The ultimate result of the proposed approach will be treatment for rheumatoid arthritis and possibly other diseases caused by the hyperactivity of TNF-a. The approach is to manipulate the alternative splicing pathways of the TNF-a receptors (TNFR) pre- mRNA through the application of a technology established in the laboratory of Ryszard Kole and licensed by Ercole Biotech. In this technology, chemically modified antisense oligonucleotides alter the expression of splice-variants either by inducing exon skipping, exon inclusion, intron inclusion or a combination of the three. This project will focus on the TNF-a receptor 2 (TNFR2). First, we propose to induce skipping of both exon 7 and exon 8 simultaneously by the oligonucleotides targeted to these exons, and thus upregulate a soluble natural splice variant of the receptor ?7/8. The ? 7/8 splice variant is secreted into the bloodstream where it binds TNF-a with high affinity and interferes with the propagation of the TNF-a signal by the cellular, full-length, membrane bound TNFR. Second, we propose, a simpler treatment, to induce skipping of exon 7 only, which will induce an isoform (? 7) predicted to behave similarly to ? 7/8 but which has not yet been characterized. This two-pronged approach will allow us to explore both the feasibility of simultaneous two exon skipping by oligonucleotides and the functionality of the ? 7 isoform of TNFR2, to select the best approach for the anti-TNF-a treatment. The oligonucleotides used in this work will be locked nucleic acids (LNA). Initial work will be carried out in cultured mouse and human liver hepatocytes, as an in vitro model of splicing in the liver. The in vivo experiments will be carried out in mice. The specific aims of this project are: 1) To induce, in vitro, in murine and human hepatocytes, skipping of exons 7 and 8 or exon 7 alone in TNFR2 pre-mRNA, thus upregulating TNFR2 ? 7/8 or ? 7 proteins. 2) To induce, in vivo, in mice, skipping of exons 7 and 8 or exon 7 alone in TNFR2 pre-mRNA, thus up-regulating TNFR2 ? 7/8 or ? 7 proteins. 3) To determine the extent of anti-TNF-a activity following up-regulation of ? 7 and ? 7/8 in vivo in mice Successful completion of the research proposed in this application will lead to development of novel drugs for rheumatoid arthritis and other inflammatory diseases. These drugs will reduce the inflammatory effects of TNF-alpha and in that they will be similar to an existing drug Enbrel(r). However, we anticipate that Ercole drugs, because of their novel design and mechanism of action, will be more effective, longer lasting and less costly than Enbrel(r) and may benefit patients who never see significant improvement with current treatments
