1. ¹Research and Development, Sangamo BioSciences, Richmond, CA
2. ²Research, MaxCyte, Inc., Gaithersburg, MD
3. ³Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA

^|[ast]|I am a full-time employee of Sangamo BioSciences.

Abstract

The identification of the chemokine receptor CCR5 as the major co-receptor required for HIV entry into CD4 T-cells has made the targeted disruption of CCR5 an attractive potential therapeutic approach for treating HIV-infected patients. Yet, no current method permits the efficient therapeutic disruption of a chosen gene in the human genome. We have developed designed zinc-finger protein nucleases (ZFNs) that specifically target the coding sequence of the CCR5 gene. Using standard delivery methods, we have shown these ZFNs can efficiently stimulate the formation of a double strand break (DSB) in the CCR5 locus to allow the natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), to subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in frameshift mutations, leading to permanent disruption in >10% of K562 cells.

To take this technology toward clinical application we sought a delivery mode that could transiently deliver the ZFNs to large numbers of primary human T-cells without disrupting their normal cellular functions. In this regard we tested the ability of a scalable, flow-based electroporation device to deliver the CCR5-ZFNs at high efficiency to K562 cells and primary human T-cells. This closed, sterile system allows for consistent delivery of the CCR5- ZFN expression plasmid to >1|[times]|10e9 cells. Initial experiments showed this approach could disrupt >20% of the CCR5 alleles in K562 cells and >5% in primary human T-cells. The frequency of gene disruption observed supports the examination of this approach as a possible method for the therapeutic modification of isolated patient cells to generate HIV-resistant T-cells. We will present data on the development of this approach in model systems and in primary human T-cells.