GENETIC THERAPY OF FABRY DISEASE

我们已经进行了一系列关于基因的临床前研究。 治疗法布里病。重组逆转录病毒基因转移载体的构建 被设计成工程师高效地转导细胞和表达 人类α-半乳糖A的活性。观察到了酶的矫正 培养的皮肤成纤维细胞和B细胞系 法布里病。校正后的细胞分泌大量的α- Gal A进入培养基中。分泌的酶被 未矫正的旁观者细胞。我们还实现了酶促校正 骨髓抽吸物获得的CD34+和祖细胞集落细胞 来自法布里病患者的。一种重组融合蛋白已经被 将框架内的α-GalA酶与羧基- 末端八个氨基酸的加成称为旗肽。此序列 在表达时具有特异性和抗原性。可以用以下方法检测到 一种抗体。这一融合蛋白将允许纯化额外的和 α-GalA的细胞内形式，并提供摄取的直接证据 进入旁观者细胞和溶酶体位置。我们还在制作 带有可选择标记的逆转录病毒载体可通过以下方式增加表达 改变病毒或α-GalA结构或通过对转导的 移植前的细胞。我们还在进行实验，以 优化外周血中动员的CD34+细胞的转导。 我们目前正在生产一种符合GMP的高滴度临床级载体 Fabry病基因治疗试验的条件。

We have performed a series of preclinical investigations concerning gene therapy for Fabry disease. A recombinant retroviral gene transfer vector was designed that engineers efficient transduction of cells and expression of human alpha-Gal A activity. Enzymatic correction was observed in cultured skin fibroblasts and B cell lines derived from patients with Fabry disease. Corrected cells secreted significant quantities of alpha- Gal A into the culture medium. The secreted enzyme is taken up by uncorrected bystander cells. We have also achieved enzymatic correction in CD34+ and progenitor colony cells obtained from bone-marrow aspirates from patients with Fabry disease. A recombinant fusion protein has been engineered that combines the alpha-Gal A enzyme in frame with a carboxy- terminal eight amino acid addition called the FLAG peptide. This sequence is specific and antigenic when it is expressed. It can be detected with an antibody. This fusion protein will permit purification of extra- and intracellular forms of alpha-Gal A and provide direct evidence of uptake into bystander cells and lysosomal location. We are also producing retroviral vectors with selectable markers to increase expression by altering the viral or alpha-Gal A constructs or by sorting the transduced cells prior to implantation. We are also conducting experiments to optimize the transduction of CD34+ cells mobilized in peripheral blood. We are currently producing a high-titer clinical-grade vector under GMP conditions for gene therapy trials in Fabry disease.