NANOBODY rescues adRP phenotype in a P23H adRP cell model, suggesting therapeutic potential

Abstract

Purpose : Treatment for autosomal dominant retinitis pigmentosa (adRP) by gene therapy is challenging, due to genetic heterogeneity. We used innovative NANOBODY® technology to test our hypothesis that nanobodies (Nbs), due to their nano-scale size, would efficiently stabilize P23H mutant RHO and restore rhodopsin homeostasis without cellular toxicity in an in vitro HEK293 cell model of RP. We also characterized the thermodynamics and crystal structure of the Nb-RHO complexes to assess the binding stability and identify the interface between the Nb and its target.

Methods : 29 Nbs were raised against native bovine rod outer segments (ROS). After confirming their binding affinity to native bovine ROS by gel shift assay, selected Nbs were expressed intracellularly in bovine wild type (WT)-RHO cells, or P23H-RHO HEK293 cells. Accumulation of mutant RHO by the Nbs was evaluated by Western blot and UV-visible spectroscopy, relative to controls with non-binding Nb. The stability of the antigen-antibody interactions was analyzed by spectroscopy at different temperatures and pH in light or dark conditions over the period of 11 days. Crystal structures of the Nb-RHO complexes were collected and analyzed along with the results from alanine-scanning mutagenesis in Nb-complementarity-determining regions.

Results : Among the 29 Nbs, 4 Nbs showed a gel shift, indicative of binding to native bovine ROS in a dose-dependent manner. Nb-RHO complexes maintained an equilibrium between ground and active states for 11 days after bleaching in the dark, at 4 ^oC or room temperature (RT). Under light conditions, the complexes were still stable at 4 ^oC, but gradually bleached and degraded at RT. While P23H-RHO levels were lower compared to WT-RHO by Western blot, intracellular co-expression of Nb in P23H cells significantly increased RHO protein levels (P<0.05), relative to the non-binding Nb control. Crystal structure and alanine-scanning results revealed the binding epitopes of Nb to RHO in its extracellular loop II and the N-terminal tail, explaining the action mechanism on mutant P23H stabilization.

Conclusions : Intracellular expression of Nbs rescues the mutant RHO phenotype in an in vitro HEK293 adRP model, suggesting therapeutic potential of the nanobody technology. Since there are few treatment options for RHO-adRP, NANOBODY technology might offer a new treatment option regardless of a patient’s genetic background.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.