The maintenance of correct stage- and cell-specific alternative splicing patterns is a key feature of normal cell functioning. A large fraction of disease-associated mutations affect proper pre-mRNA splicing, and alternative splicing is frequently deregulated in tumors, where cancer-specific splice variants seem to directly contribute to multiple aspects of the aberrant phenotype. Furthermore, many genes that play a pivotal role in tumor development can potentially express multiple isoforms with often antagonistic functions. We focused on different prominent examples of antagonistic functions for our studies: Bcl-x, expressing pro- and anti-apoptotic isoforms (Bcl-xs/Bcl-xL), vascular endothelial growth factor (VEGF), leading to a pro- and anti-angiogenic family of proteins (VEGF/VEGFb) and STAT3, where alternative splicing results in a dominant negative regulator of transcription (STAT3-alfa/STAT3-beta).
Redirection of alternative splicing presents an attractive way to specifically treat and manage aberrant splicing in tumors. We developed a new generation of compounds termed ESSENCE (Exon-Specific Splicing Enhancement by small Chimeric Effectors), based on peptide:antisense chimeras to effectively and specifically re-direct endogenous alternative splicing events. A combinatorial approach by systematic modification of multiple parameters was adopted to optimize the compounds activity and the choice of target site(s), the length and orientation of the various moieties and the specific peptide sequence were optimized. Furthermore, the central question of efficient cell delivery and activity of the compounds in living cells was addressed by monitoring the splicing activity of transfectedminigene constructs, where expression of a reporter gene (EYFP or luciferase) depends on the desired splicing event.
The ability of our compounds to modulate different types of splicing events was tested using therapeutically relevant targets, Bcl-x, STAT3, VEGF and BRCA1, which recapitulate the most commonly observed alternative splicing events, alternative 3’ and 5’ and exon skipping.
By redirecting alternative splicing patterns of cancer-related genes, we verify a new class of chimeric compounds as a universal tool for splicing modulation not just to down-regulate but also to force the expression of variants with strong potential anti-tumoral properties.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA