Characterization of a Novel Mak-deficient Rat Model of Retinal Degeneration and Phenotype Rescue Using Adeno-Associated Virus-Based Gene Therapy

Abstract

Purpose : Mutations in male germ cell associated kinase (MAK) are known to cause autosomal recessive retinitis pigmentosa in humans, but animal models of disease are critically lacking. Here, we report the generation of a novel Mak-deficient rat, characterize the retinal degeneration using in vivo imaging and histology, and demonstrate phenotype rescue using adeno-associated virus serotype 5 (AAV5)-based gene therapy.

Methods : A novel Mak-deficient rat model was created on a Sprague Dawley background using CRISPR-based genome editing technology. Heterozygous and homozygous Mak-deficient rats were evaluated at 1, 3, and 6 months of age (N≥3 per time point) using fundus photography, image-guided optical coherence tomography, and electroretinography. OCT-based retinal thickness measurements were recorded, and average thicknesses in mutant and heterozygote animals compared by unpaired T-tests. Animals were then sacrificed, and eyes enucleated and fixed for immunohistochemical analysis with cell specific markers. One-month-old Mak-deficient rats (N=4) were treated with subretinal injection of AAV (serotype 5) vector containing the wild-type retinal MAK gene (DTVRF-AAV5-MAK) or storage buffer control in the fellow eye and sacrificed at two months post-injection for analysis.

Results : By 1-month of age, Mak-deficient rats had moderate retinal thinning compared to heterozygote animals (average total retinal thickness 154 µm vs. 236 µm respectively, P<0.001), with predominance of loss in the outer nuclear layer (average thickness 28 µm vs. 81 µm, P<0.001). By 3-months of age, the outer nuclear layer (ONL) and ellipsoid zone were no longer visible on OCT (total retinal thickness 112 µm versus 206 µm, P<0.001), and the ERG signal was nonrecordable. By 6 months of age, there was complete loss of the ONL on both OCT and histology. Rats treated with DTVRF-AAV5-MAK had preservation of the ONL in the region of the subretinal bleb relative to buffer-injected fellow eyes and to uninjected, age-matched controls.

Conclusions : Mak-deficient rats demonstrate progressive photoreceptor degeneration that can be mitigated by AAV-mediated MAK gene replacement therapy. This new animal model provides a potentially powerful tool to elucidate molecular mechanisms of MAK-associated retinal degeneration and to develop effective gene and cell replacement therapies.

This is a 2020 ARVO Annual Meeting abstract.