Improving targeted nuclease-mediated gene correction in human hematopoietic stem cells

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Project Summary/Abstract The recent emergence of targeted nucleases (such as Zinc-Finger Nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system) allows for site-specific gene modification in cells. Nucleases induce a double-stranded break (DSB) in the DNA, stimulating repair via one of the two pathways: error-prone non-homologous end joining (NHEJ), or precise homology-directed repair (HDR), when a donor template is available. A donor template can be supplied exogenously to allow the cells to correct disease-causing mutations. If used in hematopoietic stem cells (HSCs), this technique could provide long-term self-renewing population to generate a life-long supply of healthy (corrected) cells. However, clinical translation of this approach is impeded by high cytotoxicity in human hematopoietic stem and progenitor cells (HSPCs) associated with nuclease and donor template delivery, subop- timal HDR levels, and low HDR to NHEJ ratio in the primitive HSC population. Therefore, this project is aimed at (1) studying the mechanisms for increased toxicity in human HSCs and minimizing cell death, (2) improving HDR to NHEJ ratio, and (3) studying what governs repair pathway choice (HDR vs. NHEJ) in human HSCs, and how to increase the levels of HDR-mediated DSB repair in HSCs. Preliminary experiments have identified that tran- sient overexpression of BCL2 during nuclease and donor template delivery decreases toxicity and improves cells viability. The studies in Aim 1 of this project are designed to decipher the mechanisms of BCL2 action, to test its effect on gene modification of HSCs, and to assess its safety by conducting in vivo experiments. The studies in Aim 2 will focus on improving the HDR/NHEJ ratio by controlling DNA repair pathway choice in cell cycle-de- pendent manner using two independent approaches: first, by temporarily synchronizing the cells in S/G2 phases of cell cycle when HDR is known to occur, and second, by minimizing nuclease activity during G1 phase of cell cycle (which usually leads to NHEJ) through the addition of cell-cycle specific degradation signal to the nuclease. Since the majority of HSCs are in G0/G1 phase of cell cycle, studies in Aim 3 will attempt to initiate HDR in G1 by manipulating the formation of a protein complex that affects the DNA DSB repair pathway choice. This project is unique in its opportunity to combine the basic mechanistic study of DNA repair in human HSCs with development of translational methods for improving targeted gene correction in HSCs, which can result in an advancement over the current treatment options. Successful completion of the proposed aims can potentially be the necessary component for enhancing nuclease-based gene therapy to be clinically viable for monogenic diseases of the blood.