megaTAL-mediated Gene Editing at the CCR5 locus

Thesis (Ph.D.)--University of Washington, 2016-08

Engineered nucleases can be used to induce double stranded breaks at specific sites in the genome to facilitate therapeutic gene disruption and/or gene insertion. Here, we report the insertion of a selection marker following gene disruption, which allows us to specifically enrich for gene-edited CD4 T cells in the disease-context of Human Immunodeficiency Virus (HIV). HIV infection remains a substantial health problem worldwide. The C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV entry into CD4+ T cells and therefore represents a potential therapeutic target for gene disruption. Our current work uses the CCR5-targeting megaTAL nuclease to achieve site specific DNA cleavage. We demonstrated highly efficient CCR5 targeting in primary human CD4+ T cells in vitro (70-90% disruption). Subsequently, we tested the protective effects of megaTAL treatment of human CD4+ T cells in NOD/SCID/γc-null mice challenged with HIV-1. We observed a 100-fold increase of megaTAL-treated cells compared to untreated controls during an active in vivo infection demonstrating the functionality of this approach. Next we coupled CCR5-disruption with the insertion of a drug-resistance gene to enable efficient selection of only CCR5-modified T cells. The mutant human dihydrofolate reductase (mDHFR) chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We combined megaTAL-treatment with the delivery of a DNA donor template using Adeno-associated virus (AAV) to insert mDHFR constructs at the CCR5 locus. Primary human CD4+ T cells transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing mDHFR flanked by 0.8kb CCR5 homology arms produced MTX-resistant CD4+ cells that also lack CCR5. These gene-modified cells exhibited between five to six-fold enrichment after chemoselection in 0.1uM MTX compared to unmodified controls ex vivo. In conclusion, CCR5-megaTAL produces very high levels of gene-disrupted human CD4+ T-cells and protects these cells from subsequent HIV infection in vivo. Furthermore, targeted insertion of drug-resistance genes followed by selection results in the enrichment of gene-modified primary T cells. To our knowledge we are the first to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted insertion at the CCR5 locus.