Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with cGMP-grade AAV vectors.

Abstract

Purpose : The purpose of this study is to characterize a novel CRISPR-Cas9-generated rat model of Pde6b-associated retinal degeneration and test the ability of clinical-grade AAV vectors (serotypes 1 and 5) to deliver PDE6B to rat photoreceptor cells and mitigate disease progression.

Methods : Pde6b-null rats were generated on a Sprague-Dawley background via CRISPR-Cas9 genome editing (Cyogen Biosciences). Small guide RNA pairs and in vitro transcribed Cas9 mRNA were co-injected into fertilized eggs for generation of animals. The genotypes of founder animals and subsequent matings were verified via PCR and sequencing analysis. Animals were evaluated at post-natal days 14 (P14), 30, 60 and 90 via optical coherence tomography (OCT), electroretinography (ERG), and immunohistochemistry with histological quantification of outer nuclear layer (ONL) cell density and thickness at each time point. For gene augmentation experiments, Pde6b-null animals received a single subretinal injection (3ml) of either AAV2/1-PDE6B or AAV2/5-PDE6B (total dose of 2x10⁹ vg) into one eye. The fellow eye was used as a vehicle-injected control. A cohort of wild-type animals were also treated with each vector to confirm delivery and lack of overexpression toxicity. ONL measurements were compared in a masked fashion to determine efficacy of gene augmentation.

Results : Compared to wild-type and heterozygous animals, Pde6b-null animals displayed early onset by P14 and rapid photoreceptor degeneration that resulted in blindness (i.e., non-recordable ERG) by P30 and near complete loss of photoreceptors by P60. Delivery of AAV2/1-PDE6B or AAV2/5-PDE6B to wild-type rats showed that AAV2/1-PDE6B drives more full-length human PDE6B protein than AAV2/5-PDE6B. No evidence of overexpression toxicity was detected for either vector. In Pde6b-null rats, eyes that received AAV2/1-PDE6B displayed a delay in photoreceptor cell loss compared to buffer-treated controls and eyes treated with AAV2/5-PDE6B.

Conclusions : These preclinical data will be used to guide development of a gene therapy vector for treatment of PDE6B-associated retinitis pigmentosa. In addition to its utility for evaluating the efficacy of gene augmentation vectors, rapid retinal degeneration resulting in near complete photoreceptor loss by 2 months of age makes the Pde6b-null rat ideal for evaluating autologous stem cell-mediated photoreceptor replacement.

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