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表达盒有效地纳入病毒载体基因输送系统中的能力大大扩展了SHRNA技术的研究和临床应用。基于HIV-1和ADENO相关病毒（AAV）载体是体内和体外基因递送领域的两个领先载体。基于AAV的载体在体内的基因递送方面非常有效，特别是在肝脏，心脏，肌肉和大脑等非分裂细胞中具有长期基因表达（在猴子中长达6年，现在在人类中为3.7年）。这些非整合载体的包装能力为5 kb，在靶向分裂细胞（例如干细胞，癌细胞和体内快速生长的细胞）时，并不是首选的载体。另一方面，基于HIV-1的载体有效地转导并维持各种干细胞中的转基因表达，包括早期胚胎和造血干细胞。低矢量滴度，体内基因输送的局限性以及生物安全问题是新的HIV-1矢量系统的弱点。综上所述，这两个强大的病毒基因输送系统相互补充，并提供了用于研究和临床应用的大多数目标器官的能力。为了响应RFA-HL-05-019，“该RFA的目的是刺激（1）理解...，（2）评估...和（3）确定目标组织摄取的最佳输送方法。”本着R21的精神，我们建议优化两个良好的病毒载体输送系统（Lenti＆AAV），以用于SHRNA的递送，并将这些试剂提供给研究社区的体外和体内SHRNA递送。该R21的主要目的是为研究界生成新颖的AAV和慢病毒载体，以利用体外和体内的shRNA盒有效递送。具体而言，我们建议开发通过将进化为SCAAV向量获得的细胞类型/受体特异性AAV突变体，以在体内进行基因递送。新颖的AAV突变体，有选择地（a）传递原始细胞系，包括气道，肝细胞或骨骼肌细胞； （b）遍历屏障上皮/内皮细胞；或（c）将分离出靶组织的靶组织，并以shRNA递送为特征。此外，我们还将开发一种通用的第三代稳定的HIV-1载体生产细胞系，这将促进携带shRNA表达盒的大量高滴度自我激活（SIN）HIV-1载体的产生。为此，我们将利用翻转重组酶系统作为将SHRNA表达盒纳入集成在第三代高滴度生产细胞系中的HIV-1载体中的手段。这种方法有助于扩大安全的HIV-1载体产生，增加了在临床试验中使用表达SHRNA的HIV-1载体的可能性。长期目标是改善现有的AAV和HIV载体，并为shRNA表达盒提供新的递送试剂。