Abstract
Purpose :
Rhodopsin (RHO) mutations account about 25~30% of adRP. Although a single P23H mutation in the RHO gene is known to be the most common cause of adRP in USA, more than 150 mutations have been identified. Group I intron-based trans-spicing ribozyme enables to reprogram target RNA into gene of interest through RNA replacement. Here, we developed a mutation-independent therapeutic strategy based on specific trans-splicing ribozyme that can replace, and thus edit mutant RHO RNA with functional RHO RNA using AAV delivery system.
Methods :
Target site of RHO RNA was first identified by in vitro mapping. To improve the trans-splicing specificity and efficacy, we optimized RHO-targeting ribozyme through modification of ribozyme structure. To verify in vivo function, AAV vector encoding the optimal ribozyme was constructed and delivered by subretinal injection into the eye of human P23H knock-in mice. After administration, scotopic ERG was measured on dark-adapted mice and retinas were dissected and processed for H&E staining, RT-PCR, and qPCR.
Results :
Sequencing analysis of trans-splicing sites through mapping revealed that the most efficient target site occurred at the 5’ UTR of RHO RNA. We modified and optimized the structure of the ribozyme targeting the most accessible site of target RHO RNA. We observed that various mutant RHO RNAs were successfully targeted and replaced with WT RHO RNA by the optimized ribozyme. When assessed the electrical response function of eye in human P23H knock-in mice model, the b-wave amplitudes significantly increased in eyes injected by AAV encoding the ribozyme as compared to those of control-injected eyes. Molecular and cellular analysis of retina tissue showed that P23H RNA is replaced with WT RHO with high efficacy and fidelity and ONL appears significantly thicker in the ribozyme-treated eyes.
Conclusions :
Various mutant RHO RNAs were trans-spliced with high fidelity and restored to WT RHO by RHO-targeting ribozyme in cells. The ribozyme effectively prevents the degeneration of photoreceptor cells and preserve their electrical response function in P23H knock-in mice through specifically targeting and trans-splicing of target RHO RNA. These results suggest that RNA replacement based on RHO-targeting ribozyme could be potent and mutation-independent therapeutic strategy for RHO-adRP.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.