Next Generation of Recombinant AAV Serotype Vectors for Gene Therapy

描述(由申请人提供)： 项目概述/摘要这个多PI方案的主要目的是评估我们开发的下一代重组腺相关病毒(AAV)载体在人类肝病特别是血友病的大小动物模型中的安全性和有效性以及宿主对其的免疫应答。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，并被用于一些疾病的基因治疗的I/II期临床试验。然而，需要相对较大的载体剂量才能达到治疗效果。大剂量的载体也会引发免疫反应，因为很大一部分载体不能有效地运输到细胞核，并成为宿主细胞蛋白酶体机械降解的目标。我们最近的研究对AAV细胞内运输的关键步骤有了深入的了解，并导致了能够以较低剂量高效转导的新型AAV载体的开发。我们将检验以下假设：a.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血清型载体中相应的酪氨酸残基突变将导致小鼠和犬肝细胞的高效转导。B.新型杆状病毒系统产生的rAAV载体，其特征是具有更高的VP1衣壳蛋白化学计量含量，将在靶组织中显示出更好的转导特性。C.酪氨酸突变AAV载体将引起宿主细胞免疫反应减弱，并在较低剂量下提供治疗益处。1.构建含多种酪氨酸突变的AAV2载体，阐明最有效的载体在体内外的转导机制，并与AAV8和AAV5载体进行比较分析。2.以Sf9为基础的下一代稳定细胞系的建立，用于生产高感染性的替代血清型的rAAV。3.优化酪氨酸突变型AAV2、AAV8和AAV5血清型载体治疗小鼠和犬血友病B，并评价载体和凝血因子IX转基因产物的免疫应答。从这些研究中获得的知识不仅将有助于阐明AAV与宿主细胞的相互作用，而且还将适用于进一步改进重组AAV载体，使其在人类肝病尤其是血友病的基因治疗中具有潜在的应用价值。 公共卫生相关性： 项目简介这项建议的主要目的是开发下一代载体，利用这些载体可以将治疗性基因安全地传递给患有出血性疾病的患者，这种疾病称为血友病B。这些载体来自一种未引起已知疾病的病毒，因此预计将更加安全。因此，这种用于血友病潜在治疗和治愈的载体的开发与公共卫生相关。

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. PUBLIC HEALTH RELEVANCE: Project Narrative The main aim of this proposal is to develop the next generation of vectors with which a therapeutic gene can be safely delivered to patients with a bleeding disorder called hemophilia B. These vectors are derived from a virus that causes no known disease, and is therefore, expected to be safer. The development of such a vector for the potential treatment and cure of hemophilia therefore has relevance to public health.