COLLABORATIVE PROJECTS ON MINORITY HEALTH--PROJECT I

这个项目的最终目标是治愈镰状细胞病(SCD)。 基因改变后的自体骨髓移植 以腺相关病毒(AAV)为载体的造血干细胞。这个 AAV载体将携带特殊设计的抗病β-珠蛋白 编码抑制镰状血红蛋白的多肽的基因(BAs) (HBS)聚合物的形成，因此，抑制红细胞的病态。 BAS-珠蛋白基因受基因座控制区(LCR)调控 特异性地引导高水平人珠蛋白表达序列 在红系细胞中。高级别所需的最小LCR序列 将通过构建不同的AAV HS 2 BAS-珠蛋白来确定表达 载体，并在转基因小鼠中分析这些结构。构造 在这个严格的测试中直接高水平的表达将是 用来制造病毒的。 最初，AAVHS2BAS-珠蛋白病毒库将用于感染 将在长期培养中保持的人类骨髓--启动培养 项目2中所述的细胞(LTC-IC)检测。 造血干细胞的感染将通过测定 形成BFU-E(突发形成单位-红系)的LTC-C的部分 含有BAS-珠蛋白序列。碱性珠蛋白多肽也将被 在来自LTC-IC的BFU-E中定量。当满足以下条件时 干细胞高效感染和高水平表达 转移的珠蛋白基因被定义，SCD患者的骨髓将被 感染。SCD骨髓LTC-IC来源的BFU-E将检测BAS-E 珠蛋白DNA和多肽。BAS活动将通过以下方式进行量化 在Fibach液体培养系统中扩增红系祖细胞 测定制备的溶血物中HBS聚合物形成的抑制作用 从这些细胞中。此外，从扩增的液体培养中提取的红系细胞 将在体外除氧，以评价抗病作用。 当转移的珠蛋白基因的抗病特性是 在体外证明，将启动临床试验。最初， 仅有人类白细胞抗原相合异基因供者的患者S将接受同种异体移植 因此，一种可行的替代疗法是 可用。感染的条件将与那些 确定长期骨髓培养物有效感染 然而，针对基因的实验将被放大。 心理治疗。移植后每周将采集血样。 并对BAS多肽水平进行定量测定。抗病 活性将通过测量HBS聚合物的抑制来评估 从红系细胞制备的溶血物的形成 来自扩展的文化。最终，这种疗法的疗效将是 由SCD的严重病理矫正程度决定。

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.