Next Generation of Recombinant AAV Serotype For Gene Therapy

 产品说明：该多PI提案的主要目的是：继续定义通过我们的下一代重组腺相关病毒（AAV）载体改善肝细胞转导的潜在机制;将这些研究扩展到从文库富集的具有高肝向性的新型AAV 2变体，以及基于AAV 3的载体，其在人肝细胞和非人灵长类动物肝脏中显示出高功效;并评价血友病B基因治疗的疗效和宿主免疫应答。将确定在小型和大型动物模型中具有高效临床试验的候选载体，特别强调 体内基因转移到人/灵长类动物肝细胞和免疫生物学。目前的应用程序包括跨学科的方法，应使我们能够开发上级下一代载体的肝脏定向人类基因治疗，在很大程度上借鉴专业知识，在佛罗里达大学的AAV载体生物学，免疫学的AAV基因转移，和动物模型研究。我们将测试以下假设：a。衣壳序列中的微小变化可以部分地通过试验后机制实质上改善肝脏基因转移，从而允许我们设计基于上级AAV 2和AAV 3的载体。B.分子进化和衣壳工程的组合将产生具有人/灵长类动物肝细胞的高转导效率和减少的MHC I抗原呈递的载体。C. Captain修饰的AAV载体将以低载体剂量和降低的免疫原性提供治疗性F.IX表达。 具体目标1：进一步优化和比较基于新型AAV 2和AAV 3的载体的体内性能，并确定负责稳健肝脏基因转移的机制。具体目标二：鉴定用于高效体内基因转移至人肝细胞的新型衣壳变体，并将其引入先进的杆状病毒生产系统。具体目标3：在血友病动物模型和非人灵长类动物中测试改进的载体，并确定其免疫原性。这些研究很有可能产生用于人类肝脏基因转移的上级载体。

 DESCRIPTION: The main aims of this multiple-PI proposal are: to continue to define the underlying mechanisms of improved hepatocyte transduction by our next generation of recombinant adeno-associated virus (AAV) vectors; to extend these studies to novel AAV2 variants with high liver tropism, enriched from a library, and to AAV3-based vectors, which show high efficacy in human hepatocytes and non-human primate liver; and to evaluate the efficacy and host immune response in gene therapy for hemophilia B. Candidate vectors for clinical trial with high efficacy in small and large animal models will be identified, with particular emphasis on in vivo gene transfer to human/primate hepatocytes and immunobiology. The current application encompassing an interdisciplinary approach should allow us to develop superior next generation vectors for liver-directed human gene therapy, drawing heavily from expertise at the University of Florida in AAV vector biology, immunology of AAV gene transfer, and animal model studies. We will test the following hypotheses: a. Minor changes in the capsid sequence can substantially improve liver gene transfer, in part through postentry mechanisms, allowing us to design superior AAV2- and AAV3-based vectors. b. A combination of molecular evolution and capsid engineering will result in vectors with high transduction efficiency of human/primate hepatocytes and reduced MHC I antigen presentation. c. Capsid-modified AAV vectors will provide therapeutic F.IX expression at low vector doses and with reduced immunogenicity. The following three Specific Aims will be pursued: Specific Aim 1: Further optimize and compare in vivo performance of novel AAV2- and AAV3-based vectors, and define the mechanisms responsible for the robust liver gene transfer. Specific Aim 2: Identify novel capsid variants for high-efficiency in vivo gene transfer to human hepatocytes and adopt these into an advanced baculovirus production system. Specific Aim 3: Test improved vectors in animal models of hemophilia and in non-human primates, and define their immunogenicity. These investigations have a high chance of yielding superior vectors for liver gene transfer in humans.