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年的培训计划，旨在发展基因治疗的学术生涯。主要研究者已单独完成博士学位。和医学博士程序.她是在儿科和医学委员会认证，以及在儿科/血液肿瘤学委员会合格。K 01赠款将有助于扩大申请人的技能，在基因治疗中使用多种资源在鲍威尔基因治疗中心在佛罗里达大学。 该计划将促进基因治疗的命令，因为它适用于造血干细胞。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，也担任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，由成功的科学家和临床医生组成，隶属于UF，他们将提供珠蛋白基因调控，骨髓环境和临床模型开发方面的指导和培训。K 01的研究部分将专注于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）酪氨酸至苯丙氨酸的系统突变和（2）DNA改组，其涉及不同AAV血清型的衣壳氨基酸的随机化。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测试新AAV衣壳的转导效率，并选择最佳载体来设计抗镰状2-珠蛋白基因治疗构建体。有两个具体目标：1。创建具有工程化衣壳的AAV载体，其促进人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-球蛋白的优化腺相关病毒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化AAV基因治疗载体。 在UF鲍威尔基因治疗部门提供了一个理想的设置，这个K 01基因治疗培训，通过将来自多个资源的专业知识纳入量身定制的计划。在UF的环境将最大限度地提高申请人的潜力，建立一个职业生涯作为一个独立的翻译调查。

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.