Redirection of Fas splicing as a cancer therapy

DESCRIPTION (provided by applicant): The goal of this proposal is to develop specific RNA-based Lassos to redirect splicing of medically important receptors and demonstrate efficacy in cell culture and animal models. RNA Lassos are novel, proprietary antisense molecules that, instead of cleaving a complementary RNA target, form a topological linkage with it and thereby block gene expression. The ability to redirect splicing could allow one to convert any membrane bound receptor into a soluble decoy. To establish proof-of-principle for this approach we have chosen Fas, a key receptor in apoptosis that has been extensively studied. The Fas pre-mRNA is known to be alternatively spliced. One of the main splice variants skips exon 6, resulting in a soluble receptor that cannot transduce the killing signal and has the added benefit of protecting neighboring cells from death by neutralizing Fas ligand. We propose to target RNA Lassos to the intron 5/exon 6 splice junction region to cause exon 6 skipping. We will test the extent of splicing redirection in a lymphocyte tissue culture model by RT-PCR and immuno-detection. The ability of Fas Lassos to protect tissue culture cells from Fas ligand-induced death will be evaluated by apoptosis assays. In Phase II, we will evaluate the ability of Fas Lassos to protect primary T cells from cancer cells expressing Fas ligand in tissue culture as well as a mouse model.