Moreau-Gaudry F, Ged C, Barbot C, Mazurier F, Boiron J M, Bensidhoum M, Reiffers J, de Verneuil H
Département de Biochimie Médicale et Biologie Moléculaire, Université de Bordeaux II, France.
Hum Gene Ther. 1995 Jan;6(1):13-20. doi: 10.1089/hum.1995.6.1-13.
Congenital erythropoietic porphyria (CEP) is a genetic disease characterized by an overproduction and accumulation of porphyrins in bone marrow. The enzyme defect concerns uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the heme biosynthetic pathway. It is the most severe porphyria and the treatment is largely symptomatic: gene therapy would represent a great therapeutic improvement. As a step toward the development of an effective gene therapy, we have constructed two retroviral vectors, LUSN and pMFG-US (with and without the selectable marker Neo), containing a full-length human cDNA for UROIIIS. Recombinant retroviruses were obtained by transfection of the LUSN or pMFG-US plasmid into the amphotropic packaging cell line psi CRIP. For each construct, three different producing clones were selected for their high titer (LUSN) or for their ability to express the message at a high level (pMFG-US). In vitro amplification of genomic DNA from target tissue demonstrated the presence of vector sequences. Murine fibroblasts infected in vitro expressed the human enzyme efficiently, as indicated by RNA and enzymatic studies. Retroviral-mediated gene transfer was then used to introduce the UROIIIS cDNA into human deficient cells. Enzyme activity was increased from 2% (deficient fibroblasts) to 121-274% of the normal value for the different clones. Transduced cells selected with G418 presented an 18-fold increase in enzyme activity compared to the normal cells. Furthermore, high gene transfer rate into peripheral blood progenitor cells (PBPB) was documented by in vitro amplification (PCR). These results demonstrate the potential usefulness of somatic gene therapy for the treatment of CEP.
先天性红细胞生成性卟啉病（CEP）是一种遗传性疾病，其特征是卟啉在骨髓中过度产生和积累。酶缺陷涉及尿卟啉原III合酶（UROIIIS），它是血红素生物合成途径的第四个酶。这是最严重的卟啉病，治疗主要是对症治疗：基因治疗将代表巨大的治疗进展。作为朝着开发有效基因治疗迈出的一步，我们构建了两种逆转录病毒载体，LUSN和pMFG-US（有和没有选择标记Neo），它们包含UROIIIS的全长人cDNA。通过将LUSN或pMFG-US质粒转染到嗜异性包装细胞系psi CRIP中获得重组逆转录病毒。对于每个构建体，选择三个不同的产生克隆，因其高滴度（LUSN）或因其高水平表达的能力（pMFG-US）。来自靶组织的基因组DNA的体外扩增证明了载体序列的存在。如RNA和酶学研究所表明的，体外感染的小鼠成纤维细胞有效表达人酶。然后使用逆转录病毒介导的基因转移将UROIIIS cDNA引入人缺陷细胞中。对于不同的克隆，酶活性从2％（缺陷成纤维细胞）增加到正常值的121-274％。与正常细胞相比，用G418选择的转导细胞的酶活性增加了18倍。此外，通过体外扩增（PCR）记录了向外周血祖细胞（PBPB）的高基因转移率。这些结果证明了体细胞基因治疗在治疗CEP中的潜在有用性。
