Gene Transfer into Hematopoietic Stem Cells

基因转移至造血干细胞

• 批准号: 7146862
• 负责人: Fabio Candotti
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146862
• 关键词: AIDS; HIV infections; biological models; biotechnology; blood disorder; bone marrow; cell differentiation; cell population study; cord blood; cytokine; disease /disorder model; gene therapy; genetic manipulation; genetic regulation; growth factor; hematopoiesis; hematopoietic stem cells; hematopoietic tissue transplantation; human subject; laboratory mouse; leukemia; microarray technology; model design /development; sheep; stem cell transplantation; thalassemia; therapy design /development; tissue /cell culture; transfection /expression vector; virus integration

A variety of inherited and acquired human diseases affecting the hematopoietic stem cell (HSC) can potentially be treated with genetic manipulation of the patients' HSCs. Potential applications of this technology include therapeutic approaches for common blood disorders (e.g. thalassemia), malignancies (e.g. leukemia) and infectious diseases of the blood cells, such as HIV-1 infection/AIDS. However, with a few exceptions, gene transfer into HSCs of large animal models and humans has thus far proven to be difficult and inefficient. By improving our knowledge of the biological characteristics of the human HSC we will likely gain important insights that will allow us to improve the efficiency of gene transfer in and correction of this elusive target cell. Under this project we have established in vitro and in vivo animal models of human hematopoiesis using hematopoietic progenitors obtained from cord blood, bone marrow or peripheral blood of volunteer donors. These cells are cultured in vitro in a variety of cytokines and growth factors to identify conditions that allow their survival in the absence of differentiation. In addition, the gene expression profile of these cells is studied by microarray analysis. The cells are then subjected to gene transfer using viral vectors that allow for integration of the transferred gene and allowed to differentiate into mature cell lineages in vitro (methylcellulose progenitor colonies) or in vivo using mouse or sheep animal models. The pattern of viral integration sites is studied to verify if trends indicating preferential genomic locations can be identified. These experiments allow comparison of cells with putative hematopoietic stem cell activity isolated from various sources (e.g. cord blood vs. bone marrow vs. peripheral blood) and testing of vectors and gene transfer conditions that efficiently target these cells. We have also developed an in vitro model of retroviral insertional oncogenesis that can be applied to murine and human hematopoietic stem cells to study the effects of genetic manipulation with retroviral vectors. These experiments will also allow to test the efficacy of safety modification of gene transfer vector that can be transferred to future human trials.

可以通过患者的HSC的遗传操纵来治疗各种影响造血干细胞（HSC）的遗传和获得的人类疾病。这项技术的潜在应用包括常见血液疾病（例如，thalassya），恶性肿瘤（例如白血病）和血细胞感染性疾病（例如HIV-1感染/艾滋病）的治疗方法。但是，除少数例外，迄今为止，基因转移到大型动物模型和人类的HSC中是困难和效率低下的。通过提高我们对人类HSC生物学特征的了解，我们可能会获得重要的见解，从而使我们能够提高基因转移的效率，并纠正该难以捉摸的靶细胞。在这个项目下，我们使用从绳索血液，骨髓或志愿供体的外周血中获得的造血祖细胞建立了人类造血的体外和体内动物模型。这些细胞在各种细胞因子和生长因子中在体外进行培养，以鉴定允许在没有分化的情况下存活的疾病。另外，通过微阵列分析研究了这些细胞的基因表达谱。然后，使用病毒载体对细胞进行基因转移，该病毒载体允许在体外（甲基纤维素祖细胞菌落）或使用小鼠或绵羊动物模型的体外分化为成熟的细胞谱系。研究了病毒整合位点的模式，以验证是否可以识别表明优先基因组位置的趋势。这些实验允许将细胞与假定的造血干细胞活性进行比较，这些干细胞活性是从各种来源分离的（例如脐带血与骨髓与外周血），并测试矢量和基因转移条件，有效地靶向这些细胞。我们还开发了一种逆转录病毒插入肿瘤发生的体外模型，可以应用于鼠和人类造血干细胞，以研究使用逆转录病毒载体的遗传操纵的影响。这些实验还将允许测试可以转移到未来人类试验的基因转移载体的安全性修饰的功效。