Novel Viral Vector Delivery for Efficient ShRNA Expression

用于高效 ShRNA 表达的新型病毒载体递送

• 批准号: 7295732
• 负责人: TAL KAFRI
• 金额: $ 17.72万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7295732
• 关键词: Applications Grants; Bioluminescence; Brain; Cell Line; Cells; Clinical Trials; Communities; Complement; Dependovirus; Development; Embryo; Endothelial Cells; Epithelial; Factor IX; Gel; Gene Delivery; Gene Expression; Gene Targeting; Generations; Genetic Recombination; HIV; HIV-1; Half-Life; Hand; Hematopoietic stem cells; Hepatocyte; Human; Image; In Vitro; Interphase Cell; Lentivirus Vector; Liver; Luciferases; Mediating; Methods; Monkeys; Mus; Muscle Fibers; Myocardium; Organ; Polymerase Chain Reaction; Process; Production; Purpose; RNA Interference; Reagent; Regulation; Reporter; Reporter Genes; Research; Satellite Viruses; Serum; Stem cells; System; Technology; Tetracycline; Tetracyclines; Tissues; Transgenes; Viral Vector; adeno-associated viral vector; base; cancer cell; cell type; clinical application; directed evolution; gene delivery system; gene therapy; improved; in vivo; mutant; novel; receptor; recombinase; response; rev Protein; scale up; small hairpin RNA; tissue tropism; transduction efficiency; transgene expression; uptake; vector; viral gene delivery

DESCRIPTION (provided by applicant): The ability to efficiently incorporate shRNA expression cassettes into viral vector gene delivery systems significantly expands the spectrum of research and clinical applications of the shRNA technology. HIV-1 and Adeno Associated Virus (AAV) based vectors are two of the leading vectors in the arena of in vivo and in vitro gene delivery. AAV based vectors are highly efficient at gene delivery in vivo, specifically in non-dividing cells such as liver, heart, muscle, and brain with long-term gene expression (up to 6 yr. in monkey and now 3.7 yr in human). These non-integrating vectors have a packaging capacity of 5 kb and have not been the vector of choice when targeting dividing cells such as stem cells, cancer cells, and rapidly growing cells in vivo. On the other hand, the integrating HIV-1 based vectors efficiently transduce and maintain transgene expression in various stem cells including early embryos, and hematopoietic stem cells. Low vector titers, limitations in in vivo gene delivery, and biosafety concerns are noted weaknesses of the newer HIV-1 vector system. Taken together, these two robust viral gene delivery systems complement each other and provide ability to transduce most target organs for both research and clinical applications. In response to the RFA- HL-05-019, "The purpose of this RFA is to stimulate research towards (1) understanding ..., (2) assessing..., and (3) determining optimal delivery methods for uptake by the target tissues." and in the spirit of a R21, we propose to optimize two well established viral vector delivery systems (Lenti & AAV) for shRNA delivery and provide these reagents to the research community for utilization in both in vitro and in vivo shRNA delivery. The primary objective of this R21 is to generate novel AAV and Lentiviral vectors for the research community to exploit efficient delivery of shRNA cassettes both in vitro and in vivo. Specifically, we propose to develop cell type/receptor-specific AAV mutants obtained through directed evolution into scAAV vectors for gene delivery in vivo. Novel AAV mutants that selectively (a) transduce primary cell lines including airway, hepatocytes, or skeletal muscle cells; (b) traverse barrier epithelial/endothelial cells; or (c) transduce target tissue in vivo will be isolated and characterized for shRNA delivery. In addition, we will develop a universal third generation stable HIV-1 vector producer cell line, which will facilitate the production of large amounts of high titer self-inactivating (SIN) HIV-1 vectors carrying shRNA expression cassettes. To this end we will employ the FLIP recombinase system as a means to incorporate shRNA expression cassettes into an HIV- 1 vector integrated in a third generation high titer producer cell line. This approach, which facilitates scaling up safe HIV-1 vector production, increases the likelihood of employing shRNA-expressing HIV-1 vectors in clinical trials. The long-term objective is to improve on existing AAV and HIV vectors and provide novel delivery reagents for shRNA expression cassettes.

描述（由申请人提供）：将shRNA表达盒有效整合到病毒载体基因递送系统中的能力显着扩展了shRNA技术的研究和临床应用范围。基于 HIV-1 和腺相关病毒 (AAV) 的载体是体内和体外基因传递领域的两种主要载体。基于 AAV 的载体在体内基因传递方面非常高效，特别是在非分裂细胞中，例如肝脏、心脏、肌肉和大脑，具有长期基因表达（在猴子中可达 6 岁，在人类中可达 3.7 岁）。这些非整合载体的包装容量为 5 kb，在靶向干细胞、癌细胞和体内快速生长细胞等分裂细胞时，并不是首选载体。另一方面，基于HIV-1的整合载体可有效转导并维持各种干细胞（包括早期胚胎和造血干细胞）中的转基因表达。载体滴度低、体内基因传递的局限性以及生物安全问题是新型 HIV-1 载体系统的弱点。总而言之，这两种强大的病毒基因传递系统相辅相成，并为研究和临床应用提供转导大多数靶器官的能力。针对 RFA-HL-05-019，“本 RFA 的目的是促进研究：(1) 理解……，(2) 评估……，以及 (3) 确定目标组织摄取的最佳递送方法。”本着 R21 的精神，我们建议优化两个完善的 shRNA 递送病毒载体递送系统（Lenti 和 AAV），并将这些试剂提供给研究界，用于体外和体内 shRNA 递送。该 R21 的主要目标是为研究界生成新型 AAV 和慢病毒载体，以在体外和体内有效地递送 shRNA 盒。具体来说，我们建议开发通过定向进化为 scAAV 载体获得的细胞类型/受体特异性 AAV 突变体，用于体内基因传递。新型 AAV 突变体选择性 (a) 转导原代细胞系，包括气道、肝细胞或骨骼肌细胞； (b) 穿过屏障上皮/内皮细胞； (c) 体内转导靶组织将被分离并表征以用于shRNA递送。此外，我们将开发通用的第三代稳定HIV-1载体生产细胞系，这将有助于生产大量携带shRNA表达盒的高滴度自失活（SIN）HIV-1载体。为此，我们将采用 FLIP 重组酶系统作为将 shRNA 表达盒整合到整合到第三代高效价生产细胞系中的 HIV-1 载体中的方法。这种方法有利于扩大安全的 HIV-1 载体生产规模，增加了在临床试验中使用表达 shRNA 的 HIV-1 载体的可能性。长期目标是改进现有的 AAV 和 HIV 载体，并为 shRNA 表达盒提供新型递送试剂。