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

项目总结/摘要 最近出现的靶向核酸酶（如锌指核酸酶（ZFN）、转录激活因子（TRANSCTION ACTIVATOR））， 类似效应核酸酶（TALEN）和重复的规则间隔短回文重复序列 （CRISPR）/Cas9系统）允许细胞中的位点特异性基因修饰。核酸酶诱导双链 DNA断裂（DSB），通过两种途径之一刺激修复：易错非同源末端连接 （NHEJ）或精确同源定向修复（HDR）。供体模板可以 外源性地提供，以允许细胞纠正致病突变。如果用于造血干细胞 细胞（HSC），这项技术可以提供长期自我更新的人口，以产生终身供应的造血干细胞。 健康的（正确的）细胞。然而，这种方法的临床转化受到人类高细胞毒性的阻碍， 与核酸酶和供体模板递送相关的造血干细胞和祖细胞（HSPC），亚基- 原始HSC群体中的最低HDR水平和低HDR与NHEJ比率。因此，该项目旨在 (1)研究人类HSC中毒性增加的机制并最大限度地减少细胞死亡，（2）改善HDR 与NHEJ比率，以及（3）研究在人类HSC中是什么支配修复途径选择（HDR与NHEJ），以及如何 以增加HSC中HDR介导的DSB修复水平。初步实验表明，trans- 在核酸酶和供体模板递送过程中BCL 2的沉默过表达降低了毒性并改善了细胞 生存能力该项目目标1中的研究旨在破译BCL 2的作用机制，测试其在细胞内的作用。 对HSC基因修饰的影响，并通过体内实验评估其安全性。中的研究 目的2将集中于通过控制细胞周期中DNA修复途径的选择来提高HDR/NHEJ比率。 采用两种独立的方法：首先，通过暂时同步S/G2期细胞 第二，通过使细胞G1期期间的核酸酶活性最小化， 通过将细胞周期特异性降解信号添加到核酸酶来改变细胞周期（其通常导致NHEJ）。 由于大多数HSC处于细胞周期的G 0/G1期，目的3中的研究将尝试在G1期启动HDR 通过操纵影响DNA DSB修复途径选择的蛋白质复合物的形成。 这个项目是独一无二的机会，联合收割机的基本机制研究DNA修复在人类HSC 随着用于改善HSC中靶向基因校正的翻译方法的发展， 这是对现有治疗方案的一种进步。成功完成拟议目标可能会 成为增强基于核酸酶的基因治疗以使单基因治疗在临床上可行的必要组成部分 血液疾病。

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.