Novel Viral Vector Delivery for Efficient ShRNA Expression

描述（由申请人提供）：有效地将shRNA表达盒掺入病毒载体基因递送系统的能力显著扩展了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递送的竞技场中的两种主要载体。基于AAV的载体在体内基因递送方面非常有效，特别是在非分裂细胞如肝、心脏、肌肉和脑中具有长期基因表达（长达6年）。在猴子中，现在在人类中为3.7岁）。这些非整合载体具有5 kb的包装容量，并且当靶向分裂细胞如干细胞、癌细胞和体内快速生长的细胞时，不是选择的载体。另一方面，整合的基于HIV-1的载体在各种干细胞（包括早期胚胎和造血干细胞）中有效地维持和维持转基因表达。较新的HIV-1载体系统的弱点是载体滴度低、体内基因递送的局限性和生物安全问题。总的来说，这两种强大的病毒基因递送系统相互补充，并为研究和临床应用提供了覆盖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）了解......，(2)评估和（3）确定被靶组织摄取的最佳递送方法。“本着R21的精神，我们建议优化两种成熟的病毒载体递送系统（Lenti和AAV）用于shRNA递送，并将这些试剂提供给研究团体用于体外和体内shRNA递送。该R21的主要目的是为研究界产生新的AAV和慢病毒载体，以利用shRNA盒在体外和体内的有效递送。具体而言，我们建议开发通过定向进化获得的细胞类型/受体特异性AAV突变体，使其成为用于体内基因递送的scAAV载体。将分离并表征选择性（a）穿过包括气道、肝细胞或骨骼肌细胞的原代细胞系;（B）穿过屏障上皮/内皮细胞;或（c）穿过体内靶组织的新型AAV突变体用于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.