In Vivo Hematopoietic Stem Cell Gene Therapy of Beta-Thalassemia

 描述(由申请人提供) 造血干细胞是感染性疾病、遗传性疾病和癌症基因治疗的重要靶点。与目前使用的方法相比，体内HSCs基因组编辑具有重大优势，这些方法包括从患者身上收集HSCs，它们的体外培养/转导，以及重新移植到骨髓调节患者体内。我们开发了一种新的体内HSC转导方法，该方法基于GCSF/AMD3100介导的HSC从骨髓到外周血流的动员和静脉注射HSC靶向的辅助性腺病毒载体(HD-Ad5/35)。这项建议的中心目标是增加体内稳定转导HD-Ad5/35的HSCs的百分比，同时避免潜在的遗传毒性。我们计划通过i)优化HSC动员和体内HSC转导，ii)通过刺激同源重组来优化转基因整合，以及iii)将其整合到HD-Ad5/35载体系统中，以允许体内选择或扩增稳定转导的细胞。在这些研究的基础上，我们将构建和测试HD-Ad5/35载体，用于体内基因治疗b-地中海贫血。

 DESCRIPTION (provided by applicant) Hematopoietic stem cells (HSCs) are an important target for the gene therapy of infectious diseases, genetic disorders, and cancer. In vivo genome editing of HSCs has major advantages over currently used approaches that involve the collection of HSCs from patients, their in vitro culture/transduction, and retransplantation into myelo-conditioned patients. We have developed a new in vivo approach for HSC transduction, which is based on the GCSF/AMD3100-mediated mobilization of HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an HSC-targeting, helper-dependent adenovirus vector (HD-Ad5/35). The central goal of this proposal is to increase the percentage of stably in vivo HD-Ad5/35 - transduced HSCs while avoiding potential genotoxicity. We plan to achieve this through i) optimization of HSC mobilization and in vivo HSC transduction, ii) optimization of transgene integration through stimulation of homologous recombination, and iii) incorporation into HD- Ad5/35 vectors of systems that allow for the in vivo selection or expansion of stably transduced cells. Based on these studies, we will generate and test HD-Ad5/35 vectors for in vivo gene therapy of b-thalassemia.