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编码治疗基因的新型腺相关病毒（AAV）载体作为抗HIV的治疗。艾滋病毒感染仍然是全世界人类社会的一个重大祸害。由于控制艾滋病毒传播及其在感染后的持续存在的努力几乎没有取得成功，因此迫切需要采取创新办法。基于基因的疗法提供了预防和治愈艾滋病毒感染的希望，可能同时进行，这取决于所包含的遗传因素。最近的临床试验表明，当疾病靶标与AAV血清型组织嗜性匹配时，AAV是非常安全和有效的。然而，迄今为止，还没有描述具有足够效率的AAV用于治疗性改变主要HIV靶T细胞。使用分子技术的定向进化方法具有创建设计者AAV载体的潜力。基于我们自己和其他人的工作，我们假设定向进化可以用于创建能够有效感染T细胞，特别是已经被HIV感染的T细胞的新型AAV衣壳。此外，我们假设进化的AAV可以被工程化以提供针对HIV的治疗基因。这个假设将被测试如下：目标1。确定AAV衣壳基因的定向进化是否可以在未感染和HIV感染的淋巴细胞中产生具有增强的基因转移效率的病毒。我们将结合联合收割机基于PCR的诱变与遗传改组以获得杂合AAV cap基因，并且将选择所得的新病毒文库以鉴定H9 T细胞或慢性HIV感染的H9 T细胞的能力。在HIV-H9细胞上的选择可以产生可以特异性感染HIV感染的细胞或感染和未感染的细胞的AAV衣壳。为了分离对HIV感染的细胞具有特异性的衣壳，将使用亲本未感染的H9 T细胞进行阴性选择。然后将研究新型衣壳变体在亲本和HIV-H9 T细胞中的转导效率。目标2.研究新型AAV病毒是否可以介导基因转移，以保护幼稚淋巴细胞和/或改变急性或慢性感染淋巴细胞的HIV感染。成功的AAV将在有和没有小分子HIV抑制剂的情况下用于HIV定向的shRNA基因转移方法进行评估。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，而且还为选择和未来鉴定可用于HIV检测和/或治疗干预的新细胞表面表位或生物标志物提供了潜力。拟议的研究还提供了一个机会，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。 公共卫生相关性：最近的临床试验已经证明了腺相关病毒（AAV）载体介导基因治疗的安全性、能力和功效。我们建议使用AAV的定向分子进化来定制HIV和T细胞特异性AAV载体，以便用基于基因的疗法治疗HIV感染。

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. PUBLIC HEALTH RELEVANCE: Recent clinical trials have demonstrated the safety, capacity, and efficacy of adeno-associated viral (AAV) vectors to mediate gene therapy. We propose to use directed molecular evolution of AAV to tailor make HIV and T cell-specific AAV vectors in order to treat HIV infection with gene-based therapeutics.