GENE TRANSFER TO FETAL AND NEONATAL HSC POPULATIONS

尽管大力开发治疗镰状细胞性贫血(SCA)的有效方法，但这种疾病仍然与显著的发病率和死亡率有关。SCA影响0.2%的非裔美国儿童和年轻人。为了使未来基于基因治疗的SCA策略获得成功：1)自我更新干细胞的转导必须高效，2)转导细胞必须具有选择性或增殖优势，3)基因输送载体必须在个体的一生中产生稳定的、治疗性的珠蛋白基因表达水平。我们的目标是开发有效地将基因转移到胎儿肝脏的方法，并且已经表明，无论是在腹膜内还是在肝脏内直接注射病毒或非病毒载体，都可以实现高水平的基因表达。我们将重点关注高增殖的HSC在小鼠胎肝中的转导。我们的第一个假设是，直接在子宫内输送基因转移载体将导致比体外实现更多数量的HSC的转导，并且不会破坏这些早期HSC的微环境或生物学。我们将使用MLV和基于HIV的逆转录病毒载体以及腺相关病毒载体来确定将基因转移到全能胎儿HSC的最有效的载体系统。我们将重点关注高增殖的HSC在小鼠胎肝中的转导。我们的第一个假设是，直接在子宫内输送基因转移载体将导致比体外实现更多数量的HSC的转导，并且不会破坏这些早期HSC的微环境或生物学。我们将使用MLV和基于HIV的逆转录病毒载体以及腺相关病毒载体来确定将基因转移到全能胎儿HSCs的最有效载体系统。我们的第二个假设是，经子宫肌醇注射为快速筛选新的珠蛋白基因载体提供了一个有效的模型。我们将提供人类伽马或贝塔基因表达。直接高水平表达珠蛋白的伽马或贝塔珠蛋白载体的治疗效果将在贝塔地中海贫血和镰状细胞性贫血的小鼠模型中进行测试。产生高水平珠蛋白基因表达的载体将减少红细胞的病态，并赋予转导的红细胞生存优势。这些研究还将确定转导的造血干细胞及其后代在个体发育过程中的命运。

Despite intensive efforts to develop effective therapy for sickle cell anemia (SCA), this disease continues to be associated with significant morbidity and mortality. SCA affects 0.2% of African American children and young adults. In order for future gene therapy-based strategies for SCA to be successful: 1) transduction of self-renewing stem cells must be highly efficient, 2) transduced cells must have a selective or proliferative advantage, and 3) gene delivery vectors must produce stable, therapeutic levels of globin gene expression over the lifetime of the individual. Our goal is to develop procedures for efficient gene transfer into fetal liver and have already shown that high-level gene expression may be achieved following either intraperitoneal or direct intrahepatic injection of viral or non-viral vectors. We will focus on the transduction of highly proliferative HSC in the murine fetal liver. Our first hypothesis is that direct in utero delivery of gene transfer vectors will result in the transduction of higher numbers of HSCs than can be achieved in vitro, and without disrupting either the microenvironment or biology of these early HSC. We will determine the most efficient vector system for gene transfer into totipotent fetal HSC using MLV- and HIV-based retroviral vectors, and adeno-associated viral vectors. We will focus on the transduction of highly proliferative HSC in the murine fetal liver. Our first hypothesis is that direct in utero delivery of gene transfer vectors will result in the transduction of higher numbers of HSCs than can be achieved in vitro, and without disrupting either the microenvironment or biology of these early HSC. We will determine the most efficient vector system for gene transfer into totipotent fetal HSCs using MLV- and HIV- based retroviral vectors, and adeno-associated viral vectors. Our second hypothesis is that transuterine injection provides an efficient model for rapidly screening novel globin gene vectors. We will deliver human gamma or beta gene expression. The therapeutic efficacy of gamma or beta globin vectors that direct high-level expression of globin will be tested in murine models of beta thalassemia and sickle cell anemia. Vectors that produce high-level globin gene expression will reduce red cell sickling and confer a survival advantage of transduced red cells. These studies will also define the fate of transduced hematopoietic stem cells and their progeny during ontogeny.