Inhibition of collagenase type I expression by psoralen antisense oligonucleotides in dermal fibroblasts

Type I collagenase plays an important role in both tumor metastasis and the remodeling of connective tissue in normal human skin, during wound healing, for example, and may participate in the pathophysiology of some dermatological diseases such as skin cancer and a chronic blistering disease, recessive dystrophic epidermolysis bullosa. In an effort specifically to inhibit collagenase expression, we have designed phosphorothioate antisense oligonucleotides, linked at the 5' ends with photoreactive 4'-(hydroxyethoxymethyl)-4,5',8-trimethyl-psoralen (HMT), and directed them against the 5' end of the collagenase mRNA. Two antisense-HMT molecules targeting a region overlapping the initiation codon were compared. Only one contained the HMT moiety targeting a 5'TpA on its complementary sense strand, and we observed greater than 50-fold improvement on the cross-linking of this antisense oligonucleotide to its target sequence after ultraviolet A (UVA) irradiation. Likewise, sequence complementary to the 5'TpA target was also required to demonstrate specific inhibition of in vitro translation of collagenase mRNA. Tissue culture experiments, conducted by incubation of collagenase-specific antisense-HMT oligonucleotides with fibroblasts in monolayer or in 3-dimensional dermal equivalents, showed lowered collagenase levels 24 h after UVA irradiation as compared to controls. Initial screening of antisense oligomers for specific hybridization and photo-cross-linking is a useful step in the design of antisense oligonucleotides, and allowed us to design an HMT-linked antisense phosphorothioate oligonucleotide that specifically inhibits the expression of fibroblastic collagenase.