Novel Viral Vector Delivery Efficient ShRNA Expression

描述（由申请人提供）：有效地将shRNA表达盒纳入病毒载体基因传递系统的能力大大扩展了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的载体在体内的基因传递效率很高，特别是在具有长期基因表达的非分裂细胞中，如肝脏、心脏、肌肉和大脑（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容量为5kb，并不是针对分裂细胞（如干细胞、癌细胞和体内快速生长的细胞）的载体选择。另一方面，整合的基于HIV-1的载体在包括早期胚胎和造血干细胞在内的各种干细胞中有效地转导和维持转基因表达。低载体滴度，体内基因传递的局限性，以及生物安全问题是新的HIV-1载体系统的弱点。总之，这两种强大的病毒基因传递系统相辅相成，为研究和临床应用提供了转导大多数靶器官的能力。针对RFA- HL-05-019，“本RFA的目的是促进对(1)理解的研究……(2)评估……(3)确定目标组织摄取的最佳递送方法。”本着R21的精神，我们建议优化两种完善的shRNA递送病毒载体系统（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的主要目的是为研究团体产生新的AAV和慢病毒载体，以在体外和体内有效地递送shRNA磁带。具体来说，我们建议将通过定向进化获得的细胞类型/受体特异性AAV突变体发展为体内基因递送的scAAV载体。新型AAV突变体选择性地(a)转导原代细胞系，包括气道细胞、肝细胞或骨骼肌细胞；(b)穿过屏障上皮/内皮细胞；或(c)在体内转导目标组织将被分离并表征为shRNA递送。此外，我们将开发一种通用的第三代稳定的HIV-1载体生产细胞系，这将有助于生产大量高滴度的携带shRNA表达盒的自灭活（SIN） HIV-1载体。为此，我们将采用FLIP重组酶系统作为一种手段，将shRNA表达盒整合到整合在第三代高滴度生产细胞系中的HIV- 1载体中。这种方法有助于扩大安全的HIV-1载体生产，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表达盒提供新的递送试剂。

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.