Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase
| Autor: | Nicholas Hubbard, David J. Rawlings, Teresa Einhaus, Christopher W. Peterson, Biswajit Paul, Hans-Peter Kiem, Guillermo S. Romano Ibarra, Alexander Astrakhan |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
lcsh:QH426-470 Genetic enhancement Transgene Mutant lentiviral vectors Biology chemoselection Article methotrexate Homology directed repair 03 medical and health sciences dihydrofolate reductase Gene knockin Dihydrofolate reductase Genetics cancer lcsh:QH573-671 T cell gene therapy Molecular Biology Gene lcsh:Cytology gene editing HIV adeno-associated viruses Transfection Molecular biology 3. Good health lcsh:Genetics homology-directed repair 030104 developmental biology biology.protein Molecular Medicine |
| Zdroj: | Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 9, Iss, Pp 347-357 (2018) |
| ISSN: | 2329-0501 |
| Popis: | 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. Keywords: gene editing, chemoselection, T cell gene therapy, lentiviral vectors, homology-directed repair, methotrexate, dihydrofolate reductase, adeno-associated viruses, HIV, cancer |
| Databáze: | OpenAIRE |
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