Methods and compositions for improving the assembly of adeno-associated viruses (AAVs)
View Patent ↗Described herein are methods and compositions for modifying cells to improve the assembly of adeno-associated viruses (AAVs).
1 . A method for improving the assembly of adeno-associated virus (AAV) in a cell, the method comprising:
increasing expression or activity of Hsc/Hsp70 or a co-factor thereof, vacuolar-specific H+ ATPase, and/or cyclin-dependent kinase 2 in the cell to produce a modified cell;
infecting the modified cell with an AAV vector to produce an infected modified cell;
culturing the infected modified cell; and
collecting the assembled AAV.
2 . The method of claim 1 , wherein the amount of assembled AAV collected is greater than the amount of assembled AAV collected following AAV infection of a cell that lacks the modification.
3 . The method of claim 1 , wherein the method results in an increase in titer of the AAV as compared to a cell without an increase in expression or activity of Hsc/Hsp70 or a co-factor thereof, vacuolar-specific H+ ATPase, and/or cyclin-dependent kinase 2.
4 . A method for improving the assembly of adeno-associated virus (AAV), the method comprising:
producing AAV from a modified cell with the AAV, wherein the cell has been modified to exhibit an increase or decrease in one or more genes or the protein encoded therefrom selected from CHMP7, CEP72, CNOT6, SLC9A6, PPHLN1, ATF7IP, SETDB1, GPR89B, KIF16B, ATAD3A, BAG2, STUB1, DNAJA1, DNAJC7, HSPA8, HSPAIA, CDK1, CDK2, CHCHD2, C1QBP, DPYSL5, FXR1, FXR2, IPO5, LBR, MTPN, NPM3, NPM1, PPL, SNX3, UBE2O, SAE1, CCDC124, GNB1, RABIA, GNB4, RPL23, CKB, SRP9, UCHL1, and TBCB; and
collecting the assembled AAV.
5 . The method of claim 4 , wherein the modified cell is a genetically engineered cell.
6 . The method of claim 5 , wherein the genetically engineered cell comprises knockout, knockdown, over-expression, or combinations thereof.
7 . The method of claim 4 , wherein the modified cell comprises a chemical compound that increases or decreases the one or more genes or the proteins encoded therefrom.
8 . The method of claim 7 , wherein the chemical compound is selected from the group consisting of Cdk1 inhibitor IV, Cdk2 inhibitor II, apoptozole, ML-792, BML282, NSC348884, FDNB, and Bafilomycin A1.
9 . A cell-free culture system for assembling adeno-associated virus comprising:
culture media; and
at least two proteins, or nucleic acids encoding the at least two proteins, selected from CHMP7, CEP72, CNOT6, SLC9A6, PPHLN1, ATF7IP, SETDB1, GPR89B, KIF16B, ATAD3A, BAG2, STUB1, DNAJA1, DNAJC7, HSPA8, HSPAIA, CDK1, CDK2, CHCHD2, C1QBP, DPYSL5, FXR1, FXR2, IPO5, LBR, MTPN, NPM3, NPM1, PPL, SNX3, UBE2O, SAE1, CCDC124, GNB1, RABIA, GNB4, RPL23, CKB, SRP9, UCHL1, and TBCB.
10 . The cell free culture system of claim 9 , comprising at least three proteins or nucleic acids encoding the at least three proteins.
11 . A cell line comprising:
one or more mutations in Hsc/Hsp70 or a co-factor thereof, vacuolar-specific H+ ATPase, and/or cyclin-dependent kinase 2; and/or
one or more exogenous constructs expressing Hsc/Hsp70 or a co-factor thereof, vacuolar-specific H+ ATPase, and/or cyclin-dependent kinase 2.
12 . The cell line of claim 11 , wherein the mutation comprises a knockout mutation.
13 . The cell line of claim 11 , wherein the mutation comprises a knockdown mutation.
14 . The cell line of claim 11 , wherein the one or more exogenous constructs is a recombinant construct.
15 . An article of manufacture for improving the assembly of adeno-associated virus (AAV) in a cell, comprising at least one member from at least two of (a), (b) and of (c):
(a) Hsc/Hsp70 or a co-factor thereof, a nucleic acid encoding Hsc/Hsp70 or a co-factor thereof, or a compound that modulates Hsc/Hsp70 or a co-factor thereof;
(b) vacuolar-specific H+ ATPase, a nucleic acid encoding vacuolar-specific H+ ATPase, or a compound that modulates vacuolar-specific H+ ATPase; and
(c) cyclin-dependent kinase 2, a nucleic acid encoding cyclin-dependent kinase 2, or a compound that modulates cyclin-dependent kinase 2.
16 . The article of manufacture of claim 15 , wherein the compound that modulates Hsc/Hsp70 or a co-factor thereof is apoptozole.
17 . The article of manufacture of claim 15 , wherein the compound that modulates vacuolar-specific H+ ATPase is Bafilomycin A1.
18 . The article of manufacture of claim 15 , wherein the compound that modulates cyclin-dependent kinase 2 is Cdk2 inhibitor II.