Molecular evolution of AAV vectors for anti-HIV gene therapy

用于抗 HIV 基因治疗的 AAV 载体的分子进化

基本信息

批准号：
8210652
负责人：
Katherine Julie Excoffon
金额：
$ 19.33万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8210652
关键词：
Address Adenoviruses Autoimmunity Binding Biological Markers Biology Capsid Cell surface Cells Cellular biology Clinical Trials Complex Data Dependovirus Detection Development Directed Molecular Evolution Disease Engineering Epitopes Evolution Future Gene Transduction Agent Gene Transfer Genes Genetic Goals Growth HIV HIV Infections HIV therapy Highly Active Antiretroviral Therapy Human Hybrids Immune Infection Knowledge Lead Libraries Literature Lymphocyte Malignant Neoplasms Mediating Membrane Molecular Molecular Evolution Mutagenesis Pathogenicity Patients Pharmaceutical Preparations Production Recombinant Proteins Research Resistance Safety Serotyping Societies T-Lymphocyte Techniques Testing Therapeutic Therapeutic Agents Therapeutic Intervention Toxic effect Tropism Variant Viral Viral Load result Virus Work Zidovudine adeno-associated viral vector base cellular transduction design directed evolution gene therapy genetic element improved inhibitor/antagonist innovation insight novel novel virus prevent small hairpin RNA small molecule success therapeutic gene therapeutic target tissue tropism transduction efficiency vector virus tropism

项目摘要

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.

描述（由申请人提供）：该项目的远距离目标是使用一种新型的腺相关病毒（AAV）载体编码治疗基因作为针对HIV的治疗方法。艾滋病毒感染仍然是全球人类社会的重大疫病。由于试图控制艾滋病毒的传播及其对感染的持久性几乎没有成功，因此迫切需要创新的方法。基于基因的疗法为预防和治愈HIV感染提供了希望，这取决于纳入的遗传元素。最近的临床试验表明，当疾病靶标与AAV神经型组织的tropism匹配时，AAV非常安全有效。然而，迄今为止，尚无具有足够效率的AAV来描述用于原发性HIV靶标T细胞的治疗改变。使用分子技术的定向进化方法具有创建设计器AAV矢量的潜力。基于我们自己和他人的工作，我们假设可以使用定向的进化来创建能够有效感染T细胞的新型AAV衣壳，尤其是已经被HIV感染的T细胞。此外，我们假设可以设计出进化的AAV来对HIV产生治疗基因。该假设将如下检验：目标1。确定AAV CAPSID基因的定向演化是否可以在未感染和HIV感染的HIV感染淋巴细胞中产生具有增强基因转移效率的病毒。我们将将基于PCR的诱变与遗传改组相结合以获得杂化AAV帽基因，并将选择所得的新型病毒库以转导H9 T细胞或慢性HIV感染的HIV感染的H9 T细胞的能力。在HIV-H9细胞上的选择可能会产生AAV衣壳，这些囊膜可能会特别感染感染HIV的细胞，或者感染和未感染的细胞。为了隔离针对HIV感染的细胞的衣壳，将使用父母未感染的H9 T细胞进行阴性选择。然后，将研究新型的衣壳变体，以提高亲本和HIV-H9 T细胞的转导效率。目的2。研究新型AAV病毒是否可以介导基因转移，以保护天真的淋巴细胞和/或改变急性或长期感染淋巴细胞的生产性HIV感染。成功的AAV将评估为HIV指导的SHRNA基因转移方法，具有或没有小分子HIV抑制剂。能够选择性感染HIV感染的细胞的新型AAV衣壳的发现不仅为新的基因治疗矢量提供了潜力，还为选择和未来鉴定新细胞表面表位或生物标志物的选择和未来鉴定，可用于HIV检测和/或治疗干预。拟议的研究还提供了一个机会来研究病毒共感染的复杂后果，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：最近的临床试验证明了与腺相关病毒（AAV）向量介导基因治疗的安全性，能力和功效。我们建议使用AAV的定向分子进化来量身定制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。