Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th–40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3′ pre–trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors. Mol Cancer Ther; 10(2); 233–41. ©2011 AACR.