Abstract B106: Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection

Engineered nucleases can be used to induce double stranded breaks at specific sites in the genome to facilitate therapeutic gene disruption or gene insertion. Gene insertion strategies may have a significant advantage over traditional lentiviral gene therapy since we have much greater control over transgene integration site, copy number, and expression. However, a major limitation in the clinical application of gene insertion technologies is the low efficiency of homology driven repair (HDR) achieved in hematopoietic cells. Here, we report the knock-in of a selection marker, which allows us to enrich for modified CD4 T cells, and to achieve clinically relevant levels of gene edited cell populations in the disease-context of HIV. Human Immunodeficiency Virus (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 represents an alternative therapeutic target. Early clinical trials using CCR5-targeting zinc finger nucleases demonstrated transient control of HIV infection in the course of antiretroviral treatment interruption (Tebas, NEJM, 2014). Our current work improves on these advances by combining a high level of CCR5 gene disruption with preferential selection of gene-modified cells using a megaTAL nuclease. The CCR5-targeting megaTAL, combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve site-specific DNA cleavage. This nuclease produces highly efficient CCR5 disruption in primary human CD4+ T cells in vitro (70-90%). To test the protective effects of gene disruption, primary human CD4+ T cells treated with CCR5-megaTAL were transplanted into NOD/SCID/γc-null (NSG) ‘humanized’ mice and 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, to overcome the decline of HIV-resistant cells, observed in the clinical trials to date, we proposed to couple CCR5 gene-disruption with gene insertion of mutant human dihydrofolate reductase (mDHFR). The mDHFR chemoselection system has been used to render cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX). We first tested our experimental approach by transducing cells with a lentiviral vector encoding a mDHFR cassette followed by chemoselection in MTX at 0.2uM. This approach resulted in a six-fold enrichment of gene modified primary CD4+ T cells ex vivo . For gene insertion, combining megaTAL treatment with adeno-associated virus (AAV) transduction produces very high rates of homology-driven repair (HDR) in primary human T cells. Hence, we merged the megaTAL nuclease and AAV gene-delivery treatments to integrate mDHFR into the CCR5 locus, thus producing a population of MTX-resistant CD4+ cells that also lack CCR5. Primary human CD4+ T cells were transfected with CCR5-megaTAL mRNA and transduced with AAV6 containing a mutant DHFR donor template flanked by 0.8kb CCR5 homology arms. They demonstrated a greater than five-fold enrichment in 0.1uM MTX compared to untreated controls ex vivo . Finally, we are assessing the therapeutic potential of our approach in NSG mice. In conclusion, the CCR5-megaTAL nuclease platform produces very high levels of gene-modified CD4+ T-cells and protects these cells from subsequent HIV infection in vivo . Furthermore, combining targeted integration and chemical selection results in the specific selection of gene modified primary human T cells. To our knowledge we are the first group to report MTX-mediated chemoselection and expansion of CD4+ T cells following targeted integration at the CCR5 locus. Eventually this system could be used to select for modified cells in patients using previously established protocols for MTX administration in the clinic. Citation Format: Biswajit Paul, Guillermo Romano Ibarra, David J. Rawlings, Hans-Peter Kiem. Substantial enrichment of HIV- resistant CD4 T cells by combining the targeted integration of a resistance-cassette at the CCR5 locus with chemoselection [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B106.