TARGETING THE VIRUS ASSEMBLY AND ENTRY PATHWAYS FOR ANTI-HIV GENE THERAPY

描述（由申请方提供）：靶向HIV进入和组装途径有望开发抗HIV基因治疗载体，因为在生命周期的多个阶段破坏病毒可能会限制耐药突变体的出现。近年来，人类CD 34+干细胞转导和移植的研究进展为抗HIV基因治疗开辟了道路。因此，这些进展促使人们寻找新的和有效的基因治疗靶点来抑制HIV复制。虽然已经测试了针对病毒和细胞因子的si/shRNAs，但是对于可以充当HIV抑制剂的显性阴性（DN）病毒或细胞蛋白的研究数量有限。在这方面，HIV-1的Gag和包膜蛋白仍然是抗HIV基因治疗的有吸引力但尚未开发的靶标。Gag和包膜蛋白在病毒生命周期的完成中发挥重要作用，即分别通过促进HIV颗粒组装/出芽或允许感染细胞。值得注意的是，病毒组装不仅需要病毒Gag蛋白，而且需要许多宿主细胞因子如Tsg 101、阿利克斯、GGA、Arfs、POSH、AP-1、AP-3蛋白等来完成病毒体形态发生。因此，上述基因的显性阴性形式无论是单独还是组合都有可能被开发为靶向HIV-1复制的有力工具。因此，通过多种方式靶向HIV复制将具有不仅阻止病毒快速传播而且限制耐药分离株出现的优势。 公共卫生相关性：艾滋病毒的快速变异性质和与常规药物治疗相关的不良反应要求开发替代治疗干预措施。化疗的一个明显的替代方案是基因治疗。最近在CD 34+干细胞的遗传操作方面的进展，沿着能够将所需基因递送至非分裂细胞的慢病毒载体的开发，使得基于HIV的基因治疗成为现实的可能性。利用显性阴性（DN）HIV蛋白靶向HIV生命周期是一种有吸引力的基因治疗方法。在这方面，HIV-1的Gag和包膜（Env）蛋白仍然是一个有吸引力但未开发的靶标，因为Gag和Env蛋白都是病毒生命周期的必要组分，并且单独或组合使用这些基因的显性阴性形式尚未被彻底研究用于抗HIV基因治疗。在这项提案中，我们的目标是识别和设计单独和组合表达DN Gag和Env蛋白的病毒载体，并测试它们抑制WT HIV复制的潜力。我们还建议，以确定是否主机的HIV-1的组装和释放的重要因素，如Tsg 101和阿利克斯可以在细胞中的DN对应物的表达为目标，并开发为新的基因治疗靶点。这种方法不仅有助于确定艾滋病毒干预的新治疗靶点，还有助于破译已知和未知宿主因子在病毒复制周期中的作用。

DESCRIPTION (provided by applicant): Targeting the HIV entry and assembly pathways holds promise for development of anti- HIV gene therapy vectors as disrupting the virus at multiple stages in the life cycle will likely limit the emergence of resistant mutants. Moreover, recent advances in human CD34+ stem cell transduction and transplantation have paved a way for anti-HIV gene therapy in the near future. These advances have thus prompted a search for new and potent gene therapy targets for suppression of HIV replication. While si/shRNAs against viral and cellular factors have been tested there are a limited number of studies on dominant negative (DN) viral or cellular proteins that can act as HIV inhibitors. In this regard, the Gag and Envelope proteins of HIV-1 remain an attractive yet unexploited target for anti-HIV gene therapy. Both the Gag and envelope proteins play essential roles in completion of the viral life cycle namely via promoting HIV particle assembly/budding or allowing infection into cells respectively. Notably, virus assembly not only requires the viral Gag protein but also numerous host cell factors like Tsg101, Alix, GGAs, Arfs, POSH, AP-1, AP-3 proteins etc to complete virion morphogenesis. Hence, dominant negative forms of the above genes either individually or in combination have the potential to be developed as powerful tools to target HIV-1 replication. Thus, targeting HIV replication via multiple ways will have the advantage of not only halting virus spread rapidly but also restrict the emergence of resistant isolates. PUBLIC HEALTH RELEVANCE: The rapidly mutating nature of HIV and the adverse effects associated with routine drug therapy solicits the development of alternative therapeutic interventions. One of the obvious alternatives to chemotherapy is gene therapy. Recent advances in genetic manipulation of CD34+ stem cells, along with the development of lentiviral vectors capable of delivering desired genes to non-dividing cells has made HIV-based gene therapy a realistic possibility. An attractive gene therapy approach is to utilize dominant negative (DN) HIV proteins to target HIV life cycle. In this regard the Gag and Envelope (Env) proteins of HIV-1 remain an attractive yet unexploited target because both the Gag and Env proteins are essential components of the virus life cycle and the use of dominant negative forms of these genes individually or in combination have not been thoroughly investigated for anti-HIV gene therapy. In this proposal we aim to identify and design viral vectors expressing DN Gag and Env proteins individually and in combination and test their potential to inhibit WT HIV replication. We also propose to determine whether host factors important for HIV-1 assembly and release like Tsg101 and Alix can be targeted in cells by expression of their DN counterparts and developed as novel gene therapy targets. This approach will not only help identify novel therapeutic targets for HIV intervention but also help decipher the role of known and unknown host factors in the virus replication cycle.