Allele-specific Homing Endonuclease (HE) Gene Editing Induces Rod Function in a P23H Transgenic Swine Model of Retinitis Pigmentosa

Abstract

Purpose : Autosomal dominant forms of retinitis pigmentosa (adRP) present as prime candidates for gene editing in heterozygous patients. If the dominant mutant allele can be specifically targeted, the remaining functional allele should maintain or restore functional vision. We previously implemented an allele-specific HE approach, targeting the human P23H rhodopsin mutation in a transgenic swine model of adRP (TgP23H hRHO). This study we utilize a modified HE, replicate our previous results and establish a minimal effective dose for this HE in a large cohort of TgP23H and WT littermates (n = 22 and 18).

Methods : We treated one eye of TgP23H and WT piglets with a subretinal injections of a 930 nt I-CreI based HE, designed to target the P23H hRHO allele. The HE was packaged in a self-complementary AAV vector (scAAV5) and we injected ~50 ul between postnatal day (P) 3 and 7. Each eye received one of 4 viral titers: 2x10⁹, 6x10⁹, 2x10¹⁰, and 6x10¹⁰ vg. To approximate the extent of AAV transfection across the retina, scAAV-GFP was coinjected at a 3:1 ratio (scAAV-HE:scAAV-GFP). Retinal function was assessed using ffERG prior to injection and at regular intervals through 24 weeks post injection. Retinal health and morphology were monitored using spectral domain Optical Coherence Tomography (OCT), fundus imaging and ocular clinical evaluation.

Results : Dark-adapted, untreated Tg retinas have no scotopic b-wave ERG response to the dimmest flash (0.001 cd/m²). Every HE-treated Tg retina at the two highest viral titers induced a significant rod isolated ffERG b-wave response, which did not decline over time (X amplitude: ~30-40 mV, n=7 and 9). The two lower titers induced a b-wave response in fewer treated eyes. Photopic ffERG responses (20 cd/m² background; ≥0.1 cd/m²) were similar between treated and control retinas. Correlations of morphological and OCT analyses are underway. Treated TgP23H retinas tolerate all viral titers without morphological changes or immune responses. Treated WT retinas show some inflammation at the highest viral titer.

Conclusions : Treatment with scAAV-HE, induces scoptopic function in TgP23H retinas and the minimum effective dose is between 6x10⁹ and 2x10¹⁰. As this HE targets the human rhodopsin mutation, our results indicate that the approach could prevent rod photoreceptor degeneration in human RP patients.

This is a 2020 ARVO Annual Meeting abstract.