Abstract
Purpose :
Aniridia is a congenital panocular disease caused by mutations leading to haploinsufficiency of the transcription factor Paired Box Protein 6 (PAX6). Small-activating RNAs (saRNA) have been used to increase expression of genes in a variety of different contexts. Here we hypothesised that saRNAs could be used to increase the levels of the wild-type PAX6 transcript and rescue the phenotype of PAX6+/- cells.
Methods :
A panel of bioinformatically designed saRNAs were designed to target different regions of the PAX6 promoter. These were then tested in three human cell lines of clinical relevance: human Telomerase-immortalised Corneal Epithelial cells (hTCEpi), human Telomerase-immortalised Limbal Epithelial Stem cells, (L-TSC) and an L-TSC PAX6+/- cell line with a CRISPR/Cas9 introduced premature termination codon mutation. After saRNA treatment the RNA and protein levels of PAX6 were quantified by 2-step RT-qPCR and Western Blot respectively. For the top saRNA candidates the expression levels of genes that are downstream targets of PAX6 were quantified and the ability of saRNAs to rescue cell behaviour phenotypes of cell detachment and migration on the PAX6+/- cells were assessed.
Results :
In hTCEpi, most of saRNA showed a trend towards upregulation of PAX6 transcript levels, with three reaching a statistically significant 2-fold or greater increase (n=3, p<0.05). One of these candidate saRNAs displayed significant upregulation (2.0±0.5 fold change, n=3, p<0.05) across all three (hTCEpi, L-TSC and L-TSC PAX6+/-) lines . Steady-state whole cell extract PAX6 protein levels did not precisely match the transcript abundance data. However, in the PAX6+/- L-TSC line that was developed to resemble the haploinsufficiency phenotype, the gene expression of four downstream targets of PAX6 was rescued, and phenotypic correction of aberrant cell adhesion and motility behaviour was achieved through saRNA treatment.
Conclusions :
Our results support the hypothesis that increasing PAX6 transcript levels with saRNAs can rescue aspects of phenotype of PAX6+/- cells. We were able to identify at least one saRNA that shows promise for future investigation towards a possible saRNA therapy for aniridia.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.