Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing

The use of antisense 2′-deoxyoligonucleotides as agents for specific downregulation of gene expression is now well established (1). Most commonly, the oligonucleotides base-pair with an mRNA target and mediate its destruction by RNase H, an enzyme that destroys the RNA in a DNA/RNA duplex. Numerous clinical trials evaluating the use of downregulating antisense oligonucleotides for the treatment of cancer and other diseases are now underway (2). The oligonucleotide drug Vitravene, the first of such drugs to be approved, is used to treat cytomegalovirus retinitis. More recently, antisense compounds are being developed for manipulation of alternative splicing patterns. In this way, the ratio of different splice variants can be altered, and the function of a gene changed. Considering that there are approximately 30,000 human genes and that up to 60% are alternatively spliced, this new antisense approach may have far-reaching implications in the treatment of a variety of diseases. Oligonucleotides can be used to silence mutations that cause aberrant splicing, thus restoring correct splicing and function of the defective gene. This is important since close to 50% of genetic disorders are caused by mutations that cause defects in premRNA splicing. Thus, targeting splicing with antisense oligonucleotides significantly extends the clinical potential of these compounds. The requirements for oligonucleotides that shift splicing are different from those for oligonucleotides used to achieve downregulation. Specifically, they must not activate RNase H, which would destroy the pre-mRNA target before it could be spliced. They must also be able to effectively compete with splicing factors for access to target pre-mRNAs that reside within the nuclei of cells. Several types of modified synthetic oligonucleotides fit these criteria. For example, oligonucleotides with modifications to the 2′ position, such as 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE) and 2′O-aminopropyl, are RNase H inactive and generally have higher nuclease resistance and affinity for target sequences than their 2′-deoxy counterparts. Oligonucleotides with backbones based on morpholino, peptide nucleic acid (PNA), phosphoramidate and methylphosphonate derivatives of nucleotides have similar characteristics (2). Modifications to the bases, which increase the affinity of oligonucleotides for their target, may facilitate favorable antisense activity in the context of splicing (2). The above modifications represent the second or third generation of compounds superior to the initially extensively studied phosphorothioate oligodeoxynucleotides, which frequently exhibited nonspecific, non-antisense effects (1).