Using pseudo gene therapy to assess cone responses during rod degeneration and following genetic rescue

Abstract

Purpose : Retinitis Pigmentosa 45 (RP45) is caused by mutations in the β subunit of the rod photoreceptor specific cGMP-gated channel (CNGB1). In this disease, rods then cones die and the retina undergoes significant remodeling. RP45 is amenable to gene therapy, as has been demonstrated during early treatment (see Koch 2012, Michalakis 2014, and Petersen-Jones 2018). However, the time of genetic rescue has been implicated as critical for retinal gene therapy success (Gardiner et al, 2019). Two outstanding questions in gene therapy of RP are: 1) how does cone-dependent visual signaling depend on the amount of rod loss; and 2) how is cone-dependent visual signaling impacted by genetic rescue of rods from degeneration?

Methods : To answer these questions, we utilize a Cngb1 functional knockout (fKO), in which a Neolox cassette prevents expression of Cngb1; administration of tamoxifen induces cre expression, removing the Neolox cassette to yield normal Cngb1 expression. This “pseudo gene therapy” allows investigation of the neurophysiological consequences of the rescue. The approach enables a best-case scenario for gene therapy that is not subject to experimental variability associated with viral capsid, dose, or injection.
We assessed cone-driven visual responses in retinal ganglion cells (RGCs) of treated and untreated Cngb1-fKO and control mice using large-scale multi-electrode arrays. At one month intervals, we measured spatiotemporal receptive field (RF) structure, contrast response functions and information transmission across diverse RGC types. The amount of rod and cone degeneration was quantified immunohistochemically and correlated with RGC function.

Results : Preliminary results suggest that receptive field structure was relatively insensitive to rod loss, but cone-mediated response precision and information transmission sharply declined with rod loss. Early rescue at <30% photoreceptor loss halted retinal degeneration and preserved cone function, but later rescue did not stop cell death, and cone-mediated visual responses continued to deteriorate.

Conclusions : These results indicate that information transmission may be a more sensitive assay than RF structure to quantify RGC physiology. Furthermore, early therapy is substantially more likely to preserve useful cone-mediated vision, and highlights the importance that timely intervention is critical for therapeutic success.

This is a 2020 ARVO Annual Meeting abstract.