HEMOPOIETIC STEM CELL SELF RENEWAL AND RETINOID ANTAGONISTS

逆转录病毒载体治疗遗传性疾病的研究进展 涉及造血干细胞（HSC）的研究进展非常缓慢。一 主要的障碍之一是， 载体优先感染有丝分裂活跃的细胞，但正常的 静止的HSC在体外被刺激分裂时， 自身分化成更成熟的谱系定向干细胞 克隆寿命有限正是这些血统 增殖的祖细胞表现出有限的寿命和差的 长期骨髓再生能力，优先感染 逆转录病毒载体。因此，成功地使用逆转录病毒载体， 正确的HSC遗传性疾病需要自我更新的HSC， 优选地被这些载体扩增和转导。我们希望 确定特定的文化条件，将促进自我， 更新的HSC在体外，并将导致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， 造血干细胞视黄酸受体活性 抑制了他们的血统承诺，增强了他们的自我更新。我们 具体目标如下： 具体目的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SCF依赖性小鼠细胞系（命名为EML）的能力 通过用显性阴性转导正常小鼠骨髓获得 RA受体构建体，在受照射的小鼠中作为干细胞在体内起作用 同基因或在SCID小鼠中。 特定目的II）确定增强细胞增殖的最佳体外条件。 小鼠和人类造血干细胞的自我更新。高度 将在体外培养鼠和人HSC的富集级分 造血干细胞生长因子 以及显示RA受体拮抗作用的合成类视色素。的 这些培养物中HSC的自我更新将通过各种方法进行评估。 技术. 特定目的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 在特定目标II中确定的培养条件下，我们将感染自身 逆转录病毒载体更新造血干细胞 特异性标志物，并评估成功转导的效率 这些HSC和这些载体。 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，将对基因工程具有广泛的适用性。 治疗患有各种不同遗传疾病的患者， 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.