Next Generation of Recombinant AAV Serotype For Gene Therapy

 描述：这个多PI建议的主要目的是：继续确定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中丰富的具有高肝脏趋向性的新的AAV2变体，以及基于AAV3的载体，这些载体在人类肝细胞和非人类灵长类肝脏中显示出高效；以及评估血友病B的基因治疗的有效性和宿主免疫反应；将确定在小动物和大动物模型上进行高效临床试验的候选载体，特别强调 体内基因转移到人/灵长类肝细胞和免疫生物学。目前的应用包括跨学科的方法，应该允许我们开发优越的下一代载体，用于肝脏导向的人类基因治疗，大量借鉴佛罗里达大学在AAV载体生物学、AAV基因转移的免疫学和动物模型研究方面的专业知识。我们将检验以下假设：a.衣壳序列的微小变化可以显著改善肝脏基因转移，部分是通过进入后机制，使我们能够设计更好的基于AAV2和AAV3的载体。B.分子进化和衣壳工程的结合将导致人/灵长类肝细胞具有高转导效率的载体，并减少MHC I抗原递呈。C.衣壳修饰的AAV载体将在低载体剂量下提供治疗性的F.IX表达，并降低免疫原性。具体目标1：进一步优化和比较基于AAV2和AAV3的新型载体的体内性能，并确定导致强大的肝脏基因转移的机制。具体目标2：鉴定新的衣壳变异体，用于体内高效地将基因转移到人肝细胞中，并将其应用于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类灵长类动物中测试改进的载体，并确定它们的免疫原性。这些研究很有可能为人类的肝脏基因转移产生更好的载体。

 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.