Abstract
Purpose :
Multiple blinding disorders are associated with chromophore deficiency due to mutations blocking its production in the retinal pigmented epithelium (RPE). The cellular retinaldehyde binding protein CRALBP is expressed in the RPE and Müller cells and plays an important role in facilitating chromophore recycling. We generated mono- and super high molecular weight (SHMW)- nanoparticles of CRALBP and investigated their ability to deliver chromophore to the retina and restore photoreceptor function in chromophore-deficient RPE65 knockout mice.
Methods :
His-tagged recombinant WT and di-cysteine mutant CRALBP mutant with 20-fold increased photostability in vitro were isolated from E. Coli and loaded with 9-cis retinal. Isolated RPE65 KO retinas were incubated in nanoparticle solution in darkness for 4 hours. The function of rod photoreceptors was then evaluated by transretinal recordings. For in vivo experiments, 2 ml nanoparticle or control PBS solution was delivered to the eye by intravitreal injection. The mice were kept in darkness and the function of their rods was evaluated by electroretinography 1 day and 4 days post-injection.
Results :
We observed robust recovery of rod maximal response and sensitivity in RPE65 KO mouse retinas treated with mono WT or mutant CRALBP nanoparticles. The recovery was even better in retinas treated with SHMW nanoparticles. The recovery with WT and mutant CRALBP nanoparticles was comparable for both mono- and SHMW samples. We also observed robust recovery of RPE65 KO mice’s scotopic ERG responses in vivo with all four nanoparticle types. Notably, the recovery of rod responses persisted for up to 4 days post-injection. Finally, we evaluated by in vivo ERG recordings the rod recovery from a bright >90% bleach in nanoparticle-treated RPE65 KO and WT control mice. Surprisingly, despite the lack of a functional visual cycle, nanoparticle-treated RPE65 KO mice recovered their rod sensitivity significantly faster than WT mice, indicating storage of nanoparticle-bound excess 9-cis retinal.
Conclusions :
CRALBP nanoparticles can deliver efficiently 9-cis retinal to chromophore-deficient isolated retinas and intact eyes and promotes robust rod pigment regeneration both in the dark and following a complete bleach. These results demonstrate the potential of our approach for treating retinal disorders linked to chromophore deficiency.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.