A Mutation-Independent CRISPR/Cas9-based ‘Knockout and Replace’ Strategy to Treat Rhodopsin-Associated Autosomal Dominant Retinitis Pigmentosa

Abstract

Purpose : Rhodopsin-associated autosomal dominant retinitis pigmentosa (RHO-adRP) is an inherited retinal disease that results in blindness due to photoreceptor degeneration. Over 150 mutations in the RHO gene are known to cause RHO-adRP. Using a dual AAV system, Editas Medicine is exploring potential therapy with a highly efficient CRISPR/Cas9 to knockout aberrant endogenous rhodopsin and replace it with an exogenous functional rhodopsin (KO&R).

Methods : The specificity of a human and non-human primate (NHP) cross-reactive Cas9 guide was tested using human retina explants transduced with the KO&R. Pharmacokinetics and pharmacodynamics of the KO&R were assessed in humanized mRho^hRHO/+ mice. Efficacy was measured in NHPs by comparing knockout (KO) only and KO&R versions of the dual AAV system delivered via subretinal injection. A miniRHO promoter was used to drive Cas9 as well as RHO expression while restricting editing and replacement of RHO expression to the rod photoreceptors. On- and off-target editing was assessed using Next Generation Sequencing. Endogenous and replacement RHO levels were quantified using NanoString and tandem mass spectrometry. Morphological and functional readouts were assessed by histopathology and electroretinography.

Results : No off-target editing was observed in human retina explants after transduction with the KO&R. The KO&R achieved maximal levels approximately 6 weeks post-dose and remained stable for at least 13 weeks post-dose in mRho^hRHO/+ mice. The KO&R levels displayed a dose response reaching a plateau at a dose of 6E12 vg/ml. Studies in NHP demonstrated nearly 100% knockout of endogenous RHO, and replacement RHO produced over 30% of normal RHO protein. The KO&R-injected eyes showed restoration of RHO expression in the outer segments and retention of normal photoreceptor structure and function (ERG analysis) compared to the KO-injected eye.

Conclusions : Editas characterized and demonstrated high efficacy of a CRISPR/Cas9-based KO&R therapeutic strategy for RHO-adRP. The experimental therapy is mutation-agnostic and could be a potential one-time treatment to permanently suppress the toxic gain of function associated with mutated RHO. The efficacy of our KO&R gene editing therapy in NHP supports continued advancement of this approach toward treating patients.

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