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

描述(由申请者提供)：本计划是一项为期5年的培训计划，旨在发展基因治疗的学术生涯。首席研究员分别完成了博士和医学博士课程。她拥有儿科和内科的董事会认证，以及儿科/血液肿瘤学的董事会资格。K01基金将帮助扩大申请者的技能，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。这项计划将促进基因治疗的指挥，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。这位导师是细胞和分子治疗部门的负责人，也是密歇根大学综合临床研究中心的助理主任。申请者和导师已经建立了一个由UF附属的有成就的科学家和临床医生组成的内部咨询委员会，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的研究部分将专注于AAV衣壳工程，以促进在造血干细胞中表达治疗水平的β-珠蛋白。新的AAV衣壳将通过两种方法产生：(1)系统地将酪氨酸突变为苯丙氨酸；(2)DNA改组，涉及不同AAV血清型衣壳氨基酸的随机化。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病2-珠蛋白基因治疗构建体。有两个特定的目标：1.创建一种具有工程衣壳的AAV载体，该载体可促进人和小鼠造血干细胞中的高水平基因表达。2.确定优化的表达抗镰状2-珠蛋白的AAV载体能否纠正转基因小鼠的镰状细胞病。如果成功，这将是首个治疗性抗镰状AAV基因治疗载体。密歇根大学的鲍威尔基因治疗部通过将来自多个资源的专业知识整合到一个定制的计划中，为K01基因治疗培训提供了理想的环境。UF的环境将最大限度地发挥申请人作为独立翻译调查员建立职业生涯的潜力。 公共卫生相关性：这项建议的主要目的是确定是否可以通过改造AAV衣壳来促进造血干细胞中的治疗性基因输送。重组AAV载体目前正用于多种人类疾病的I/II期临床试验。我们提出的研究有望产生新的有用信息，进一步开发这些载体用于治疗血液疾病。(摘要结束)

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)