Maturation of synaptic ribbons and synaptogenesis occur after CaBP4 gene augmentation therapy

Abstract

Purpose : Widespread immaturity of photoreceptor synaptic ribbons has been described resulting from several genetic mutations that involve calcium signaling in the first retinal synapse. We examined dogs, and retinal samples from dogs affected by a CaBP4 mutation with and without gene augmentation therapy to determine whether introduction of functional CaBP4 could allow for completion of synaptic ribbon maturation and formation of normal synapses.

Methods : Samples were collected from eyes of 4 control, 2 untreated CaBP4^-/-, and 7 CaBP4^-/- dogs that had undergone subretinal AAV-mediated gene augmentation therapy. Dogs were evaluated for retinal structure and visual function using optical coherence tomography (OCT), electroretinography (ERG), visual behavior testing, immunohistochemistry (IHC) and transmission electron microscopy (TEM). For animals treated with gene therapy IHC and TEM were compared between treated and untreated retinal regions.

Results : Young CaBP4^-/- dogs had a thin outer plexiform layer (OPL) with immature synaptic ribbons, rare synaptic triads and stretched bipolar cell pedicles with severely reduced levels of GPR179. OPL thickness was 8.2±0.15 µm in unaffected vs 2.9±0.2 µm in affected dogs and ribbon length was shorter in affected animals at 227±14 nm when compared to WT at 642±27 nm. The ERG b-wave was absent and they showed reduced visual performance, which was more severe in scotopic conditions. Retinal degeneration was progressive and initially involved only the outer retina. Treatment with gene therapy resulted in thickening of the OPL, recovery of GPR179 immunolabeling, increased expression of PKCa and maturation of ribbons. Ribbons were significantly shorter in untreated retinal regions of affected animals 245±10 nm (n=171) when compared to treated regions 604±15 nm. The OPL thickness had increased thickness in treated regions (6.8±0.2 µm in treated vs 1.8±0.5 µm in untreated regions). Retinal degeneration was halted in treated retinal regions, the ERG b-wave was recovered and visual performance improved.

Conclusions : Restoration of calcium signaling in CaBP4 deficient dogs by gene augmentation therapy leads to maturation of synaptic ribbons, expansion of the outer plexiform layer, and reformation of synaptic machinery showing that even in mature animals there is remarkable plasticity in the first retinal synapse.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.