Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering

Targeted genome engineering is a powerful method to create specific modifications at chromosomal loci. This technique makes it feasible to precisely alter DNA sequences by introducing a specific DNA double-strand break, which is repaired by the natural cellular machinery. These double strand breaks are induced by engineered chimeric nucleases – either zinc finger nucleases (ZFNs) or Tal effector nucleases (TALENs) – and depending on the experimental design, can result in gene disruption, gene correction or targeted transgene integration. In this thesis, I present two applications of this approach in the context of two prevalent human diseases, HIV infection and sickle cell disease. HIV infects CD4+ T-cells by binding to the CD4 receptor and either the CCR5 or CXCR4 co-receptor on the surface of those cells. Previously, ZFNs were described that create gene specific knockouts of CCR5, protecting cells against CCR5-tropic (R5) HIV, but not against CXCR4-tropic (X4) HIV. I hypothesized that combining ZFN-mediated CCR5 disruption with targeted integration of a cassette of anti-HIV genes would confer higher levels of resistance against R5-tropic virus and also be protective against X4-tropic virus. In a T-cell reporter line, I showed that CCR5 disruption alone conferred 16-fold protection against R5-tropic virus but had no effect against X4-tropic HIV. In contrast, CCR5 disruption, combined with targeted gene integration into that locus, of the anti-HIV restriction factors human-rhesus hybrid TRIM5α, APOBEC3G D128K and Rev M10 was completely protective against both viral tropisms. Sickle cell disease is caused by a point mutation in the β-globin gene, and I sought to correct this mutation by synthesizing TALENs specific for that site. The β-globin TALENs stimulated integration of therapeutic β-globin cDNA in approximately 20% of cells prior to selection. Using FDA-approved drugs to select for modified cells, I showed virtually complete enrichment of targeted cells. Furthermore, I used the β-globin TALENs to target GFP to the β-globin start codon and designed TALENs to target tdTomato to the start codon of the γ-globin gene, upregulation of which is a goal of sickle cell disease pharmacotherapy. In this way, I developed an endogenous dual promoter reporter system and screened for drugs that preferentially upregulated γ-globin.