COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT I

该项目的最终目标是治愈镰状细胞病（SCD） 通过自体骨髓移植后的遗传改变， 用腺相关病毒（AAV）载体转染造血干细胞。 的 AAV载体将携带特别设计的抗镰状化β-珠蛋白 编码抑制镰状血红蛋白的多肽的基因（BAS (HbS)聚合物形成，并因此抑制红细胞镰状化。 BAS-珠蛋白基因受基因座控制区（LCR）调控 特异性指导高水平OS人珠蛋白表达的序列 在红细胞中。 高水平所需的最小LCR序列 表达将通过构建各种AAVHS 2 BAS球蛋白来确定 载体并在转基因小鼠中测定这些构建体。 构建体 在该严格测定中指导高水平表达将 用于生产病毒。 最初，将使用AAV HS 2 BAS-球蛋白病毒储备液感染 将在长期培养中维持的人骨髓 细胞（LTC-IC）试验，如项目2中所述。的效率 造血干细胞的感染将通过测定 形成BFU-E（爆发形成单位-红细胞）的LTC-C部分 含有BAS-珠蛋白序列。 BAS-珠蛋白多肽也将被 在衍生自LTC-IC的BFU-E中定量。 当需要条件时 干细胞的有效感染和高水平表达 转移的珠蛋白基因被定义，来自SCD患者的骨髓将被 infect. 将测定来源于SCD骨髓的LTC-IC的BFU-E的BAS- 珠蛋白DNA和多肽。 BAS活动将通过以下方式进行定量： 在Fibach液体培养系统中扩增红系祖细胞， 测量制备的溶血产物中HbS聚合物形成的抑制 从这些细胞。 此外，来自扩增液体培养物的红系细胞 将在体外脱氧以评估抗镰状化作用。 当转移的珠蛋白基因的抗镰状化特性被破坏时， 在体外证明，将启动临床试验。 起初， 只有HLA匹配的异基因供者才能接受移植。 感染的自体骨髓，这样一个可行的替代疗法， available. 感染的条件将与那些 确定长期骨髓培养物的有效感染 如上所述;然而，实验将按比例扩大基因 疗法 移植后每周采集血样 并定量BAS多肽的水平。 抗镰状化 通过测量HbS聚合物的抑制来评估活性 在从获得的红系细胞制备的溶血产物中形成 从扩大的文化。 最终，该疗法的疗效将是 这取决于SCD严重病理学的矫正程度。

The ultimate goal of this project is to cure Sickle Cell Disease (SCD) by autologous bone marrow transplantation after genetic alteration of hematopoietic stem cells with Adeno-Associated Virus (AAV) vectors. The AAV vectors will carry specially designed, anti-sickling beta-globin genes (BAS) which encode polypeptides that inhibit sickle hemoglobin (HbS) polymer formation and, therefore, inhibit erythrocyte sickling. The BAS-globin genes will be regulated by Locus Control Region (LCR) sequence that direct high level os human globin expression specifically in erythroid cells. The minimal LCR sequences required for high level expression will be determined by constructing various AAV HS 2 BAS-globin vectors and assaying these constructs in transgenic mice. Constructs that direct high levels of expression in this stringent assay will be utilized to produce virus. Initially, the AAV HS 2 BAS-globin viral stocks will be used to infect human marrow that will be maintained in the long term culture-initiating cell (LTC-IC) assay as described in Project 2. The efficiency of infection of hematopoietic stem cells will be assessed by determining the fraction of LTC-C that form BFU-E (burst Forming Units-Erythroid) containing BAS-globin sequence. BAS-globin polypeptides will also be quantitated in BFU-E derived from LTC-IC. When conditions required for efficient infection of stem cells and high level expression of transferred globin genes are defined, marrow from SCD patients will be infect. BFU-E derived from LTC-IC of SCD marrow will be assayed for BAS- globin DNA and polypeptides. BAS activity will be quantitated by expanding erythroid progenitors in Fibach's liquid culture system and measuring the inhibition of HbS polymer formation in hemolysates prepared from these cells. Also, erythroid cells from expanded liquid cultures will be deoxygenated in vitro to evaluate anti-sickling effects. When the anti-sickling properties of transferred globin genes are demonstrated in vitro, clinical trials will be initiated. Initially, only patient s with HLA matched allogeneic donor will be transplanted wit infected, autologous marrow so that a viable alternative therapy is available. The conditions for infection will be identical to those determined for efficient infection of the long term marrow cultures described above; however, the experiments will be scaled up for gene therapy. Blood samples will be obtained each week post-transplantation and the levels of BAS polypeptides will be quantitated. Anti-sickling activity will be assessed by measuring the inhibition of HbS polymer formation in hemolysates prepared from erythroid cells that are obtained from expanded cultures. Ultimately, the efficacy of the therapy will be determined by the degree of correction of the severe pathology of SCD.