US 10,889,834 B2
Methods and compositions for enhancing targeted transgene integration
Gregory J. Cost, Richmond, CA (US); and Thomas Wechsler, Richmond, CA (US)
Assigned to Sangamo Therapeutics, Inc., Brisbane, CA (US)
Filed by Sangamo BioSciences, Inc., Richmond, CA (US)
Filed on Dec. 14, 2015, as Appl. No. 14/967,885.
Claims priority of provisional application 62/092,160, filed on Dec. 15, 2014.
Prior Publication US 2016/0168593 A1, Jun. 16, 2016
Int. Cl. C12N 15/90 (2006.01); A61K 31/4745 (2006.01); A61K 31/7048 (2006.01); A61K 48/00 (2006.01)
CPC C12N 15/907 (2013.01) [A61K 31/4745 (2013.01); A61K 31/7048 (2013.01); A61K 48/00 (2013.01); C12N 2750/14143 (2013.01)] 11 Claims
 
1. A method of integrating a transgene encoding a protein in a forward orientation into a selected endogenous genomic locus of an isolated population of liver or hematopoietic stem cells, the method comprising:
treating the isolated liver or hematopoietic stem cells with at least one topoisomerase inhibitor, the at least one topoisomerase inhibitor comprising camptothecin or etoposide;
introducing an AAV vector into the treated population of isolated liver or hematopoietic stem cells, the AAV vector comprising regions of homology to the selected endogenous genomic locus flanking the transgene, wherein the population of liver or hematopoietic stem cells is grown in the presence of the at least one topoisomerase inhibitor, such that the transgene is integrated into the selected endogenous locus in a forward orientation and expression of the transgene is driven an endogenous promoter and further wherein the transgene is integrated into the selected endogenous genomic locus and the protein is expressed from the endogenous promoter at levels increased at least 5-fold as compared to cells not grown in the presence of the at least one topoisomerase inhibitor.