Novel Viral Vector Delivery Efficient ShRNA Expression

描述(由申请人提供)：将shRNA表达盒有效地整合到病毒载体基因传递系统中的能力显著扩展了shRNA技术的研究和临床应用的范围。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方面非常高效，特别是在肝、心脏、肌肉和脑等长期基因表达的未分裂细胞中(长达6年。在猴子身上，现在是在人类3.7岁)。这些非整合载体的包装容量为5kb，在针对干细胞、癌细胞和体内快速生长的细胞等分裂细胞时并不是首选的载体。另一方面，基于HIV-1的整合载体可以有效地转导和维持转基因在包括早期胚胎和造血干细胞在内的各种干细胞中的表达。较新的HIV-1载体系统的弱点是低载体滴度、体内基因传递的限制以及生物安全方面的担忧。综上所述，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。作为对RFA-HL-05-019的回应，“本RFA的目的是促进研究(1)了解……，(2)评估……和(3)确定目标组织摄取的最佳给药方法。”本着R21的精神，我们建议优化两种成熟的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的主要目标是为研究界创造新的AAV和慢病毒载体，以利用体外和体内有效的shRNA盒递送。具体地说，我们建议开发细胞类型/受体特异性AAV突变体，通过定向进化获得单链AAV载体，用于体内基因传递。选择性地(A)转导原代细胞系包括呼吸道、肝细胞或骨骼肌细胞；(B)穿越屏障上皮/内皮细胞；或(C)转导体内靶组织的新型AAV突变体将被分离并鉴定为shRNA递送。此外，我们还将开发一种通用的第三代稳定的HIV-1载体产生细胞系，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，我们将使用翻转重组酶系统作为一种手段，将shRNA表达框整合到第三代高滴度生产细胞系中整合的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.