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

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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.