Gene Replacement Therapy in Hematopoietic Stem Cells

描述(由申请人提供)： 该计划的总体目标是通过造血干细胞的基因修饰为纠正骨髓源性细胞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来实现安全和有效地将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力。其具体目标是1)确定在保持最佳归巢和移植能力的同时，提高HSC体外病毒转导效率的策略2)确定非消融性调节方案和体内选择方案，以增强转导细胞的植入，这些方案有效但最大限度地减少暴露于基因毒性物质的影响；3)确定使用病毒介导的基因转移的最佳策略，以纠正两种遗传性血液疾病：X连锁慢性肉芽肿性疾病(X-CGD)和Fanconi贫血A和C组的表型。实验方法包括使用体外培养系统、NOD/SCID鼠-人异种移植，以及以前通过基因打靶方法产生的X-CGD和Fanconi贫血的小鼠模型。这些目标的落实将由3个项目和4个核心单位分担。这一建议来自一组研究人员，他们在干细胞生物学、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拥有不同但互补的经验。这些目标的实现将允许将这项基础工作转化为开发有效的病毒介导的遗传性血液疾病基因转移的临床方案。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的洞察，以及体外操纵它们的能力。

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.