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

基于 ZFN 的造血干细胞 CCR5 基因破坏对 HIV 的保护

基本信息

批准号：
8413587
负责人：
Premlata Shankar
金额：
$ 18.86万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8413587
关键词：
Acquired Immunodeficiency Syndrome Antiviral Agents Attention Autologous Back Berlin Binding CCR5 gene CD34 gene CD4 Positive T Lymphocytes Catalytic Domain Catalytic RNA Cell Death Cells Chemokine (C-C Motif) Receptor 5 Clinical Trials DNA Development Disease Dose Drug resistance Education Emerging Technologies Engineering Exposure to Gene Expression Profile Gene Silencing Gene Targeting Gene-Modified Generations Genes Genetic Genome Genome engineering Goals HIV HIV Infections HIV Receptors Hematopoietic stem cells Highly Active Antiretroviral Therapy Immune In Vitro Infection Interruption Intervention Knowledge Lead Life Lipids Mediating Membrane Messenger RNA Methods Modeling Modification Molecular Mus Mutation Nucleic Acids Patients Pharmaceutical Preparations Plasmid Cloning Vector Population Protein Tyrosine Kinase RNA Interference Receptor Protein-Tyrosine Kinases Reporting Research Resistance Series Site Site-Directed Mutagenesis Small Interfering RNA Specificity Stem cells T-Cell Development T-Lymphocyte Technology Testing Therapeutic Time Toxic effect Toxicity Tests Transcription Coactivator Transplantation Viral Load result Viral Vector Virus Virus Diseases Zinc Fingers aptamer base chemokine receptor design gene therapy in vivo knock-down leukemia mRNA Expression macrophage monocyte mouse model nanoparticle novel nuclease protein expression receptor reconstitution targeted delivery therapeutic target uptake

项目摘要

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.

描述（由申请人提供）：使用锌指核酸酶和 TALEN 进行基因组编辑已成为一种有前途的技术，用于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，因此正在进行研究破坏 T 细胞和 CD34+ 造血干细胞中的基因，以保护细胞或其后代免受病毒感染。到目前为止，病毒和质粒载体已用于 ZFN/TALEN 的递送。然而，这会导致核酸酶的长期表达，这可能是有害的，因为越来越多的研究已经记录了许多工程 ZFN 的剂量依赖性毒性，大概是由于在脱靶时产生了意外的双链断裂网站。从 mRNA 瞬时表达 ZFN/TALEN 可能提供一种可行的替代方案，因为短期暴露于核酸酶足以引起靶基因的永久修饰。在此提案中，mRNA 的 ZFN 或 TALEN 表达将用作破坏 CD34+ 造血干细胞 (HSC) 中 CCR5 的策略。为了克服 ZFN/TALEN mRNA 递送至 CD34+ HSC 的障碍（这些细胞是通过常规方法难以转染的细胞），我们将开发脂质纳米颗粒，该纳米颗粒展示先前描述的 DNA 适体 sgc8，该适体与细胞上表达的蛋白酪氨酸激酶 7 结合。人源化 BLT 小鼠将用 ZFN/TALEN 修饰的 CD34+ HSPC 进行重组，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颖且安全的方法来利用 ZFN/TALEN 进行 HIV 基因治疗，甚至提供一种将分子直接递送到体内动员干细胞的潜在方法。公共健康相关性：通过定制锌指核酸酶 (ZFN) 进行基因破坏是一项新兴技术，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。该研究的主要目标是利用 ZFN 破坏 CD34+ 造血干细胞中的 HIV 共受体 CCR5，作为基因疗法来产生 HIV 抗性后代。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。