CCR5 knockout HSC as an anti-HIV therapy

Paula M. Cannon, Childrens Hospital, Los Angeles
Molecular Biology

Genetic engineering of hematopoietic stem cells (HSC) could potentially be used to generate HIV-resistant cells in all of the lineages that are susceptible to HIV infection (e.g. T lymphocytes, monocyte/macrophages). The expansion of the pool of resistant cells over time is expected to increase the ability of a patient to limit viremia and slow HIV spread, and our clinical experience at Childrens Hospital Los Angeles suggests that there will be selection for such cells in the face of an active HIV infection. To date, gene therapy approaches for HIV that target HSC have focused exclusively on the addition of genetic material to cells in order to confer resistance. The panel of anti-HIV genes that have been used includes transdominant viral proteins, siRNAs, antisense constructs and single chain antibodies. Although these strategies have been highly effective at suppressing HIV replication in tissue culture, the requirement for high efficiency HSC engineering, together with the long-term expression of a foreign gene at an ectopic site, provide challenges for the clinical setting. In response to these concerns, we are exploring an alternate strategy to confer HIV-resistance to HSC progeny, based on disrupting the CCR5 co-receptor gene through transient engineering with zinc finger nucleases (ZFNs). These chimeric proteins are able to bind to a specific sequence in the CCR5 locus and introduce a double-stranded break that effectively disrupts CCR5 expression, while simultaneously introducing a marker gene that can facilitate experimental analyses. Since individuals homozygous for a natural CCR5 deletion show marked resistance to HIV infection without any adverse hematological effects, disruption of CCR5 is expected to be a safe but effective anti-HIV approach. In this proposal we aim to develop the technology necessary to allow us to efficiently and safely engineer human HSC with ZFNs. We will compare two different methods of transient gene delivery of the ZFN to HSC to identify the most efficient method to engineer human HSC. Subsequently, we will examine the consequences of this genome editing for HSC survival and activity, using both in vitro assays of HSC function and NOD/SCID/IL2Rγnull mice transplanted with the engineered HSC. Finally, we will assess whether the loss of CCR5 from a population of cells can indeed limit HIV infection, and whether the CCR5-negative progeny have a survival advantage in the face of such HIV infection, using both cell culture infection of HSC-derived macrophages, as well as HIV challenge of HSC transplanted NOD/SCID/IL2Rγ mice.