GENE REPLACEMENT THERAPY IN HEMATOPOIETIC STEM CELLS

(改编自申请人的摘要)该计划的总体目标是为通过造血干细胞的基因修饰纠正骨髓源性细胞遗传病奠定坚实的实验基础。目前的目标集中在使用重组逆转录病毒、慢病毒和腺相关病毒(AAV)载体来实现有效地将功能基因转移到原始造血干细胞，同时保持其最大的造血潜力。其具体目标是1)检测细胞周期、纤维连接蛋白介导的黏附和细胞因子调节因子对造血细胞功能和病毒转导的关系；2)确定AAV和慢病毒衍生的重组病毒载体是否可用于小鼠和人类造血细胞高效而稳定的基因转移；3)确定使用病毒介导的基因转移来纠正两种遗传性血液疾病(X连锁慢性肉芽肿病和范可尼贫血C组)的最佳策略；以及4)评估非消融性预处理方案和使用蛋白质标记基因或耐药基因进行转导细胞的选择。实验方法包括使用体外培养系统，NOD/SCID鼠-人异种移植，以及先前通过基因打靶方法产生的X-CGD和FANC的小鼠模型。这些目标的落实将由4个项目和3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、慢病毒和AAV介导的基因转移、分子遗传学、病毒学、骨髓移植和新生儿学方面具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。

(Adapted from the applicant's abstract) The overall goal of this program is to develop a strong experimental foundation for the correction of inherited disease of bone marrow-derived cells by genetic modification of hematopoietic stem cells. Current objectives are focused on the use of recombinant retroviral, lentiviral, and adeno-associated virus (AAV) vectors to achieve efficient transfer of functional genes into primitive hematopoietic stem cells while maintaining their maximal hematopoietic potential. The specific aims are to 1) examine the relationship between cell cycle, fibronectin-mediated adhesion, and cytokine regulators on hematopoietic cell function and viral transduction; 2) determine whether recombinant viral vectors derived from AAV and lentivirus can be used for efficient and stable gene transfer in murine and human hematopoietic cells; 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 group C (FanC); and 4) evaluate non- ablative conditioning regimens and selection of transduced cells using maker proteins or drug resistance genes. Experimental approaches include the use of in vitro culture systems, NOD/SCID mouse-human xenografts, and murine models of X-CGD and FanC previously generated by gene targeting approaches. The implementation of these aims will be shared among 4 projects and 3 core units. This proposal draws from a group of investigators with diverse but complementary experience in stem cell biology, retrovirus-, lentivirus-, and AAV-mediated gene transfer, molecular genetics, virology, bone marrow transplantation, and neonatology. 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.