GENE REPLACEMENT THERAPY IN HEMATOPOIETIC STEM CELLS

（改编自申请人摘要）本项目的总体目标是为通过造血干细胞的遗传修饰矫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逆转录病毒，慢病毒和腺相关病毒（AAV）载体，以实现有效的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能。 具体目的是：1）研究细胞周期、纤连蛋白介导的粘附和细胞因子调节剂对造血细胞功能和病毒转导的关系; 2）确定来自AAV和慢病毒的重组病毒载体是否可用于在小鼠和人造血细胞中有效和稳定的基因转移; 3）确定使用病毒介导的基因转移来纠正两种遗传性血液病（X连锁慢性肉芽肿病（X-CGD）和范可尼贫血C组（FanC））的表型的最佳策略;和4）评价非消融性预处理方案和使用标记蛋白或药物抗性基因选择转导的细胞。 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FanC小鼠模型。 这些目标的实现将由4个项目和3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、慢病毒和AAV介导的基因转移、分子遗传学、病毒学、骨髓移植和肿瘤学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。

(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.