Molecular evolution of AAV vectors for anti-HIV gene therapy

描述（由申请人提供）：该项目的长期目标是使用一种新的腺相关病毒（AAV）载体编码治疗基因来治疗HIV。艾滋病毒感染仍然是全世界人类社会的一大祸害。由于控制艾滋病毒传播及其在感染后持续存在的努力收效甚微，因此迫切需要创新的办法。基于基因的治疗方法为预防和治愈艾滋病毒感染提供了希望，这可能是同时发生的，这取决于所结合的遗传因素。最近的临床试验表明，当疾病靶点与AAV血清型组织亲和性相匹配时，AAV是非常安全有效的。然而，到目前为止，还没有一种具有足够效率的AAV被描述为对HIV主要靶点T细胞进行治疗性改变。使用分子技术的定向进化方法有可能创建设计AAV载体。基于我们自己和其他人的工作，我们假设定向进化可以用来创造新的AAV衣壳，能够有效地感染T细胞，特别是已经被HIV感染的T细胞。此外，我们假设进化的aav可以被改造成一种抗HIV的治疗基因。这一假设将被检验如下：目的1。确定AAV衣壳基因的定向进化是否能在未感染和hiv感染淋巴细胞中产生具有更高基因转移效率的病毒。我们将结合基于PCR的诱变和基因重组来获得AAV杂交帽基因，并选择新病毒文库来转导H9 T细胞或慢性hiv感染的H9 T细胞。对HIV-H9细胞的选择可能产生AAV衣壳，这种衣壳可以特异性地感染hiv感染的细胞，或者感染和未感染的细胞。为了分离出针对hiv感染细胞的特异性衣壳，将使用亲代未感染的H9 T细胞进行负选择。新的衣壳变异将在亲本和HIV-H9 T细胞中进行转导效率的研究。目标2。目的：探讨新型AAV病毒是否能介导基因转移以保护初始淋巴细胞和/或改变急性或慢性感染淋巴细胞的生产性HIV感染。成功的aav将用于评估HIV定向shRNA基因转移方法，无论是否使用小分子HIV抑制剂。能够选择性感染HIV感染细胞的新型AAV衣壳的发现，不仅为新的基因治疗载体提供了潜力，而且为选择和未来鉴定新的细胞表面表位或生物标志物，用于HIV检测和/或治疗干预提供了可能。拟议的研究还提供了一个机会来调查病毒合并感染的复杂后果，并可能导致对病毒相互作用，致病性和治疗策略的新见解。

DESCRIPTION (provided by applicant): The long range goal of this project is to use a novel adeno-associated virus (AAV) vector encoding therapeutic genes as a treatment against HIV. HIV infection remains a major blight on human society worldwide. Because attempts to control the spread of HIV and its persistence upon infection have yielded few successes, innovative approaches are urgently needed. Gene-based therapeutics offer hope to both prevent and cure HIV infection, potentially simultaneously, depending on the genetic elements incorporated. Recent clinical trials have shown that AAV is extremely safe and efficacious when the disease target is matched with AAV-serotype tissue tropism. To date, however, no AAV with sufficient efficiency has been described for therapeutic alteration of the primary HIV target, T cells. Directed evolutionary approaches using molecular techniques have the potential to create designer AAV vectors. Based on the work of ourselves and others, we hypothesize that directed evolution can be used to create novel AAV capsids capable of efficiently infecting T cells, and in particular, T cells already infected by HIV. Furthermore, we hypothesize that the evolved AAVs can then be engineered to deliver a therapeutic gene against HIV. This hypothesis will be tested as follows: Aim 1. To determine whether directed evolution of the AAV capsid gene can generate viruses with enhanced gene transfer efficiency in uninfected and HIV-infected lymphocytes. We will combine PCR- based mutagenesis with genetic shuffling to obtain hybrid AAV cap genes, and the resulting library of novel viruses will be selected for the ability to transduce H9 T cells or chronically HIV-infected H9 T cells. Selection on HIV-H9 cells may yield AAV capsids that can specifically infect HIV-infected cells, or both infected and uninfected cells. To isolate capsids that are specific for HIV-infected cells, negative selection will be performed using parental uninfected H9 T cells. Novel capsid variants will then be investigated for transduction efficiency in parental- and HIV-H9 T cells. Aim 2. To investigate whether novel AAV viruses can mediate gene transfer in order to protect naive lymphocytes and/or alter productive HIV infection of acutely or chronically infected lymphocytes. Successful AAVs will be evaluated for HIV-directed shRNA gene transfer approaches, with and without small molecule HIV inhibitors. The discovery of novel AAV capsids able to selectively infect HIV-infected cells offers the potential not only for new gene therapy vectors, but also for the selection and future identification of new cell surface epitopes or biomarkers useful for HIV detection and/or therapeutic intervention. The proposed research also provides an opportunity to investigate the complex consequences of viral co-infection and may lead to novel insights into viral interactions, pathogenicity, and therapeutic strategies.