Gene augmentation for adRP using rhodopsin genomic loci DNA nanoparticles.

Abstract

Purpose : Gene therapy for autosomal dominant retinitis pigmentosa (adRP) is challenged by the dominant inheritance condition. We have begun to use intact genomic DNA with its full complement of endogenous regulatory elements for gene augmentation. In this study, we investigated the possibility of whether the delivery of a single full-length of rhodopsin gene can overcome the dominant negative effects of the mutated rhodopsin in a P23H knock-in heterozygous mouse model (P23H^+/-).

Methods : We compacted linearized full-length human and mouse rhodopsin DNA into CK30PEG-TAT nanoparticles (NPs) (gRHO-NP and gRho-NP) following the established protocol and subretinally delivered them to the animal at 0.3 μL (2μg)/eye at postnatal day 3 (P3), respectively. We assayed the animal at post-injection 3 months (PI-3m) and PI-5m to evaluate the rhodopsin protein expression and structural and functional improvements by western blot (n=4-6), immunohistochemistry (n=4-6), electroretinography (ERG) (n=8-12), and small RNA sequencing (sRNA-seq) (n=4-6).

Results : At PI-3m, our results showed that P23H^+/- mice in both gRHO and gRho NPs treated expressed at ~20% of WT rhodopsin levels compared to that of 5-10% in age-matched untreated or saline treated mice. Outer nuclear layer (ONL) exhibited 4-7 rows of nuclei in NPs treated compared to 3-5 rows in untreated or saline treated mice. Rod-response ERG (0.01 cd s/m²) were ~45% of WT levels in NPs treated compared to that of 30% in untreated or saline treated controls. At PI-5m, we found that the rhodopsin expression in untreated or saline treated mice were barely detectable. In contrast, the NPs treated mice resulted in 5-15% levels of the WT rhodopsin. ONL decreased to 1-3 rows in NPs treated mice compared to 0-1 row of nuclei in untreated or saline treated mice. Rod-response ERG reduced to ~25% of WT levels compared to that of ~0-5% in untreated or saline treated mice. Significant differences were observed comparing the NPs treated with untreated or saline treated mice at each time point (p<0.05). To study post-transcriptional regulation and the expression patterns after the injection, sRNA-seq was performed. More insight into biological response and whole transcriptome analysis in expression level is currently underway.

Conclusions : Our results indicated that delivering an intact genomic locus as a transgene may have a greater chance of success for the treatment of adRP caused by P23H mutation.

This is a 2020 ARVO Annual Meeting abstract.