Developing More Efficient Protective Therapies Against HIV: Combining MegaTAL Nuclease-Driven Genome Engineering with a Resistance Cassette

摘要

Background: Human Immunodeficiency Virus (HIV) infection remains a substantial health problem worldwide. The human C-C chemokine receptor 5 (CCR5) gene, which encodes a co-receptor required for HIV entry into CD4+ T cells, is a promising alternative therapeutic target. Early clinical trials using CCR5-disrupting zinc finger nucleases in patients have demonstrated sustained functional control of HIV during antiretroviral treatment interruption. However, two limitations of current gene editing required to achieve therapeutic benefit remain unaddressed. These are (a) need for higher levels of CCR5-disruption and (b) preferential selection of gene modified cells. Methods: The CCR5-targeting megaTAL is a novel nuclease architecture that combines a LAGLIDADG homing endonuclease scaffold with an eleven repeat transcription activator-like (TAL) effector array to achieve efficient site specific cleavage. We are coupling megaTAL nuclease treatment with drug selection in order to disrupt the CCR5 locus, and select modified CD4+ T-cells to achieve therapeutically relevant levels of HIV-protected cells. The mutant human dihydrofolate reductase (DHFR) construct renders cells resistant to lymphotoxic concentrations of the drug methotrexate (MTX) at 0.02uM. For optimal cell viability we deliver nucleases via mRNA and selection-constructs via adeno-associated virus (AAV). Results: Electroporation with megaTAL mRNA demonstrated robust CCR5 disruption: 95% in GHOST-Hi5 cell lines and 70-90% in human CD4+ T-cells. Gene-modified human T-cells were transplanted into NOD/SCID/?c-null 'humanized' mice and subsequently challenged with HIV-1 infection. CCR5-null modified cells preferentially survived during active HIV infection in vivo (100 fold increase). Initially, the HIV plasma viremia was significantly lower in the nuclease-treated mice. However, the virus levels rebounded over time. We hope to address this by selectively transplanting cells that have been CCR5 disrupted and hence protected from any infection. In our preliminary experiments, primary T-cells lentivirally transduced with a Tyr-22-DHFR cassette at 15% efficiency showed an enrichment of >90% over 7 days in 0.02uM methotrexate. More recently, we have demonstrated AAV-mediated targeted gene insertion followed by chemoselection, and reached >50% enriched populations of human CD4 T cells. Conclusions: 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. We are able to knock-in a selection cassette at the CCR5 disruption-site and specifically enrich for gene-modified cells.