Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase.

ABSTRACT: Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression-significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4⁺ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion

SUBMITTER: Paul B 

PROVIDER: S-EPMC6054698 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature