Generation of splice switching oligonucleotides targeting the Cockayne syndrome group B gene product in order to change the diseased cell state

Cockayne syndrome (CS) is a severe disorder with no effective treatment. The Cockayne syndrome group B (CSB) gene is one gene responsible for CS and also causes UV sensitive syndrome (UVSS), a disorder that causes mild symptoms. How the CSB gene determines a patient’s fate is unknown, but one intriguing point is that in UVSS patient cell, there are nonsense mutations in both alleles at the same position in each upstream region of the PiggyBac transposable element derived 3 (PGBD3) inserted region. In contrast, in CS patient cells, there is at least one allele with several mutations downstream of the PGBD3 inserted region, or there are homozygous mutations in exon 1. Here, we designed and synthesized 24 splice switching oligonucleotides (SSOs) to skip exon 3 in CSB mRNA. Use of these SSOs induced a frame shift in order to generate an alternative stop codon at the upstream region of the PGBD3 invasion site. As a result, a reduction of mitochondrial membrane potential following H2O2 treatment in CS cell was recovered. It was demonstrated that up-regulation of several gene expression brought about by SSOs are related to mitochondrial dysfunction in CS cells. In this study, we investigated whether it was possible to get Cockayne syndrome (CS) cells to mimic UV sensitive syndrome (UVSS) cell by using splice switching oligonucleotides (SSOs). Thus, we needed to assess whether the states of SSO-transfected cells were close to CS or UVSS cells. One of the way to assess that, a microarray analysis was performed to define the differences in gene expression states between CS and UVSS. Gene expression states of each patient cell lines were compared, WI-28 VA13 sub 2 RA (normal) vs UVS1KO (UVSS), CS1AN (AN) vs UVS1KO (UVSS) and CS1BE (BE) vs UVS1KO (UVSS) (n=4).