The goal of the proposed research is to develop a potentially curative AIDS treatment. We hypothesize that novel cell-penetrating zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) will allow for direct gene editing in human CD4+ T-cells and CD34+ hematopoietic stem cells (HSCs), thereby bypassing concerns and difficulties posed by contemporary delivery systems. Direct application of engineered nuclease proteins will lead to biallelic disruption of CCR5/CXCR4 genes and allow for HIV-1 resistant cells to repopulate and protect the body, eventually allowing affected individuals to go off drug therapy and on to a life free of disease. Success may require combining our therapy with emerging drugs that activate latent viral reservoirs allowing them to be eliminated by the gene edited immune cells we develop here. Driving our enthusiasm is the report of an HIV-1 positive patient cured by stem cell transplant from a ?32 donor. The "Berlin patient" had no detectable levels of HIV-1 years after the procedure despite discontinuing retroviral therapy.
We aim to recapitulate this success using our innovative approach that is free of nucleic acids and viral delivery vehicles. While clinical trials using adenoviral delivery of ZFN SB-728 are currentl underway, recent data indicates that low efficiency of biallelic modification observed in phase I studies may limit its potential to patients whom are ?32 heterozygous. Significant improvements in ZFN nucleases are needed. In preliminary studies we have shown that cell-penetrating ZFN proteins can knock-out CCR5 after their application to cell lines, T-cells, and HSCs. We found that cells treated with our proteins showed a high rate of biallelic modification. In one experiment, 58% of modified cells showed disruption at both alleles. The efficiency of our approach is similar to DNA vector transfection;however, our approach provides dramatic reductions in off-target cleavage. In new preliminary data, we have developed a novel approach to cell- permeable TALENs and demonstrated gene editing at CCR5. Here we develop a family of very efficient CCR5/CXCR4 targeting nucleases using design and in vitro evolution approaches. ZFNs and TALENs will be optimized with respect to catalytic activity, sequence specificity, cell-permeability, and biallelic gene editing. Coupled with charge engineering of nucleases should allow for dramatic increases in the quantity of transduced protein delivered to cells. Because our approaches have allowed us to identify FokI nuclease variants with >15 fold improvement in activity and novel sequence specificity, we are confident that the nucleases developed herein will edit T cells and HSCs at levels exceeding that reported by viral delivery systems. The nucleases developed here will demonstrate higher specificity and improved safety profiles. We will optimize treatment of human T-cells and HSCs with purified proteins, study their transplant into immunocompromised mice, and their ability to limit HIV-1 infection. The efficiency and safety of ZFN/TALEN protein delivery will be compared to conventional delivery methods. We believe that the development of the permeable nucleases proposed here could prove to be a breakthrough in HIV-1 therapy and many other genetic diseases.
The goal of the proposed research is to develop a new and potentially curative AIDS treatment. We hypothesize that our novel evolved zinc finger nucleases and transcription activator-like effector nucleases will allow for direct gene editing of CCR5 and/or CXCR4 with purified nuclease proteins in human CD4+ T-cells and CD34+ hematopoietic stem cells, thereby bypassing concerns and difficulties posed by contemporary nuclease delivery systems. If successful, our approach will allow a reservoir of HIV-1 resistant cells to repopulate the body;over time allowing affected individuals to go off drug therapy and on to a life free of disease.