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

描述（由申请人提供）：该提案是一个为期 5 年的培训计划，旨在发展基因治疗领域的学术生涯。主要研究者已单独完成博士学位。和医学博士课程。她获得了儿科和医学委员会的认证，以及儿科/血液肿瘤学委员会的资格。 K01 赠款将有助于扩大申请人利用佛罗里达大学鲍威尔基因治疗中心的多种资源进行基因治疗的技能。 该计划将促进基因治疗应用于造血干细胞的掌握。申请人的导师是基因治疗领域公认的领导者。导师为细胞与分子治疗部主任，兼任佛罗里达大学普通临床研究中心助理主任。申请人和导师建立了一个由佛罗里达大学附属的有成就的科学家和临床医生组成的内部顾问委员会，他们将在珠蛋白基因调控、骨髓环境和临床模型开发方面提供指导和培训。 K01 的研究部分将重点关注 AAV 衣壳工程，以促进造血干细胞中治疗水平的 β-珠蛋白的表达。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 改组，其中涉及不同 AAV 血清型衣壳氨基酸的随机化。新型 AAV 衣壳的转导效率将使用表达增强型绿色荧光蛋白 (EGFP) 和荧光素酶的报告基因进行测试，并选择最佳载体来设计抗镰状化 2-珠蛋白基因治疗构建体。有两个具体目标： 1. 创建带有工程衣壳的 AAV 载体，促进人类和小鼠造血干细胞中的高水平基因表达。 2. 确定表达抗镰状细胞2-珠蛋白的优化AAV载体是否可以纠正转基因小鼠的镰状细胞病。如果成功，这将是第一个治疗性抗镰状 AAV 基因治疗载体。 佛罗里达大学的鲍威尔基因治疗系将多种资源的专业知识融入定制计划，为基因治疗 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作为独立转化研究者的职业潜力。

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.