HEMATOPOIETIC STEM CELL BIOLOGY
造血干细胞生物学
基本信息
- 批准号:6681484
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:bone marrow bone marrow transplantation cell differentiation cell growth regulation colony stimulating factor cytokine receptors gene expression gene therapy genetic mapping green fluorescent proteins growth factor hematopoietic stem cells laboratory mouse microarray technology nucleic acid sequence polymerase chain reaction receptor expression transfection /expression vector
项目摘要
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. In addition, cells highly enriched for HSC can generate cardiac myocytes when injected into the healthy tissue surrounding a myocardial infarct or when mobilized into the peripheral blood with cytokines (e.g. G-CSF and SCF). Others have shown that enriched populations of HSC can give rise to vascular, skeletal muscle, hepatic and neuronal cells. Project 1 will examine stem cell biology. Project 1: Biology of Hematopoietic Stem Cells Specific Aim 1.1: We hypothesized that the same hematopoietic stem cells that repopulated the bone marrow were responsible for the regeneration of cardiac myocytes after experimental myocardial infraction (MI). To test this hypothesis, we transplanted mice with bone marrow cells marked with a retrovirus vector containing the GFP gene. These mice then underwent a coronary artery ligation and stem and progenitor cells were mobilized into the peripheral blood with G-CSF and SCF. The infarct region was repopulated with new cells that contain the identical retrovirus markers found in peripheral blood cells. These studies are ongoing, but we conclude that the new cardiac myocytes are the progeny of cells that also give rise to peripheral blood cells. Specific Aim 1.2: We hypothesized that the repopulation of the injured regions of the heart was due to engraftment and expansion of new cells as opposed to fusion of a primitive cell with surrounding cardiac cells. To test this hypothesis we have isolated cells from the regenerating infarct and used in situ hybridization to demonstrate that the cells express proteins in primitive cardiac cells and are diploid, arguing against the fusion hypothesis. Future studies will involve mice genetically deficient in HSC to see whether G-CSF/SCF treatment can repopulate an infarct region. Specific Aim 1.3: We hypothesize that specific genes expressed in both HSC and stem cells isolated from skeletal muscle are responsible for maintaining an undifferentiated state. To test this hypothesis, we have isolated the ?side population? stem cells from mouse skeletal muscle cultures and used cDNA subtraction to generate a library of sequences expressed in these cells. The muscle stem cell transcripts will be compared to those we have identified previously in a cDNA subtraction library of HSC. We will select transcripts common to both libraries for analysis in transgenic mice. Gene transfer to HSC has recently been shown to cure Severe Combined Immune Deficiency, demonstrating that HSC gene therapy could be applied to more common diseases. 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. We have found that one important reason that gene transfer is so low is that the conventional retrovirus receptors on HSC are nearly undetectable. A second problem has been the instability of retrovirus vectors containing globin genes. Project 2 will examine these problems separately. Project 2: Gene therapy for the hemoglobinopathies Specific aim 2.1: We have evidence that the receptors of the RD114 and FeLV-C retrovirus are expressed at high levels on hematopoietic stem cells. We hypothesize that these high levels of receptor will result in a higher frequency of human HSC transduction. We begun to test this hypothesis by simultaneously transducing human HSC with a control GALV pseudotyped vector and either RD114 or FeLV-C pseudotyped vectors for transplantation into fetal sheep. Peripheral blood and bone marrow cells from the the first 5 sheep chimeras showed greater than 10 fold higher levels of transduction with RD114 or FeLV-C pseudotyped vectors compared to GALV pseudotyped vectors. This work is on going. Specific Aim 2.2: We hypothesize that stable retrovirus vectors containing globin genes linked to the promoters of genes expressed in erythroid cells can be generated that will allow expression of globin mRNA at levels adequate to treat Sickle Cell Disease and b-thalassemia. 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 generated stable retroviruses with this construct which will be evaluated in the mouse b-thalassemia model.
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用途。造血干细胞(HSC)是一种罕见的自我更新细胞群,可产生外周血中的所有细胞,使其成为遗传性造血疾病基因替代治疗的理想载体。此外,当注射到心肌梗塞周围的健康组织中或与细胞因子(例如 G-CSF 和 SCF)一起动员到外周血中时,高度富集 HSC 的细胞可以产生心肌细胞。其他研究表明,HSC 群体的富集可以产生血管、骨骼肌、肝和神经元细胞。项目 1 将研究干细胞生物学。项目 1:造血干细胞生物学 具体目标 1.1:我们假设,在实验性心肌梗塞 (MI) 后重新填充骨髓的相同造血干细胞负责心肌细胞的再生。为了验证这一假设,我们将用含有 GFP 基因的逆转录病毒载体标记的骨髓细胞移植到小鼠身上。然后对这些小鼠进行冠状动脉结扎,并用 G-CSF 和 SCF 将干细胞和祖细胞动员到外周血中。梗塞区域重新填充了新细胞,这些细胞含有与外周血细胞中发现的相同的逆转录病毒标记。这些研究正在进行中,但我们得出的结论是,新的心肌细胞是也产生外周血细胞的细胞的后代。具体目标 1.2:我们假设心脏受损区域的重新增殖是由于新细胞的植入和扩张,而不是原始细胞与周围心肌细胞的融合。为了检验这一假设,我们从再生梗塞中分离出细胞,并使用原位杂交来证明这些细胞在原始心肌细胞中表达蛋白质并且是二倍体,从而反对融合假设。未来的研究将涉及 HSC 基因缺陷的小鼠,以观察 G-CSF/SCF 治疗是否可以重新填充梗塞区域。具体目标 1.3:我们假设 HSC 和从骨骼肌分离的干细胞中表达的特定基因负责维持未分化状态。为了检验这个假设,我们分离出了“侧面种群”。小鼠骨骼肌培养物中的干细胞,并使用 cDNA 消减来生成在这些细胞中表达的序列文库。肌肉干细胞转录本将与我们之前在 HSC cDNA 消减文库中鉴定的转录本进行比较。我们将选择两个文库共有的转录本用于转基因小鼠中的分析。最近,HSC 基因转移已被证明可以治愈严重的联合免疫缺陷,这表明 HSC 基因治疗可以应用于更常见的疾病。我们希望开发一种针对镰状细胞病的基因疗法。然而,目前造血干细胞的基因转移水平太低,无法治疗这种疾病。我们发现基因转移如此之低的一个重要原因是HSC上的常规逆转录病毒受体几乎检测不到。第二个问题是含有珠蛋白基因的逆转录病毒载体的不稳定性。项目2将分别研究这些问题。项目2:血红蛋白病的基因治疗具体目标2.1:我们有证据表明RD114和FeLV-C逆转录病毒的受体在造血干细胞上高水平表达。我们假设这些高水平的受体将导致人类 HSC 转导的频率更高。我们开始通过同时转导人类 HSC 与对照 GALV 假型载体和 RD114 或 FeLV-C 假型载体以移植到胎羊中来测试这一假设。与 GALV 假型载体相比,前 5 个绵羊嵌合体的外周血和骨髓细胞显示 RD114 或 FeLV-C 假型载体的转导水平高出 10 倍以上。这项工作正在进行中。具体目标 2.2:我们假设可以生成含有与红系细胞中表达的基因启动子连接的球蛋白基因的稳定逆转录病毒载体,该载体将允许球蛋白 mRNA 的表达水平足以治疗镰状细胞病和 b 地中海贫血。我们使用转基因小鼠测定对红细胞基因启动子表达的相对水平进行的评估表明,与鸡绝缘体元件连接的AE-1启动子指导位置独立、均匀、高水平和拷贝数依赖的表达。我们已经用这种构建体生成了稳定的逆转录病毒,将在小鼠 b 地中海贫血模型中对其进行评估。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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DAVID M. BODINE其他文献
DAVID M. BODINE的其他文献
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{{ truncateString('DAVID M. BODINE', 18)}}的其他基金
VISION: ValIdated Systematic IntegratiON of epigenomic data
愿景:经过验证的表观基因组数据的系统整合
- 批准号:
9183143 - 财政年份:2016
- 资助金额:
-- - 项目类别:
VISION: ValIdated Systematic IntegratiON of epigenomic data
愿景:经过验证的表观基因组数据的系统整合
- 批准号:
9976999 - 财政年份:2016
- 资助金额:
-- - 项目类别:
Global Predictions and Tests of Hematopoietic Regulation
造血调节的整体预测和测试
- 批准号:
8912612 - 财政年份:2004
- 资助金额:
-- - 项目类别:
ENHANCER ELEMENTS IN THE HUMAN B GLOBIN GENE CLUSTER
人类 B 珠蛋白基因簇中的增强子元件
- 批准号:
3049744 - 财政年份:1986
- 资助金额:
-- - 项目类别:
ENHANCER ELEMENTS IN THE HUMAN B GLOBIN GENE CLUSTER
人类 B 珠蛋白基因簇中的增强子元件
- 批准号:
3049745 - 财政年份:1986
- 资助金额:
-- - 项目类别:
ENHANCER ELEMENTS IN THE HUMAN B GLOBIN GENE CLUSTER
人类 B 珠蛋白基因簇中的增强子元件
- 批准号:
3049743 - 财政年份:1985
- 资助金额:
-- - 项目类别:
ENHANCER ELEMENTS IN THE HUMAN B GLOBIN GENE CLUSTER
人类 B 珠蛋白基因簇中的增强子元件
- 批准号:
3049742 - 财政年份:1985
- 资助金额:
-- - 项目类别:
Improving gene transfer to provide intracellular immuniz
改善基因转移以提供细胞内免疫
- 批准号:
6988880 - 财政年份:
- 资助金额:
-- - 项目类别:
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