Next Generation of Recombinant AAV Serotype Vectors for Gene Therapy

描述（由申请人提供）： 项目摘要/摘要本多PI提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）载体在人类肝脏疾病（尤其是血友病）的小型和大型动物模型中的安全性和有效性以及宿主免疫应答。AAV载体作为更常用的逆转录病毒和腺病毒载体的替代物已经获得关注，并且在I/II期临床试验中用于许多疾病的基因治疗。然而，需要相对大的载体剂量来实现治疗益处。大的载体剂量也会引发免疫应答，因为大部分载体不能有效地运输到细胞核，并且被宿主细胞蛋白酶体机制靶向降解。我们最近的研究已经深入了解了AAV细胞内运输的关键步骤，并导致了能够在较低剂量下高效转导的新型AAV载体的开发。我们将测试以下假设：a。AAV 2衣壳中特异性酪氨酸突变的组合将进一步降低高效转导所需的载体剂量，并且AAV 8和AAV 5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.新型杆状病毒系统产生的rAAV载体，其特征在于较高的VP 1衣壳蛋白化学计量含量，将在靶组织中表现出上级转导特性。C.酪氨酸突变型AAV载体将引起降低的宿主细胞免疫应答，并在较低剂量下提供治疗益处。 将实现以下三个具体目标：1.开发含有多个酪氨酸突变的AAV 2载体，阐明体外和体内最有效载体的潜在转导机制，并与AAV 8和AAV 5载体进行比较分析。2.开发下一代基于Sf 9的稳定细胞系，用于生产替代血清型的高感染性rAAV。3.用最佳酪氨酸突变体AAV 2、AAV 8和AAV 5血清型载体治疗鼠和犬血友病B并评价对载体和凝血因子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揭示AAV-宿主细胞相互作用，而且还将适用于进一步改进重组AAV载体，以用于其在人类肝脏疾病，特别是血友病的基因治疗中的潜在用途。 公共卫生相关性： 该提案的主要目的是开发下一代载体，利用该载体可以将治疗基因安全地递送给患有称为血友病B的出血性疾病的患者。这些载体来源于一种不会引起已知疾病的病毒，因此预计更安全。因此，开发这种用于血友病的潜在治疗和治愈的载体与公共卫生相关。

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.