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

项目摘要/摘要 最近出现的靶向核酸酶(如锌指核酸酶(ZFN)、转录激活剂- 像效应核酸酶(TALEN)和成簇的规则间隔的短回文重复 (CRISPR)/Cas9系统)允许在细胞中进行特定部位的基因修饰。核酸酶诱导双链 DNA断裂(DSB)，通过两条途径之一刺激修复：容易出错的非同源末端连接 (NHEJ)，或精确同源定向修复(HDR)，当供体模板可用时。供体模板可以 以外源方式供应，使细胞能够纠正致病突变。如果用于造血干细胞 细胞(HSCs)，这项技术可以提供长期自我更新的种群，以产生终身供应 健康(已更正)细胞。然而，这种方法的临床翻译受到人类高细胞毒性的阻碍。 与核酸酶和供体模板传递相关的造血干细胞和祖细胞(HSPC)，亚单位。 原始HSC人群中的时间HDR水平和低HDR/NHEJ比率。因此，该项目旨在 (1)研究人类HSCs毒性增加和减少细胞死亡的机制；(2)改善HDR 与NHEJ的比率，以及(3)研究是什么控制了人类HSCs的修复途径选择(HDR与NHEJ)，以及如何 增加HDR介导的HSCs DSB修复水平。初步实验已证实，反式- 在核酸酶和供体模板输送过程中过表达bcl2可降低毒性并改善细胞 生存能力。本项目目标1中的研究旨在破译bcl2的作用机制，测试其 对HSCs基因修饰的影响，并通过体内实验评估其安全性。中的研究 目标2将致力于通过控制DNA修复途径的选择来提高HDR/NHEJ比率。 使用两种独立的方法以挂起的方式：第一，通过临时同步处于S/G2期的细胞 在已知发生HDR的情况下，抑制细胞周期；第二，通过最小化细胞G1期的核酸酶活性 细胞周期(通常导致NHEJ)通过将细胞周期特定的降解信号添加到核酸酶来实现。 由于大多数造血干细胞处于细胞周期的G0/G1期，目标3的研究将尝试在G1期启动HDR 通过操纵蛋白质复合体的形成，影响DNA DSB修复途径的选择。 这个项目的独特之处在于它结合了人类造血干细胞DNA修复的基本机制研究 随着翻译方法的发展，以改善HSCs中的靶向基因校正，这可能导致 与目前的治疗方案相比，这是一个进步。成功完成拟议的目标可能会 是加强以核酸酶为基础的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.