HEMOPOIETIC STEM CELL SELF RENEWAL AND RETINOID ANTAGONISTS

利用逆转录病毒载体纠正遗传性疾病的研究进展 涉及到造血干细胞(HSC)的过程一直非常缓慢。一 主要绊脚石之一与观察到的逆转录病毒 载体优先感染有丝分裂活性细胞，但正常的 静止的HSC在体外被刺激分裂时，经常 分化为更成熟、更有血统的干细胞 表现出有限的克隆寿命。正是这些血统承诺了 增殖的祖先，既表现出有限的寿命，又表现出贫穷 优先感染的长期骨髓再生能力 逆转录病毒载体。因此，成功使用逆转录病毒载体来 纠正HSC遗传病需要自我更新的HSC 通过这些载体优先扩增和转导。我们希望 确定将促进自我发展的特定文化条件 肝星状细胞在体外的更新及更有效的逆转录病毒载体 介导的HSC转导。我们的实验方法将基于 我们实验室最近的观察表明，阻止 维甲酸受体在造血干细胞中的活性 抑制他们的血统承诺，并加强他们的自我更新。我们的 具体目标如下： 特异性AIM I)确定在体内的骨髓再生能力 培养的小鼠淋巴造血EML细胞。我们希望确定 原始干细胞依赖小鼠细胞系(简称EML)的能力 通过转导具有显性阴性的正常小鼠骨髓获得的 RA受体的构建，在体内作为辐射后的干细胞发挥作用 同基因的或在SCID小鼠中。 特异性AIM II)确定最佳的体外条件 小鼠和人类造血干细胞的自我更新。高度 小鼠和人HSCs的浓缩部分将在体外培养 在不同条件下使用造血干细胞生长因子 以及表现出RA受体拮抗作用的合成维甲酸。这个 在这些文化中的造血干细胞的自我更新将由不同的 技巧。 特异性AIM III)确定优化逆转录病毒的体外条件 介导的基因转导入人造血干细胞。利用 特定目标确定的培养条件Il我们会感染自己 携带逆转录病毒载体的造血干细胞更新 特异性标记并评估成功转导的效率 这些带有这些载体的造血干细胞。 这些研究直接解决了与效率低下有关的问题 逆转录病毒载体介导的造血干细胞基因转导 细胞。我们的方法，如果成功，将对基因具有广泛的适用性。 多种不同遗传性疾病患者的治疗 造血干细胞。

Progress in utilizing retroviral vectors to correct hereditary disorders involving the hematopoietic stem cell (HSC) has been exceedingly slow. One of the major stumbling blocks relates to the observation that retroviral vectors preferentially infect mitotically active cells, but the normally quiescent HSC, when stimulated to divide in vitro, frequently commits itself to differentiate into a more mature, lineage committed stem cell exhibiting a limited clonal lifespan. It is these lineage committed proliferating progenitors exhibiting both a limited lifespan and poor long-term marrow repopulating ability that are preferentially infected by the retroviral vectors. Thus the successful use of retroviral vectors to correct HSC genetic disorders requires that self renewing HSC be preferentially expanded and transduced by these vectors. We wish to determine the specific culture conditions that will promote the self- renewal of HSC in vitro and will lead to more efficient retroviral vector mediated transduction of HSC. Our experimental approach will be based on recent observations in our laboratory indicating that blocking the activity of retinoic acid (RA) receptors in hematopoietic stem cells inhibits their lineage commitment and enhances their self renewal. Our specific goals are as follows: SPECIFIC AIM I) Determine the in vivo marrow repopulating capability of the cultured murine lymphohematopoietic EML cells. We wish to determine the capacity of primitive SCF-dependent mouse cell lines (designated EML) derived by transducing normal mouse bone marrow with a dominant negative RA receptor construct, to function in vivo as stem cells in irradiated syngeneic or in SCID mice. SPECIFIC AIM II) Determine the optimal in vitro conditions that enhance the self renewal of murine and human hematopoietic stem cells. Highly enriched fractions of murine and human HSCs will be cultured in vitro under various conditions with hematopoietic stem cell growth factors together with synthetic retinoids exhibiting RA receptor antagonism. The self renewal of HSCs in these cultures will be evaluated by various techniques. SPECIFIC AIM III) Determine in vitro conditions for optimizing retroviral mediated gene transduction into human hematopoietic stem cells. Utilizing culture conditions determined in Specific Aim Il we will infect self renewing hematopoietic stem cells with retroviral vectors harboring specific markers and assess the efficiency of successfully transducing these HSCs with these vectors. These studies directly address the problem related to inefficient retroviral vector mediated gene transduction into hematopoietic stem cells. Our approach, if successful, will have broad applicability to gene therapy of patients with a variety of different genetic disorders of hematopoietic stem cells.