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. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.
Paul, B., Ibarra, G. S. R., Hubbard, N., Einhaus, T., Astrakhan, A., Rawlings, D. J., … Peterson, C. W. (2018). Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase. Molecular Therapy - Methods and Clinical Development, 9, 347–357. https://doi.org/10.1016/j.omtm.2018.04.002