Most of modern gene discovery methods based on DNA microarrays are inherently biased toward genes with the polyA tail at the 3 end. However, the current estimates of gene transcripts are four to five times higher than the number of genes. Alternative splicing may account for some of this diversity. A growing number of examples, including apoptosis, beta-thalassemia, familial dysautonomia, FGF signaling, and spinal muscular atrophy, demonstrate that RNA isoforms have considerable biological effect. Thus, characterization of the RNA isoforms may soon become an integral part of the target validation process.
In one recent example, the correction of a splicing event gives hope to children suffering from spinal muscular atrophy (SMA). Homozygous mutation obliterates function of the survival motor neuron gene, SMN1, leading to degeneration of motor neurons of the spinal cord, causing atrophy of muscles and eventual death. Due to aberrant splicing, the backup gene, SMN2, produces only 10% of the full-length transcripts.
Treatment with valproic acid and some other histone deacetylase inhibitor drugs, is able to correct splicing and increase the amount of full-length SMN2 transcripts by several fold. The multicenter clinical trial is currently under way to assess safety and efficacy of valproic acid treatment for children with SMA.
ExonHit Therapeutics (www.exonhit.com) utilizes a database of publicly available sequences to identify alternatively spliced forms. Using this information, the company generates a series of specific exon body and junction probes and organizes them on the SpliceArrays.
By comparing RNA from several samples, the SpliceArrays are able to monitor expression of splice variants, including skipping of individual exons. The SpliceArrays are arranged by gene families (GPCR, ion channels, nuclear receptors, etc.) and contain genes that are known to produce alternatively splice transcripts, as well as genes that do not splice.
The dogma of one gene producing only one protein has been replaced by one gene producing many transcripts or proteins. Alternative splicing could result in transcripts with stop codons. These transcripts were thought to be quickly degraded. It now seems that that this degradation process is a secondary event, and many of the alternative transcripts can participate in protein translation, impacting various signaling pathways, says Richard Einstein, Ph.D., vp of research. There is little data relating to the expression patterns of splice variants. We only now can begin to catalog gene-specific splicing events and how many proteins actually result from them.
The SpliceArrays are not only able to identify alternative gene products but also provide information about differential expression and regulation of the isoforms under various conditions, their involvement in pathways, and their significance in pathology, adds Dr. Einstein.
In collaboration with Allergan (www.allergan.com), ExonHit was able to identify altered signaling pathways related to neuropathic pain. A new orally active small molecule is now being developed and has already shown a good profile in in vivo preclinical models.