Hematopoietic Stem Cell Biology

摘要：造血科的研究重点是干细胞的基础生物学，以及干细胞作为细胞和基因治疗载体的使用。造血干细胞(HSC)是一种罕见的自我更新细胞群，可在外周血中产生所有细胞，使其成为遗传性血液病基因替代治疗的理想载体。项目1：造血干细胞生物学特定目标1.1：我们已经证明Hmgb3是一种蛋白质，它既能与转录因子结合，又能与染色质结合，并使用Hmgb3基因敲除的小鼠模型来证明Hmgb3是调节最原始的造血细胞分化和增殖之间的平衡所必需的。我们已经证明，Hmgb3调节活化的HSC上c-kit受体的表达，并正在开发工具来证明Hmgb3与KIT基因座的结合。具体目标1.2：我们假设在从骨骼肌分离的HSC和干细胞中表达的特定基因负责维持未分化状态。我们已经鉴定出一个基因Asridj，它在骨骼肌干细胞、ES细胞和HSC中都有表达，在几种干细胞中也有表达。数据库。逆转录病毒将Asridj基因转移到骨髓HSC可抑制造血分化。我们现在正在开发敲除ES细胞来进一步评估Asrij的功能，这将被用来识别Asridj过表达的影响。项目2：我们想开发一种治疗镰刀细胞病的基因疗法。然而，目前向HSC转移的基因水平太低，无法治疗这种疾病，而且其他基因治疗试验中的不良事件表明，将来自珠蛋白基因座的那些强大的增强子插入基因组中是一个问题。特定目的2.1：我们已经证明RD114和FeLV-C逆转录病毒的受体在造血干细胞上高水平表达，这导致在绵羊异种移植模型中改善了向人类造血细胞的基因转移。我们正在将FeLV-C包膜改造成伪型慢病毒载体，以抑制珠蛋白载体的不稳定性，提高基因转移频率。具体目的2.2：我们假设稳定的逆转录病毒载体含有与红系细胞中表达的基因启动子相连的珠蛋白基因，允许珠蛋白mRNA在没有增强子元件的情况下以足以治疗镰刀细胞病和b地中海贫血的水平表达。我们使用转基因小鼠实验对红细胞基因启动子的相对表达水平进行了评估，结果表明，连接到鸡绝缘元件的AE-1启动子引导位置无关、均匀、高水平和拷贝数相关的表达。我们还确定了锚蛋白启动子中的一个关键调控区域，我们将对其进行修改，试图提高锚蛋白启动子的活性，以获得更高水平的珠蛋白表达。我们还证明了在Ankyrin启动子中有一个紧凑的绝缘体元件，它可以在体外和体内提供保护，防止基因沉默。我们目前正在评估其他红细胞基因的调控区域，以确定要整合到我们的珠蛋白载体中的增强剂和屏障元件。

Summary: The research of the Hematopoiesis Section is focused on the basic biology of stem cells and the use of stem cells as vehicles for cell and gene therapy. Hematopoietic stem cells (HSC) are a rare population of self-renewing cells that give rise to all cells in the peripheral blood, making them ideal vehicles for gene replacement therapy of inherited hematopoietic diseases. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We have shown that Hmgb3 is a protein the binds both to transcription factors and to chromatin and used an Hmgb3 knockout mouse model to demonstrate that Hmgb3 is required to regulate the balance between differentiation and proliferation of the most primitive hematopoietic cells. We have shown that Hmgb3 regulates the expression of c-kit receptor on active HSC and are deveoping tools to demonstrate Hmgb3 binding to the kit locus. Specific Aim 1.2: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. We have identified a gene - Asridj, which is expressed in both skeletal muscle stem cells, ES cells and HSC, as well as being in several ?stem cell? databases. Retroviral transfer of the Asridj gene into bone marrow HSC inhibits hematopoietic differentiation. We are now developing knock out ES cells to further evaluate Asrij functionwill be used to identify the effects of over expression of Asridj. Project 2: We would like to develop a gene therapy for Sickle Cell Disease. However, current levels of gene transfer to HSC are too low to treat this disease and the adverse events in other gene therapy trials point out the problem of inserting powerful enhancers like those from the globin locus into the genome. Specific aim 2.1: We have shown that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells, and that this leads to improved gene transfer to human hematopoietic cells in the sheep xenograft model. We are adapting the FeLV-C envelope to pseudotype lentivirus vectors to suppress globin vector instability and improve gene transfer frequency. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia without enhancer elements. Our evaluation of the relative level of expression of red cell gene promoters using a transgenic mouse assay has shown that the AE-1 promoter linked to a chicken insulator element directs position independent, uniform, high-level, and copy number dependent expression. We have also identified a key regulatory region in the ankyrin promoter that we will modify in an attempt to increase the activity of the ankyrin promoter to give higher levels of globin expression. We have also demonstrated a compact insulator element in the ankyrin promoter that provides protection from gene silencing in vitro and in vivo. We are currently evaluating the regulatory regions of other red cells genes to identify both enhancers and barrier elements to incorporate into our globin vectors.