Gene Replacement Therapy in Hematopoietic Stem Cells

描述（由申请人提供）： 该项目的总体目标是为通过造血干细胞的遗传修饰来纠正骨髓来源细胞的遗传性疾病奠定坚实的实验基础。目前的目标集中在使用重组肿瘤逆转录病毒，慢病毒和泡沫病毒载体，以实现安全和有效的功能基因转移到原始的小鼠和人类造血干细胞，同时保持其最大的造血潜能。具体目的是1）鉴定提高HSC离体有效病毒转导同时保持其最佳归巢和移植能力的策略2）鉴定有效但最小化暴露于遗传毒性剂的用于增强转导细胞移植的非消融性调节方案和体内选择方案，3）确定使用病毒介导的基因转移来校正两种遗传性血液病（X-连锁慢性肉芽肿病（X-CGD）和范可尼贫血A组和C组）中的表型的最佳策略。实验方法包括使用体外培养系统，NOD/SCID小鼠-人异种移植物，以及先前通过基因靶向方法产生的X-CGD和范可尼贫血的小鼠模型。这些目标的实现将由3个项目和4个核心单位共同承担。该提案来自一组在干细胞生物学、逆转录病毒和慢病毒介导的基因转移、分子遗传学、病毒学和骨髓移植方面具有不同但互补经验的研究人员。这些目标的实现将使这一基础工作转化为有效的病毒介导的基因转移遗传性血液病的临床方案的发展。在更广泛的层面上，这些研究应该提供对造血干细胞的生物学行为和体外操纵它们的能力的洞察。

DESCRIPTION (provided by applicant): The overall goal of this program is to develop a strong experimental foundation for the correction of inherited diseases of bone marrow-derived cells by genetic modification of hematopoietic stem cells. Current objectives are focused on the use of recombinant oncoretroviral, lentiviral, and foamy virus vectors to achieve safe and efficient transfer of functional genes into primitive murine and human hematopoietic stem cells while maintaining their maximal hematopoietic potential. The specific aims are to 1) identify strategies to improve efficient viral transduction of HSC ex vivo while retaining their optimal homing and engrafting capabilities 2) identify non-ablative conditioning regimens and in vivo selection protocols for enhanced engraftment of transduced cells that are effective yet minimize exposure to genotoxic agents, 3) identify optimal strategies using viral -mediated gene transfer to correct the phenotype in two inherited blood disorders, X-linked chronic granulomatous disease (X-CGD) and Fanconi anemia groups A and C. Experimental approaches include the use of in vitro culture systems, NOD/SCID mouse-human xenografts, and murine models of X-CGD and Fanconi anemia previously generated by gene targeting approaches. The implementation of these aims will be shared among 3 projects and 4 core units. This proposal draws from a group of investigators with diverse but complementary experience in stem cell biology, retrovirus- and lentivirus-mediated gene transfer, molecular genetics, virology, and bone marrow transplantation. Achievement of these goals will permit the translation of this basic work to the development of clinical protocols for effective viral-mediated gene transfer of genetic blood diseases. On a broader level, these studies should provide insight into the biologic behavior of hematopoietic stem cells and the ability to manipulate them ex vivo.