Development of AAV vectors for the phenotypic correction of sickle cell disease

描述（由申请人提供）：本提案是一个为期5年的基因治疗学术生涯发展培训计划。首席研究员分别完成了博士和医学博士学位。她是儿科和医学的董事会认证，以及儿科/血液肿瘤学的董事会资格。K01基金将利用佛罗里达大学鲍威尔基因治疗中心的多种资源，帮助扩大申请人在基因治疗方面的技能。该项目将促进基因治疗在造血干细胞上的应用。申请人的导师是基因治疗领域公认的领导者。导师是细胞和分子治疗部门的负责人，同时也是UF一般临床研究中心的助理主任。申请人和导师已经建立了一个内部咨询委员会，由佛罗里达大学附属的有成就的科学家和临床医生组成，他们将在珠蛋白基因调控、骨髓环境和临床模型的开发方面提供指导和培训。K01的研究部分将集中在AAV衣壳工程上，以促进造血干细胞中治疗水平的β -球蛋白的表达。新的AAV衣壳将通过两种方法产生：(1)酪氨酸系统突变为苯丙氨酸；(2)DNA洗牌，将不同AAV血清型的衣壳氨基酸随机化。利用表达增强型绿色荧光蛋白（EGFP）和荧光素酶的报告基因检测新型AAV衣壳的转导效率，并选择最佳载体设计抗镰状细胞2-球蛋白基因治疗构建体。有两个具体目标：1。构建具有工程衣壳的AAV载体，促进人类和小鼠造血干细胞中基因的高水平表达。2. 目的探讨表达抗镰状细胞2-珠蛋白的优化AAV载体是否能纠正转基因小鼠的镰状细胞病。如果成功，这将是首个抗镰状细胞AAV基因治疗载体。佛罗里达大学的鲍威尔基因治疗部门通过将多种资源的专业知识整合到量身定制的项目中，为基因治疗方面的K01培训提供了理想的环境。佛罗里达大学的环境将最大限度地发挥申请人的潜力，使其成为一名独立的翻译研究者。

DESCRIPTION (provided by applicant): This proposal is a 5 year training program for the development of an academic career in gene therapy. The principal investigator has separately completed Ph.D. and M.D. programs. She is board certified in Pediatrics and Medicine, as well as board eligible in Pediatric/Hematology Oncology. The K01 grant will help to expand the applicant's skills, in gene therapy using multiple resources at the Powell Gene Therapy Center at the University of Florida. This program will promote the command of gene therapy as it applies to hematopoietic stem cells. The applicant's mentor is a recognized leader in the field of gene therapy. The mentor is Chief of Division of Cellular and Molecular Therapy, and also serves as Assistant Director of the UF General Clinical Research Center. The applicant and mentor have established an internal advisory committee composed of accomplished scientist and clinicians affiliated with UF, who will provide guidance and training in globin gene regulation, bone marrow environment, and development of clinical models. The research component of the K01 will focus on AAV capsid engineering to facilitate expression of therapeutic levels of beta-globin in hematopoietic stem cells. Novel AAV capsids will be generated by two methods: (1) systematic mutation of tyrosine to phenylalanine and (2) DNA shuffling, which involves randomization of capsid amino acids of different AAV serotypes. The transduction efficiency of the novel AAV capsids will be tested using reporter genes expressing enhanced green fluorescence protein (EGFP) and luciferase, and the optimum vector selected to design an antisickling 2-globin gene therapy construct. There are two specific aims: 1. To create an AAV vector with an engineered capsid that facilitates high level gene expression in both human and mouse hematopoietic stem cells. 2. To determine whether an optimized AAV vector expressing anti-sickling 2-globin can correct sickle cell disease in transgenic mice. If successful this will be the first therapeutic anti-sickling AAV gene therapy vector. The Powell Gene Therapy Department at the UF provides an ideal setting for this K01 training in gene therapy, by incorporating expertise from multiple resources into a tailored program. The environment at the UF will maximize the applicant's potential to establish a career as an independent translational investigator. PUBLIC HEALTH RELEVANCE: The main aim of this proposal is to determine whether AAV capsid can be engineered to facilitate therapeutic gene delivery in hematopoietic stem cells. Recombinant AAV vectors are currently being used in a number of Phase I/II clinical trials for a wide variety of human diseases. Our proposed studies are expected to yield new and useful information that will further development of these vectors for the treatment of blood diseases. (End of Abstract)