammation-induced apoptosis.

[1326] Differential expression analysis revealed that EGFR expression was largely stable across cell lines and media conditions, with no significant differences between PCL1 and PCL2 at any time point barring 0 hpi on control media (Log2FC = -0.43; adjusted p- value = 5E-02). Temporal analysis indicated that on control media, EGFR levels were higher at 0 hpi than at 24 hpi in both PCLs, consistent with a transient reduction following infection, with this change being significant in PCL1 (Log2FC = -0.6; adjusted p = 9E10-04). This early suppression aligns with the known adenoviral strategy of dampening EGFR signaling to limit host inflammatory responses. In contrast, PCL2 on supplemented media exhibited a modest rebound in EGFR expression by 48 hpi (Log2FC = -0.6; adjusted p = IE-02), suggesting that under nutrient-enriched conditions EGFR activity may be partially restored or sustained during the productive phase. Consistent with this, receptor subset enrichment analyses showed that DMSO supplementation of PCL2 enriched for receptors, including EGFR, within the PI3K-Akt signaling pathway at 0 hpi relative to 24 hpi (fold enrichment = 1.6; adjusted p-value = 4E-02), and within the PI3K-Akt (fold enrichment = 3.1; adjusted p-value = 5E-03), and MAPK (fold enrichment = 2.0; adjusted p-value = 2E-02) signaling pathways at 24 hpi relative to 48 hpi, reflecting a temporal re-engagement of growth and survival signaling as infection progresses. Together, these observations support the rationale for EGFR overexpression in PCL1 on control media prior to 48 hpi, as this condition lacks the late-phase recovery seen in PCL2. Enhancing EGFR signaling may therefore offset viral downregulation, sustaining proliferative and anti-apoptotic cascades via RAS / RAF / MEK / ERK, PI3K / Akt, and JAK / STAT pathways that favor continued AAV production.

[1327] IGF1R ( I ns nl i n-like Growth Factor 1 Receptor) & INSR (Insulin Receptor)

[1328] IGF1R and INSR are homologous receptor tyrosine kinases that trigger strong PI3K / Akt and MAPK signaling in response to IGF or insulin, generally promoting metabolism, protein synthesis, cell survival, and proliferation. HeLa cells in culture often have IGF1R and can respond to growth factors in serum. Adenovirus E4orfl protein specifically exploits the InsR / IGF 1R axis: E4orfl binds the cell scaffold protein Dlgl and forms a complex that includes EGFR and InsR / IGF 1R at the plasma membrane. This leads to constitutive activation of PI3K and maintenance of high Akt activity, as well as ligand-independent signaling through Ras / ERK via EGFR. Kong et al. (2015), J. Virol. 89, 10774-10785. One outcome is sustained Myc protein levels in the nucleus, which adenovirus uses to drive transcription of genes advantageous for viral replication. Id. InsR / IGF 1R normally can negatively crosstalk with EGFR (they can moderate EGFR activity), but E4orfl by binding Dlgl appears to antagonize that restraint, allowing maximal EGFR-Ras signaling. Id. Beyond E4orfl, the strong Akt signaling from IGFIR / InsR can prevent premature apoptosis of the host cell, giving the virus more time to replicate. In infection context: While adenovirus doesn’t bind IGF1R directly, it co-opts downstream survival signaling. In a helper-virus scenario for AAV, the presence of adenovirus might activate PI3K / Akt via E4orfl and serum growth factors, which could enhance cell survival and productivity of viral vectors. On the flip side, if insulin / IGF signaling is blocked, cells may undergo apoptosis faster in response to infection. Thus, IGFIR / InsR pathways are part of the pro-survival program induced by adenovirus early during infection.

[1329] Differential expression analysis showed that IGF1R expression remained largely unchanged across cell lines under both control and supplemented media, reflecting stable baseline expression. However, expression was significantly higher in control media relative to supplemented media for PCL1 at 0 (Log2FC = -0.5; adjusted p-value = IE-02), 24 (Log2FC = -0.6; adjusted p-value = IE-02), and 48 hpi (Log2FC = -0.6; adjusted p-value = 8E-03), and for PCL2 at 0 hpi (Log2FC = -0.4; adjusted p-value = 2E-02), indicating that supplementation broadly suppresses IGF1R transcription in PCL1 throughout infection and transiently in PCL2 during early infection. Temporal comparisons further revealed that on both control and supplemented media for either PCL, IGF1R levels were higher at 0 hpi than at 24 hpi (-1.3 < Log2FCs < -1.5; adjusted p-values < 0.05), consistent with an early infection-associated reduction in receptor expression. Given that IGF1R activity promotes PI3K / Akt and MAPK signaling to support metabolism and survival, this pattern suggests a general downregulation of growth factor signaling following infection, with supplementation modulating the extent of this suppression. Accordingly, attenuation of IGF1R in PCL1 prior to 24 hpi under supplemented conditions may help rebalance this axis, reducing redundant Akt signaling and preventing excessive survival activity that competes with transcriptional reprogramming and viral production. This interpretation aligns with the known role of adenoviral E4orfl in hijacking the InsR / IGF 1R complex to sustain constitutive Akt activation and Myc stabilization, underscoring how partial dampening of IGF1R could relieve viral or metabolic overactivation and enhance process efficiency. Pathway enrichment provided additional context for this transcriptional behavior: on control media, PCL1 was enriched for receptor subsets, including IGF1R, participating in the FoxO (fold enrichment = 3.74; adjusted p-value = IE-03), mTOR (fold enrichment = 3.32; adjusted p-value = 9E-04), and HIF-1 (fold enrichment = 3.42; adjusted p-value = 3E-03) signaling pathways at 0 hpi relative to 24 hpi, while PCL2 showed enrichment for receptor subsets within PI3K-Akt (fold enrichment = 1.6; adjusted p-value = 4E-02), HIF-1 (fold enrichment = 2.75; adjusted p-value = 4E-02), and FoxO (fold enrichment = 4.00; adjusted p-value = IE-03) pathways under the same comparison. On supplemented media, PCL1 retained enrichment for mTOR signaling (fold enrichment = 3.70; adjusted p-value = 3E-06), whereas PCL2 was enriched for PI3K-Akt (fold enrichment = 1.55; adjusted p-value = 4E-02) and mTOR (fold enrichment = 3.10; adjusted p-value = 6E-4) pathways at 0 hpi relative to 24 hpi, suggesting that both cell lines transiently engage growth and metabolic signaling programs centered around IGF1R during early infection, though supplementation modifies their amplitude and composition. Given that IGF1R activity drives PI3K / Akt and MAPK cascades to support metabolism and survival, these enrichment trends imply a coordinated downregulation of growth factor-linked signaling following infection, with supplementation tempering but not eliminating this early signaling pulse. Accordingly, attenuation of IGF1R in PCL1 prior to 24 hpi under supplemented conditions could help rebalance this axis, reducing redundant Akt activity and preventing excessive survival signaling that competes with transcriptional reprogramming and viral production, underscoring how partial dampening of IGF1R could relieve viral or metabolic overactivation and enhance process efficiency.

[1330] GRB2 (Growth factor Receptor-Bound protein 2)

[1331] GRB2 is an adaptor protein (not a receptor) that links activated receptors to the RAS / MAPK pathway. It contains SH2 / SH3 domains and typically binds phospho-tyrosines on receptors like EGFR or on focal adhesion kinase, then recruits the SOS guanine nucleotide exchange factor to activate RAS. Bruder et al. (1997), J. Virol. 71, 398-404. In adenovirus infection of HeLa, GRB2 is rapidly engaged as part of the integrin-mediated entry signaling. Adenovirus uses penton base proteins with an RGD motif to bind cellular integrins, which activates focal adhesion kinase (FAK) and other kinases. This leads to recruitment of GRB2-SOS and activation of Ras and Raf. Indeed, within ~20 minutes of adenoviral attachment, the Raf / MAPK / ERK cascade is turned on, leading to transcription of IL-8 and other early response genes-. This occurs even in the presence of cycloheximide (no new proteins made), indicating it’s due to pre-existing signaling components like GRB2. Id. The functional outcome is an inflammatory signal (IL-8) that can recruit immune cells, but it may also condition the cell for viral replication. Adenovirus benefits from moderate MAPK activation: it can induce prosurvival genes via NF-KB / AP-1, yet if excessive, inflammation could harm the cell. The virus later uses E3 proteins to tone down these signals. Summary: GRB2 is essential for linking adenovirus entry to intracellular signaling. In HeLa cells, integrin engagement by adenovirus likely triggers GRB2-dependent Ras / MAPK activation, contributing to the early innate response and potentially aiding viral genome transit to the nucleus. Id. By the time AAV is being produced (with adenovirus help), these GRB2-mediated signals might influence cellular stress responses that affect viral vector yields.

[1332] Differential expression analysis indicated that GRB2 expression was stable across cell lines and media types, showing no significant differences between PCLs under baseline conditions. Temporal comparisons, however, revealed consistently higher GRB2 expression at 0 hpi relative to 24 hpi on both control and supplemented media for PCL1 and PCL2 (-0.83 < Log2FC < -0.68; p-adjusted values < 0.05), as well as greater expression at 24 hpi relative to 48 hpi in both cell lines under each media condition (-0.62 < Log2FC < -0.25; p-adjusted values < 0.05), indicating a progressive decline in GRB2 transcript abundance over the course of infection. Pathway enrichment analysis contextualized this temporal trend, showing that at 0 hpi relative to 24 hpi, PCL1 was enriched for receptor subsets including GRB2 that participate in FoxO (fold enrichment = 3.74; p-adjusted value = IE-03) and mTOR (fold enrichment = 3.32; p-adjusted value = 9E-04) signaling on control media and in mTOR (fold enrichment = 3.70; p-adjusted value = 3E-06) signaling on supplemented media, while PCL2 was enriched for receptor subsets linked to FoxO (fold enrichment = 4.00; p-adjusted value = IE-03), PI3K-Akt (fold enrichment = 1.57; p-adjusted value = 4E-02), and mTOR (fold enrichment = 3.56; p-adjusted value = 4E-04) signaling under control conditions, and PI3K-Akt (fold enrichment = 2.04; p-adjusted value = 4E-02) and mTOR (fold enrichment = 3.06; p-adjusted value = 6E-04) pathways under supplementation. Since GRB2 acts as a key adaptor coupling receptor activation to Ras / MAPK and PI3K / Akt signaling, its early enrichment within these pathways indicates strong integration of growth factor and metabolic inputs during infection onset. The subsequent decline in GRB2 expression, coinciding with Ad5 E3 and E4-mediated suppression of MAPK and NF-KB activity, likely reflects a controlled viral mechanism to reduce host stress and inflammatory signaling. In this context, attenuating GRB2 expression in PCL1 under supplemented conditions prior to 24 hpi could mimic this natural downregulation, tempering MAPK-dependent cytokine and stress responses while preserving the beneficial early Ras- and Akt-mediated survival and metabolic activation that support productive AAV biosynthesis.

[1333] Methods of Example 9

[1334] Library Preparation

[1335] The RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3 ’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).

[1336] Sequencing

[1337] The sequencing libraries were multiplexed and clustered onto an Illumina flowcell. After clustering, the flow cell was loaded onto the Illumina NovaSeq 6000 instrument according to the manufacturer's instructions. The samples were sequenced using a 2xl50bp Paired End (PE) configuration and targeting ~30 million reads / sample. Image analysis and base calling were conducted by the Illumina Control Software. Raw sequence data (.bcl files) generated from Illumina was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.

[1338] The human comprehensive gene annotation and reference genome for GRCh38.pl3 release 38 primary assembly was retrieved from Gencode in GTF and fasta format respectively. Ad5 gene annotation and reference genome was retrieved from NCBI (RefSeq accession AC_000008.1; Geninfo accession 56160529; RefSeq assembly GCA_000857865.1; GenBank assembly GCF_000857865.1; Name ViralProj 15107). AAV gene annotation and reference genome was provided by Sanofi. Both genome indexing and alignment of trimmed reads were performed using the STAR aligner (v2.7.3a). Prior to alignment, quality control of paired-end fastq reads through sequential application of FastQC (vO.11.9), Trimmomatic (v0.39) and FastQC on trimmed reads ensured removal of low-quality and adapter sequences. Following alignment, both raw and transcript-per-million (TPM) read count quantification from the binary alignment map (BAM) file was performed using HTSeq (v0.11.3), SAMtools (vl.10) and StringTie (version 2.1.1). Alignment, QC, and readcount quantification was performed using a nextflow (v#) pipeline as hosted on a docker instance of Ubuntu 18.04.

[1339] DESeq2

[1340] Differential gene expression (DE) analysis was performed via DESeq2 (vl.38.3) using the R (v4.2.2) programming language. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). Prior to testing, all genes with 0 recorded counts across samples are removed from the analysis. Using the raw count matrix and Ensembl annotations (version 104) output from the rnaseq docker nextflow pipeline (commit 179f36b) as input to the testing framework, and following normalization by relative log expression (RLE) scaling factors, DE testing was performed between choice of (i) media and (ii) cell line at each timepoint post AAV transfection, and (iii) between each timepoint post AAV transfection in a given media and cell line. The resulting 24 sets of DEGs (Bonferroni adjusted p value < 0.05) were subsequently interrogated in ligand receptor enrichment analyses.

[1341] Gene Background

[1342] To ensure biological relevance of results in the context of each DESeq2 comparison, a gene background (i.e., all expressed genes) was curated consisting of all non-DE genes with baseMean expression within or greater than the baseMean expression range of each DESeq2 comparison.

[1343] Manual curation of the Ad5 immune response network

[1344] The Ad5 immune response network was constructed by integrating host signaling and viral targeting information to contextualize the cellular processes modulated during adenovirus type 5 (Ad5) infection. A total of 45 signal transduction, cell growth and death, and immune system networks were downloaded from the KEGG database and iteratively merged to form a unified host signaling framework encompassing innate immune, stress-response, apoptotic, and metabolic regulation pathways. Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. & Morishima, K. KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45, D353-D361 (2017). Within this framework, known adenoviral targets were curated through literature review, focusing on experimentally validated interactions between Ad5-encoded proteins (E1B, E3, and E4) and host signaling components. These curated viral-host interactions were overlaid onto the merged KEGG signaling network to generate an Ad5-specific immune response network capturing the breadth of host pathways directly or indirectly targeted by viral proteins. Visualization and manual refinement were performed in Cytoscape (v3.9.1). Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498-2504 (2003). Individual pathway maps were extracted from the global network as necessary to highlight specific receptor-linked signaling routes or viral interference points referenced in the main text.

[1345] Ligand Receptor Analysis

[1346] Following the application of the DESEQ2 analysis pipeline DEG results were subset for genes annotated as a participating ligand or receptor of an LR pair as annotated in CellChat (Jin, S. et al. Inference and analysis of cell-cell communication using CellChat. Nat. Commun.

[1347] 12, 1088 (2021)), NeuronChat (Zhao, W., Johnston, K., Ren, H., Xu, X. & Nie, Q. Inferring neuron-neuron communications from single-cell transcriptomics through NeuronChat. Nature Communications 14, (2023)), Stadtmauer et al (Stadtmauer, D. J. et al. Cell type and cell signalling innovations underlying mammalian pregnancy. Nat. Ecol. Evol. 9, 1469-1486 (2025)) and Uniprot (UniProt Consortium. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49, D480-D489 (2021)). Subsequent filtering of all mapped LR pairs on the manually curated Ad5 immune response network through shared receptor EntrezIDs yielded a network-specific set of LR pairs. To account for receptors in the Ad5 immune response network that were not encompassed in this set for downstream enrichment analyses, the Ad5 immune response network was subset for uniprot identifiers associated with the uniprot keyword KW-0675 corresponding to receptor molecular function.

[1348] Corresponding enriched receptors of LR pairs and enriched pathways were found using the enrichr function of the clusterProfiler R package (v4.16.0) with default arguments. Yu, G., Wang, L.-G., Han, Y. & He, Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 16, 284-287 (2012). For receptor enrichment analyses, a total of 3 ORA were run on the receptors of LR pairs for each comparison of PCL, media supplementation, and hpi corresponding to i) bi-directional ii) up- and iii) down-regulated DE genes. In all ORAs, background was set to receptors of the Ad5 immune response network, and the set of input DE receptors was filtered for a DE ligand in the corresponding LR pairs. Additionally, LR pairs with a complex ligand were required to exhibit consistent expression directionality across all components of the ligand complex. For pathway enrichment analyses, this process was repeated using non-receptor DE genes of the Ad5 immune response network as input and the same network filtered for receptors as background. In all ORAs, pathway selection was constrained to those constituting the Ad5 immune response network. ORA results were filtered for a nominal p-value < 0.05, Benjamini -Hochberg adjusted p-value < 0.05, and q-value < 0.2. Adjusted p-values were computed using the Benjamini -Hochberg procedure, and q-values were calculated using Storey's method implemented in the qvalue R package (v2.40.0). Dabney, A. R. & Storey, J. D. Bioconductor’s qvalue package, hist (hedenfalk) (2006).

Claims

CLAIMS1. An engineered cell line in which the expression or activity of at least one of ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS1L, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, PARD6G-AS1, ACTA1, ACTC1, ADCY10, ADCY4, ADORA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2 is altered as compared to a control cell line.

2. An engineered cell line in which the expression or activity of at least one of the transcription factor genes listed in Tables 1-4 is altered as compared to a control cell line.

3. An engineered cell line in which the expression or activity of at least one of the transcription factor target genes listed in Tables 1-4 is altered as compared to a control cell line.

4. The engineered cell line of any one of claims 1-3, wherein expression or activity of at least one gene is reduced and / or expression of at least one gene is increased, as compared to the control cell line.

5. The engineered cell line of any one of claims 1-4, wherein the cell line is modified by a modulator that increases or decreases the expression and / or activity of at least one gene.

6. The engineered cell line of any one of claims 1-5, wherein the cell line comprises a genetic modification.

7. The engineered cell line of claim 5 or 6, wherein the modulator is a nuclease.

8. The engineered cell line of claim 7, wherein the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activatorlike effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

9. The engineered cell line of claim 5, wherein the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

10. The engineered cell line of claim 5, wherein the modulator comprises a template for homologous arm mediated recombination.

11. The engineered cell line of claim 5, wherein the modulator comprises an inhibitor of at least one gene selected from the group consisting of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SSI 8, THEM6, TNFAIP3, EGFR, IGF1R, INSR, and GRB2.

12. The engineered cell line of claim 11, wherein the inhibitor comprises a synthetic small molecule inhibitor.

13. The engineered cell line of claim 5, wherein the modulator comprises agonist of at least one gene selected from the group consisting of: ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1,ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNP02, T0P3A, TRIM25, TRIM72, TXLNA, UB A7, UNC5 A, VAV3, VPS 18, WASL, XRN2, and YY1.

14. The engineered cell line of claim 13, wherein the agonist comprises a synthetic small molecule agonist.

15. The engineered cell line according to any one of claims 1-14, wherein the cell line is a mammalian cell line or an insect cell line.

16. The engineered cell line of claim 15, wherein the mammalian cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or HEK293 cell line.

17. The engineered cell line of claim 16, wherein the mammalian cell line is a HeLa cell line or a human embryonic kidney (HEK) 293 cell line.

18. The engineered cell line of claim 15, wherein the insect cell line is an Sf9 cell line or a Hi5 cell line.

19. The engineered cell line according to any one of claims 1-18, wherein the cell line is a host cell line for the production of viral particles.

20. The engineered cell line according to any one of claims 1-18, wherein the cell line is a producer cell line.

21. The engineered cell line of claim 20, wherein the producer cell line further comprises stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest.

22. The engineered cell line of claim 20 or 21, wherein the producer cell line further comprises AAV helper function encoding sequences of an AAV helper virus.

23. The engineered cell line of claim 22, wherein the AAV helper virus is an adenovirus.

24. The engineered cell line of claim 23, wherein the adenovirus is Ad5 or Ad2.

25. The engineered cell line of claim 22, wherein the AAV helper function encoding sequences comprise at least one of: Ela, Elb, E2A, L4, E4, VARNA.

26. The engineered cell line according to any one of claims 1-25, wherein the titer of rAAV produced from the engineered cell line is at least about 1.5-fold higher compared to the titer of rAAV produced from a cell line in which the expression of the gene is not altered.

27. A method for identifying a gene associated with increasing the production of rAAV particles from a cell, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of : ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS IL, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SSI 8, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL20A1, GJB3, ISYNA1, PARD6G-AS1, and RP11-268 JI 5.6, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80,KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

28. The method of claim 27, further comprising determining the titer of rAAV particles expressed from the cell and comparing the titer to a reference rAAV titer, wherein the reference rAAV titer is that of rAAV particles expressed from a cell that has not been cultured in the presence of the modulator but is otherwise identical.

29. The method of claim 28, wherein if the titer of rAAV particles is increased compared to the reference rAAV titer, the gene is identified as a target for knock-down or knockout in the cell.

30. The method of claim 28, wherein if the titer of rAAV particles is decreased compared to the reference rAAV titer, the gene is identified a target for over-expression in the cell.

31. A method of modulating gene expression in a cell line for increasing the production of rAAV particles from a cell, wherein the method comprises culturing the cell in the presence of a modulator that alters the expression or activity of at least one gene selected from the group consisting of: ADM2, AURKA, BCL2, CCND1, CHD1, CREB3L3, RIGI / DDX58, ECSIT, EHHADH, ERBB2, FAS, FBXW7, FGFR3, FOXL2, GATA1, HDAC5, H0XA5, HSP90B1, IL6, ITGA2B, KAT5, LAS IL, LIF, NFKB1, NFKB2, NFKB1A, NFE2L2, NGEF, NME1, NR3C1, POLN, PPAR-A, PTEN, RELB, RPA1, RXRA, SMAD4, SS18, TCF7, TGM2, THEM6, TNFAIP3, TP63, TRPC1, DBH, ITGAM, HHEX, TXNIP, ASNS, PLEKH02, CDKN1A, C5orf64, COL2A1, GJB3, ISYNA1, PARD6G-AS1, RP11-268 JI 5.6, a transcription factor gene listed in Tables 1-4, and a transcription factor targeted gene listed in Tables 1-4, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC,C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EX0C3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, P0LE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, T0P3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, YY1, EGFR, IGF1R, INSR, and GRB2.

32. The method of any one of claims 27-31, wherein the modulator is DMSO, DMF, NMP, or dihydrolevoglucosenone (Cyrene).

33. The method of claim 32, wherein the concentration of DMSO is at least about 0.5%.

34. A method for increasing the production of rAAV particles, wherein the method comprises at least one of the following genes : DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SSI 8, THEM6, and TNFAIP3 is reduced as compared to a control cell line, and / or in which the expression of at least one of ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1 is increased as compared to a control cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

35. The method of claim 34, wherein the method increases recombinant viral titer by at least 1.5-fold compared to a method comprising a control cell.

36. The method of claim 34 or 35, wherein the modulator comprises a synthetic small molecule inhibitor of at least one of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SSI 8, THEM6, TNFAIP3, EGFR, IGF1R, INSR, and GRB2.

37. The method of claim 34 or 35, wherein the modulator comprises a synthetic small molecule agonist of at least one gene selected from the group consisting of: ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, C0PS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, and YY1.

38. The method of any one of claims 34-37, wherein the modulator is DMSO, DMF, NMP, or dihydrolevoglucosenone (Cyrene).

39. The method of claim 38, wherein the concentration of DMSO is at least about 0.5%.

40. The method of any one of claims 34-39, wherein the modulator is an inhibitor or agonist of a pathway regulated by any one of the genes listed in claim 34.

41. The method of any one of claims 34-40, wherein the modulator is a nuclease.

42. The method of claim 41, wherein the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

43. The method of any one of claims 34-42, wherein the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

44. The method of any one of claims 34-40, wherein the modulator comprises a template for homologous arm-mediated recombination.

45. The method of any one of claims 27-44, wherein the cell line is a mammalian cell line or an insect cell line.

46. The method of claim 45, wherein the mammalian cell line is a CHO cell line, Vero cell line, HeLa cell line, MDCK cell line, BHK cell line, A549 cell line, amniocyte cell line, or HEK293 cell line.

47. The method of claim 45 or 46, wherein the mammalian cell line is a HeLa cell line or a human embryonic kidney (EEK) 293 cell line.

48. The method of claim 45, wherein the insect cell line is an Sf9 cell line or a Hi5 cell line.

49. An engineered host cell line produced by a method comprising knocking down the expression or activity of at least one of the genes selected from the group consisting of: DBH, ECSIT, EHHADH, FAS, FGFR3, FOXL2, HDAC5, H0AX1, HSP90B1, ISYNA1, ITGAM, KAT5, NFKB1, NFKBIA, POLN, RELB, RIGI / DDX58, SS18, THEM6, and TNFAIP3.

50. The engineered host cell line produced by the method of claim 49, wherein the knockdown is carried out using at least one of: homology directed recombination, RNA based knock-down, and a CRISPR-Cas enzymatic method.

51. An engineered host cell line produced by a method comprising increasing the expression or activity of at least one of the genes selected from the group consisting of: ASNS, BCL2, CDKN1A, COL20A1, GATA1, GJB3, LAS1L, LIF, NFKB2, NGEF, NME1, NR3C1, RPA1, SMAD4, TRPC1, TXNIP, PARD6G-AS1, PLEKHO2, and ACTA1, ACTC1, ADCY10, ADCY4, AD0RA2A, ALKBH5, ATP1A4, ATP1B2, ATP2B2, BAIAP3, BAZ1B, BTK, CBL, CCNA1, CD14, CLTC, COPS3, CRHR2, CXADR, DAPK1, DCTN5, DYNLRB1, EME2, EXOC3L1, EYA3, EYA4, FANCA, FEN1, GAK, GCGR, GHSR, GSK3B, H2BC5, H4C7, H4C8, HAP1, HCAR1, INO80, KCNB1, LAT2, LIN7C, MAP1B, MBD4, MYH11, MYH15, MYH3, MYH7B, NAPA, NKD2, NLRP5, NPM2, NR4A3, NFRKB, PAXIP1, PIAS1, PIAS4, POLE, POLE3, POLDI, POLH, PPP4R2, PTGER3, RAB25, RAB40C, RAB44, RAD9A, REV1, RIF1, RNF111, RNF168, RNF4, RPA4, RTEL1, SCRIB, SNRNP70, SPHK1, STX3, SYT1, SYT2, SYT5, SYTL1, SYNJ2, TERF2, TNFSF10, TNPO2, TOP3A, TRIM25, TRIM72, TXLNA, UBA7, UNC5A, VAV3, VPS 18, WASL, XRN2, andYYl.

52. The engineered host cell line of claim 51, wherein increasing the expression or activity is carried out by transient delivery or stable integration of a target expression modulation construct into the host cell line using at least one of: random integration, homology directed recombination, a transposase-based recombination, a lentiviral based integration, an integrase mediated integration or a CRISPR-Cas activation method, optionally wherein the Cas comprises a dCas, such as dCas9.

53. The engineered host cell line of any one of claims 49-52, further comprising stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus.

54. The engineered host cell line of any one of claims 49-52, further comprising transiently transfected nucleic acid sequences encoding for AAV rep and / or cap, AAVITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus.

55. The engineered host cell line of claim 53 or 54, wherein the AAV helper virus is an adenovirus.

56. The engineered host cell line of claim 55, wherein the adenovirus is Ad5 or Ad2.

57. The engineered cell line of claim 56, wherein the AAV helper genes comprise at least one of: Ela, Elb, E2A, L4, E4, VA.

58. A recombinant viral particle obtained from the engineered host cell line of any one of claims 49-57.

59. The recombinant viral particle of claim 58, wherein the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle.

60. A method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: transiently transfecting the host cell line of claim 19 with one or more plasmids comprising: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid encoding a gene of interest.

61. The method of claim 60, further comprising transfecting the host cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus.

62. The method of claim 61, wherein the AAV helper virus is an adenovirus.

63. The method of claim 62, wherein the adenovirus is Ad5 or Ad2.

64. A method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: stably transfecting the producer cell line of claim 20 with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, (iii) nucleic acid encoding a gene of interest.

65. The method of claim 64, further comprising transfecting the producer cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus.

66. The method of claim 65, wherein the AAV helper virus is an adenovirus.

67. The method of claim 66, wherein the adenovirus is Ad5 or Ad2.

68. The method of any one of claims 27-48 and 60-67 wherein the rAAV comprises an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV DJ, a goat AAV, a bovine AAV, or a mouse AAV, AAV2 / 207m8, or AAV LK03, or hybrids, or variants or chimeras thereof.

69. An engineered human embryonic kidney (HEK) 293 cell line in which the expression of glyceradehyde-3 -phosphate dehydrogenase (GAPDH) is downregulated compared to a control HEK293 cell line, wherein the HEK293 cell line is a host HEK293 cell line for the production of viral particles.

70. The engineered cell line of claim 69, wherein the GAPDH is downregulated by a modulator that decreases the expression and / or activity of the GAPDH.

71. The engineered cell line of claim 69 or 70, wherein the cell line comprises a genetic modification.

72. The engineered cell line of claim 70 or 71, wherein the modulator is a nuclease.

73. The engineered cell line of claim 72, wherein the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

74. The engineered cell line of claim 70, wherein the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

75. The engineered cell line of claim 70, wherein the modulator comprises a template for homologous arm-mediated recombination.

76. The engineered cell line of claim 70, wherein the modulator comprises an inhibitor of GAPDH.

77. The engineered cell line of claim 76, wherein the inhibitor comprises a synthetic small molecule inhibitor.

78. The engineered cell line according to any one of claims 69-77, wherein the cell line is a producer cell line.

79. The engineered cell line of claim 78, wherein the producer cell line further comprises stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest.

80. The engineered cell line of claim 78 or 79, wherein the producer cell line further comprises AAV helper function encoding sequences of an AAV helper virus.

81. The engineered cell line of claim 80, wherein the AAV helper virus is an adenovirus.

82. The engineered cell line of claim 81, wherein the adenovirus is Ad5 or Ad2.

83. The engineered cell line of claim 82, wherein the AAV helper function encoding sequences comprise at least one of: Ela, Elb, E2A, L4, E4, and VA.

84. The engineered cell line according to any one of claims 69-83, wherein the titer of rAAV produced from the engineered cell line is at least about 1.5-fold higher compared to the titer of rAAV produced from a cell line in which the expression of the gene is not altered.

85. A method of downregulating gene expression of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) for increasing the production of rAAV particles from a cell,wherein the method comprises culturing the cell in the presence of a modulator that downregulates the expression or activity of GAPDH.

86. The method of claim 85, wherein the modulator is dimethylsulfoxide (DMSO), dimethylformamide (DMF), (N-methylpyrrolidone (NMP), or dihydrolevoglucosenone (Cyrene).

87. The method of claim 86, wherein the concentration of DMSO is at least about 0.5%.

88. A method for increasing the production of rAAV particles, wherein the method comprises culturing a HEK293 cell in the presence of a modulator, wherein the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) is decreased as compared to a control HEK293 cell line, under conditions that allow for production and / or secretion of the recombinant viral particles.

89. The method of claim 88, wherein the method increases recombinant viral titer by at least 1.5-fold compared to a method comprising a control HEK293 cell.

90. The method of claim 88 or 89, wherein the modulator comprises a synthetic small molecule inhibitor of GAPDH.

91. The method of claim 88 or 89, wherein the modulator is dimethyl sulfoxide (DMSO), dimethylformamide (DMF), (N-methylpyrrolidone (NMP), dihydrolevoglucosenone (Cyrene), DC-5163, 1,2,3,4,6-penta-O-galloyl-p-D-glucopyranose (PGG), or 4-octyl itaconate (4-OI).

92. The method of claim 91, wherein the concentration of DMSO is at least about 0.5%.

93. The method according to any one of claims 88-92, wherein the modulator is an inhibitor of a pathway regulated by GAPDH.

94. The method according to any one of claims 88-93, wherein the modulator is a nuclease.

95. The method of claim 94, wherein the nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a meganuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeats (CRISPR) system.

96. The method according to any one of claims 88-93, wherein the modulator is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or an antisense RNA oligonucleotide (ASO).

97. The method according to any one of claims 88-93, wherein the modulator comprises a template for homologous arm-mediated recombination.

98. An engineered HEK293 host cell line produced by a method comprising knocking down the expression or activity of glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), wherein the engineered HEK293 cell line is for the production of viral particles.

99. The engineered HEK293 host cell line produced by the method of claim 98, wherein the knockdown is carried out using at least one of: homology-directed recombination, RNA-based knock-down, and a CRISPR-Cas enzymatic method.

100. The engineered HEK293 host cell line produced by the method of claim 98 or 99, further comprising stably integrated nucleic acid sequences encoding for AAV rep and / or cap, AAV ITR, and a gene of interest and optionally AAV helper function encoding sequences of an AAV helper virus.

101. The engineered HEK293 host cell line produced by the method according to any one of claims 98-100, wherein the AAV helper virus is an adenovirus.

102. The engineered HEK293 host cell line produced by the method of claim 99, wherein the adenovirus is Ad5 or Ad2.

103. The engineered HEK293 host cell line produced by the method of claim 100, wherein the AAV helper function encoding sequences comprise at least one of: Ela, Elb, E2A, L4, E4, and VA.

104. A recombinant viral particle obtained from the engineered HEK293 host cell line produced by the method according to any one of claims 98-103.

105. The recombinant viral particle of claim 104, wherein the recombinant viral particle is a recombinant adeno-associated virus (rAAV) particle.

106. A method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: transiently transfecting the host HEK293 cell line of claim 69 with one or more plasmids comprising: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, and (iii) nucleic acid encoding a gene of interest.

107. The method of claim 106, further comprising transfecting the host HEK293 cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus.

108. The method of claim 107, wherein the AAV helper virus is an adenovirus.

109. The method of claim 108, wherein the adenovirus is Ad5 or Ad2.

110. A method for producing a recombinant adeno-associated virus (rAAV) particle, the method comprising: stably transfecting the producer HEK293 cell line of claim 78 with: (i) nucleic acids encoding AAV rep and / or AAV cap, (ii) nucleic acids encoding AAV ITRs, and (iii) nucleic acid encoding a gene of interest.

111. The method of claim 110, further comprising transfecting the producer HEK293 cell line with a plasmid comprising AAV helper function encoding sequences of an AAV helper virus.

112. The method of claim 111, wherein the AAV helper virus is an adenovirus.

113. The method of claim 112, wherein the adenovirus is Ad5 or Ad2.

114. The method according to any one of claims 85-97 and 106-109, wherein the rAAV comprises an AAV serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV DJ, a goat AAV, a bovine AAV, or a mouse AAV, AAV2 / 207m8, or AAV LK03, or hybrids, or variants or chimeras thereof.