1. ¹Dept Biochem and Molec Biol, Nippon Medical Sch, Tokyo, Japan
2. ²Div Clin Genet, Nippon Medical Sch Main Hosp, Tokyo, Japan
3. ³Div Clin and Molec Genet, Shinsyu Univ Hosp, Matsumoto, Japan
4. ⁴UPBSB, Dept Biophysics and Biochem, Sch of Sci, University of Tokyo, Tokyo, Japan
5. ⁵Dept Biochem and Molec Biol, Kanagawa Dental Collage, Yokosuka, Japan

Abstract

Vascular Ehlers-Danlos syndrome (vascular EDS, also known as EDSIV), is a life-threatening dominantly inherited disorder caused by mutations in the gene for type III procollagen (COL3A1). Affected individuals are at risk for arterial, bowel and uterine rupture. There is no effective treatment to prevent the symptom associated with vascular EDS. 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 vascular EDS. To establish a therapeutic strategy for dominant inherited diseases, we examined the feasibility of two approaches. In the first experiment, we tested RNAi mediated inhibition of the mutant allele. Approximately one third of the known mutations of vascular EDS occur at 5' donor splice sites. Therefore, if aberrantly spliced mRNA could be selectively destroyed by RNAi, it might be possible to reduce the mutant COL3A1 level and increase the relative concentration of normal triple helix. We synthesized several small interfering RNA (siRNA) molecules targeting the extra intron 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 % by transient transfection of one of the siRNA molecules. In the second experiment, we attempted to increase the concentration of normal COL3A1 mRNA, since half-dose of COL3A1 may not be sufficient to prevent vascular EDS 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 were observed. The concentration of normal COL3A1 mRNA was increased up to nearly normal level in control cells. These results strongly suggest that the combination of RNAi mediated mutant allele specific gene silencing and transcriptional activation of a normal allele is a useful strategy for phenotypic correction of dominant inherited diseases.