The pathological hallmark of scleroderma is progressive fibrosis of the skin and various internal organs caused by a deposition of excessive amount of collagen as a result of elevated production of newly synthesized collagen. To develop molecular approaches applicable for treatment of fibrotic diseases, we propose to inhibit the type I collagen synthesis using a novel strategy of gene silencing by RNA interference (RNAi). Although a double-stranded RNA (dsRNA) has specifically silenced its cognate gene in many organisms, its application to mammalian cells has been difficult due to the interferon response. With the recent discovery of small interfering RNA (siRNA), various forms of small dsRNA are now emerging as a novel class of therapeutics, which cause degradation of the target mRNA in a sequence-specific manner, thus suppressing gene expression in mammalian cells. In preliminary studies, we have shown that siRNA for the COL1A1 caused over 50% of inhibition in the Type I collagen synthesis in fibroblasts. This inhibition was specific since unrelated siRNA did not cause any decrease in the collagen synthesis. We will further evaluate several different forms of dsRNA with various lengths and sequences for their efficacy and specificity in the collagen gene silencing. Inhibition of collagen synthesis will be quantitated at mRNA and protein levels for both a1(I) and a2(I) polypeptides of the type I collagen. Once the most effective form of dsRNA is identified, we will evaluate its in vivo efficacy. RNAi gene silencing in mammalian cells is expected to be transient and to inhibit only a de novo synthesis of protein. Thus, we will utilize a skin wound-healing model that showed a transient but dramatic increase in the type I collagen synthesis. Skin is also an ideal organ for testing and developing such novel strategy, because it is accessible to administer therapeutics locally and to monitor the treated and control sites simultaneously. Several in vivo delivery methods will be developed and wounds will analyzed by molecular and histological methods. The amount of collagen in the wound area will be quantitated at mRNA and protein levels. The importance of such feasibility testing is that information gained from the skin can be extended to the treatment of scleroderma, a frequently fatal systemic disorder affecting not only skin, but also lungs, kidneys, and other internal organs, for which no effective treatment is available.
| Cesarone, Gregory; Garofalo, Cecilia; Abrams, Marc T et al. (2006) RNAi-mediated silencing of insulin receptor substrate 1 (IRS-1) enhances tamoxifen-induced cell death in MCF-7 breast cancer cells. J Cell Biochem 98:440-50 |
