The design and optimization of RNA trans-splicing molecules for skin cancer therapy.

[dystrophic epidermolysis bullosa]

Targeting tumor marker genes by RNA trans-splicing is a promising means to induce tumor cell-specific death . Using a screening system we designed RNA trans-splicing molecules (RTM ) specifically binding the pre-m RNA of SLCO 1 B 3 , a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC ). Specific trans-splicing , results in the fusion of the endogenous target mRNA of SLCO 1 B 3 and the coding sequence of the suicide gene , provided by the RTM . SLCO 1 B 3 -specific RTMs containing HSV-tk were analyzed regarding their trans-splicing potential in a heterologous context using a SLCO 1 B 3 expressing minigene (SLCO 1 B 3 -MG ). Expression of the chimeric SLCO 1 B 3 -tk was detected by semi-quantitative RT-PCR and Western blot analysis . Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene -mediated apoptosis in SLCO 1 B 3 -MG expressing cells . The lead RTM also showed its potential to facilitate a trans-splicing reaction into the endogenous SLCO 1 B 3 pre-m RNA in EB-SCC cells resulting in tk -mediated apoptosis . We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells .