Development and classification of therapeutic method for vascularEhlers-Danlos syndrome by mutation types

血管性ehers - danlos综合征（vEDS）是由COL3A1突变引起的一种危及生命的显性遗传性疾病。虽然基因治疗是治疗遗传性疾病的重要选择，但添加缺陷基因并不适用于显性疾病，如vEDS。为了建立vEDS的治疗策略，我们研究了每种突变的两种方法的可行性。首先，我们测试了RNAi介导的突变等位基因的抑制作用。我们合成了几种靶向突变mRNA特异性序列的小干扰RNA （siRNA）分子，并将其引入患者的成纤维细胞。COL3A1 mRNA的定量测定表明，剪接突变可选择性地降低突变体mRNA水平达80%，并有可能增加正常三螺旋的相对浓度。其次，我们试图增加正常COL3A1 mRNA的浓度，因为一半剂量的COL3A1可能不足以预防vEDS症状。赖氨酸氧化酶（LOX）是一种双功能蛋白，既具有控制胶原和弹性蛋白成熟的细胞外酶活性，又具有人胶原III启动子的细胞内转录激活剂活性。用突变特异性siRNA和LOX表达载体转染患者细胞后，观察到突变等位基因的特异性抑制和正常等位基因的特异性增强。

Vascular Ehlers-Danlos syndrome (vEDS), is a life-threatening dominantly inherited disorder caused by COL3A1 mutation. Although gene therapy is an important option for treatment of genetic disorders, addition of the defective gene is not applicable for dominant diseases such as vEDS. To establish a therapeutic strategy for vEDS, we examined the feasibility of two approaches in each mutation. First, we tested RNAi mediated inhibition of the mutant allele. We synthesized several small interfering RNA (siRNA) molecules targeting sequence specific for mutant mRNA and introduced into patient’s fibroblasts. Quantitative measurement of COL3A1 mRNA showed that mutant mRNA levels could be selectively decreased up to 80 % in splicing mutation and it was possible to increase the relative concentration of normal triple helix. Second, we attempted to increase theconcentration of normal COL3A1 mRNA, since half-dose of COL3A1 may not be sufficient to prevent vEDS symptoms. Lysyl oxidase(LOX)is a bifunctional protein carrying activities of both an extracellular enzyme that controls the maturation of collagen and elastin and an intracellular tranascriptional activator for the human collagen III promoter. When patient’s cells were transfected with mutant specific siRNA and LOX expression vector, specific inhibition of the mutant allele and enhancement of the normal allele wereobserved.