Cone Targeted AAV-mediated Gene Therapy Restores Cone Function in the Cngb3 Knockout Mouse, a Model of Human Achromatopsia 1

Abstract

Purpose: : Mutations in the gene encoding the beta-subunit of the cone cyclic nucleotide-gated channel (CNGB3) cause cone function loss in mammals including humans. We tested two AAV5-hCngb3 vectors with different cone targeting promoters to see if gene replacement therapy would result in restoration of cone function in the Cngb3 knockout mice, a model of human Achromatopsia 1 (ACHM1).

Methods: : Human CNGB3 cDNA in conjuction with cone-targeting promoter mCARpro or IRBP/GNAT2 was packaged into AAV serotype 5 (AAV5-mCARpro-hCngb3 or AAV5-IRBP/GNAT2-hCngb3 at10¹³ viral genome-containing particles /ml). At postnatal day 14, 1 μl of either vector was injected subretinally into one eye of groups of 20 Cngb3 knockout mice, respectively. The untreated, contralateral eyes served as controls. Dark- and light-adapted ERGs were recorded periodically from 6 weeks to 6 months after treatment. 6 months after injection, both treated and control eyes were harvested for histochemical studies.

Results: : At 6 weeks post-treatment both treated and untreated eyes of Cngb3 knockout mice showed normal rod-derived ERGs. In untreated control eyes, cone-derived ERG signals were nearly unrecordable. In both AAV5-mCAR-hCngb3 and AAV5-IRBP/GNAT2-hCngb3 treated eyes, restored light-adapted cone-derived ERG waveforms were first recorded 6 weeks after treatment and remained stable for at least 6 months. ERG amplitudes were about 2/3 of those of normal uninjected C57BL/6J mice. Immunohistochemistry showed human CNGB3 staining in the inner segments of many cones in treated eyes but not in cones from partner untreated eyes. Anti-M-cone or S-cone opsin staining also showed that S-opsins were preserved in treated eyes but not in untreated eyes of Cngb3 knockout mice.

Conclusions: : Both AAV5-mCAR-hCngb3 and AAV5-IRBP/GNAT2-hCngb3 restore cone function and prevent S-cone degeneration for at least 6 months in Cngb3 knockout mice, a model of ACHM1. However since studies in an accompanying abstract show that in addition to cones, mCARpro in AAV5 also expresses its transgene in the RPE while the IRBP/GNAT2 promoter is cone-exclusive, the latter may be preferable for future studies in humans.

Keywords: color vision • gene transfer/gene therapy • retinal degenerations: hereditary