Abstract
Purpose :
Mirtrons are an atypical subclass of RNA interference effectors that are spliced from pre-mRNA transcripts as short introns. Mirtrons have been described as a promising therapeutic strategy, thanks to their capacity of knocking down native rhodopsin whilst also providing mirtron-resistant (codon optimised) rhodopsin (RHO). The overriding aim is to develop this strategy further, generating and testing new mirtrons designed specifically for targeting the human RHO sequence.
Methods :
Nine mirtrons candidates (M1-M9) against the human RHO sequence were designed and cloned into the 5’UTR upstream of an eGFP coding sequence. To determine the RHO targeting efficiency, a dual luciferase assay was performed. For which RHO was fused to Renilla coding sequence with firefly activity used as a transfection control. HEK293 cells were seeded into 96 well culture plates. After 24h, cells were co-transfected with mirtron-GFP and dual luciferase plasmids and after 48h Renilla and Firefly luminescence were measured. Mirtron RHO knockdown was calculated by the relative luciferase ratio of Renilla/Firefly and normalised to that recorded from positive control samples that received no RHO-targeting mirtron. The best-performing mirtrons were cloned in double-mirtron constructs by oligo insertion following PstI digestion.
Results :
Nine new mirtron sequences targeting human RHO were compared side-by-side in luciferase reporter assays against two previously published sequences. Successful targeting of RHO would be indicated by knockdown of Renilla activity, which was observed for 6 of the new 9 designs. Two new mirtron designs, M6 and M7, provided significantly improved knockdown. M6 and M7 were then selected for the double-mirtron strategy, for comparison against the published double-mirtron construct to identify if further improvements in RHO knockdown can be achieved.
Conclusions :
Mirtrons are an effective mode of gene silencing and comparison of a number of designs and combinations is critical to achieving optimal target knockdown. This strategy has great potential, and it can be broadly used for the treatment of any dominantly inherited disease.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.