Next Generation of Recombinant AAV Serotype Vectors for Gene Therapy

用于基因治疗的下一代重组 AAV 血清型载体

• 批准号: 8450212
• 负责人: Roland W. Herzog
• 金额: $ 59.31万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450212
• 关键词: Adenovirus Vector; Adenoviruses; Animal Model; Applications Grants; Attention; Baculoviruses; Biology; Canis familiaris; Capsid; Capsid Proteins; Cell Communication; Cell Nucleus; Cells; DNA; Dependovirus; Development; Disease; Dose; Evaluation; Exhibits; Factor IX; Gene Transduction Agent; Genes; Helper Viruses; Hemophilia A; Hemophilia B; Hemorrhage; Hepatocyte; Herpesviridae; Human; Immune response; Immunology; In Vitro; Knockout Mice; Knowledge; Lead; Life Cycle Stages; Light; Liver; Liver diseases; Mediating; Modeling; Molecular; Mus; Muscle; Mutation; Nature; Parkinson Disease; Parvovirus; Patients; Phase; Production; Property; Public Health; Recombinant adeno-associated virus (rAAV); Retroviral Vector; Retroviridae; Safety; Serotyping; Single-Stranded DNA; Site; System; Techniques; Testing; Therapeutic; Tissues; Transgenes; Transgenic Mice; Trypsin; Tyrosine; Vaccinia virus; Viral Genome; Virus; abstracting; adeno-associated viral vector; base; cell type; clinical efficacy; comparative; gene therapy; gene therapy clinical trial; in vivo; insight; interest; latent infection; multicatalytic endopeptidase complex; mutant; next generation; novel; public health relevance; stable cell line; therapeutic gene; trafficking; transduction efficiency; transgene expression; tumor; vector

DESCRIPTION (provided by applicant): Project Summary/Abstract The main aims of this multiple-PI proposal are to evaluate the safety and eficacy of, and the host immune response to, the next generation of recombinant adeno-associated virus (AAV) vectors that we have developed, in small and large animal models of human liver diseases in general, and hemophilia in particular. AAV vectors have gained attention as an alternative to the more commonly used retrovirus and adenovirus vectors, and are in use in Phase I/II clinical trials for gene therapy of a number of diseases. However, relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus, and is targeted for degradation by the host cell proteasome machinery. Our recent studies have yielded insights into key steps in intracellular trafficking of AAV, and led to the development of novel AAV vectors that are capable of high-efficiency transduction at lower doses. We will test the following hypotheses: a. Combination of specific tyrosine mutations in AAV2 capsids will further reduce the vector dose needed for high-efficiency transduction, and corresponding mutations in tyrosine residues in AAV8 and AAV5 serotype vectors will lead high-efficiency transduction of murine and canine hepatocytes. b. Novel Baculovirus system-produced rAAV vectors, characterized by higher VP1 capsid protein stoichiometric content, will exhibit superior transduction properties in target tissues. c. Tyrosine-mutant AAV vectors will elicit a reduced host cell immune response, and provide therapeutic benefits at lower doses. The following three Specific Aims will be pursued: 1. Development of AAV2 vectors containing multiple tyrosine-mutations, elucidation of the underlying mechanism of transduction by the most efficient vector in vitro and in vivo, and comparative analysis with AAV8 and AAV5 vectors. 2. Development of the next generation of Sf9-based stable cell lines for the production of highly infectious rAAV of alternative serotypes. 3. Treatment of murine and canine hemophilia B with optimal tyrosine-mutant AAV2, AAV8, and AAV5 serotype vectors and evaluation of immune responses to vector and coagulation factor IX transgene product. The knowledge gained from these studies will not only shed light on the AAV-host cell interactions, but will also be applicable in further improvements in recombinant AAV vectors for their potential use in gene therapy of human liver diseases in general, and hemophilia in particular.

描述（由申请人提供）： 项目摘要/摘要 该多 PI 提案的主要目的是评估我们开发的小型和小型化的下一代重组腺相关病毒 (AAV) 载体的安全性和有效性以及宿主免疫反应。一般人类肝脏疾病，特别是血友病的大型动物模型。 AAV 载体作为更常用的逆转录病毒和腺病毒载体的替代品而受到关注，并用于多种疾病基因治疗的 I/II 期临床试验。然而，需要相对较大的载体剂量才能实现治疗效果。大剂量的载体也会引发免疫反应，因为很大一部分载体无法有效地运输到细胞核，并被宿主细胞蛋白酶体机制靶向降解。我们最近的研究深入了解了 AAV 细胞内运输的关键步骤，并导致了能够以较低剂量进行高效转导的新型 AAV 载体的开发。我们将测试以下假设： AAV2衣壳中特定酪氨酸突变的组合将进一步减少高效转导所需的载体剂量，而AAV8和AAV5血清型载体中酪氨酸残基的相应突变将导致鼠和犬肝细胞的高效转导。 b.杆状病毒系统生产的新型 rAAV 载体具有较高的 VP1 衣壳蛋白化学计量含量，将在靶组织中表现出优异的转导特性。 c.酪氨酸突变 AAV 载体将引起宿主细胞免疫反应减弱，并以较低剂量提供治疗效果。 将追求以下三个具体目标： 1. 开发含有多个酪氨酸突变的 AAV2 载体，阐明体外和体内最有效载体转导的基本机制，并与 AAV8 和 AAV5 载体进行比较分析。 2. 开发下一代基于 Sf9 的稳定细胞系，用于生产替代血清型的高传染性 rAAV。 3.用最佳酪氨酸突变体AAV2、AAV8和AAV5血清型载体治疗鼠和犬血友病B，并评估对载体和凝血因子IX转基因产物的免疫反应。 从这些研究中获得的知识不仅将揭示 AAV 与宿主细胞的相互作用，而且还将适用于重组 AAV 载体的进一步改进，使其在人类肝脏疾病（特别是血友病）的基因治疗中具有潜在用途。