HIV protection by ZFN-based disruption of CCR5 gene in Hematopoietic stem cells

描述(申请人提供)：用锌指核酸酶和TALENS进行基因组编辑已经成为一种很有前途的技术，可以在目标基因中诱导局部序列突变。由于CCR5是HIV的主要共同受体，因此正在进行研究，以破坏T细胞和CD34+造血干细胞的基因，以保护这些细胞或其后代免受病毒感染。到目前为止，病毒载体和质粒载体已被用于ZFN/TALEN的传递。然而，这会导致核酸酶的长期表达，这可能是潜在的有害的，因为越来越多的研究证明了许多工程ZFN的剂量依赖性毒性，推测是由于在非靶点产生意外的双链断裂。从mRNA中瞬时表达ZFN/TALEN可能提供一种可行的替代方案，因为短期接触核酸酶就足以导致目标基因的永久性修饰。在这项建议中，ZFN或TALEN的mRNA表达将被用作干扰CD34+造血干细胞(HSCs)中CCR5的一种策略。为了克服ZFN/TALEN mRNA传递到CD34+HSCs的障碍，这是传统方法难以转染的细胞，我们将开发展示先前描述的DNA适配子sgc8的脂质纳米颗粒，它与细胞上表达的蛋白酪氨酸激酶7结合。人源化的BLT小鼠将与ZFN/TALEN修饰的CD34+HSPC重组，并测试其对HIV攻击的保护作用。从这些研究中获得的知识可能会导致开发一种新的、安全的方法来利用ZFN/TALEN进行HIV基因治疗，甚至提供一种潜在的方法，将分子直接输送到体内动员的干细胞。 公共卫生相关性：定制锌指核酸酶(ZFN)基因干扰是一项新兴技术，在包括艾滋病在内的各种疾病的基因治疗中具有广阔的应用前景。这项拟议研究的主要目标是利用ZFN干扰CD34+造血干细胞中的HIV共受体CCR5，作为基因治疗来产生抗HIV的后代。由于ZFN的长期表达可能是有害的，我们计划开发一种纳米颗粒递送平台，用于在细胞中瞬时表达mRNA，以便ZFN/TALEN仅在短时间内表达，以发挥其CCR5基因修饰活性。

DESCRIPTION (provided by applicant): Genome editing with Zinc Finger nucleases and TALENs has emerged as a promising technology for inducing localized sequence mutations in targeted genes. As CCR5 is the principal co-receptor for HIV, studies are underway to disrupt the gene in T cells and CD34+ Hematopoietic stem cells to protect the cells or their progeny from viral infection. So far, viral and plasmid vectors have been used for delivery of ZFNs/TALENs. However, this results in long-term expression of the nucleases, which may be potentially harmful, as increasing number of studies have documented dose-dependent toxicity of many engineered ZFN, presumably due to the generation of unintended double-stranded breaks at off-target sites. Transient expression of ZFNs/TALENs from mRNA may provide a feasible alternative as short term exposure to the nucleases is sufficient to cause permanent modification of the targeted gene. In this proposal, ZFN or TALEN expression from mRNA will be used as a strategy to disrupt CCR5 in CD34+ hematopoietic stem cells (HSCs). To overcome the hurdle of ZFN/TALEN mRNA delivery to CD34+ HSCs, which are difficult cells to transfect by conventional methods, we will develop lipid nanoparticles displaying a previously described DNA aptamer sgc8 that binds to Protein tyrosine kinase 7, expressed on the cells. Humanized BLT mice will be reconstituted with the ZFN/TALEN-modified CD34+ HSPCs and tested for protection from HIV challenge. Knowledge gained from the studies could lead to the development of a novel and safe approach to harness ZFN/TALEN for HIV gene therapy and even provide a potential method for direct delivery of the molecules to mobilized stem cells in vivo. PUBLIC HEALTH RELEVANCE: Gene disruption by tailored Zinc Finger nuclease (ZFN) is an emerging technology with promising applications in gene therapy of various diseases including AIDS. The main goal of the proposed research is to use ZFN to disrupt the HIV co-receptor CCR5 in CD34+ hematopoietic stem cells as gene therapy to generate HIV resistant progeny. As long-term expression of ZFN can be harmful, we plan to develop a nanoparticle delivery platform for transient mRNA expression in the cells so that ZFNs/TALENs are expressed only for a short time to exert their CCR5 gene modification activity.