Natural History of the CNGB3/NRL Double Knock-Out Mouse

Abstract

 

Purpose

 

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder with an incidence of 1:30,000. Cyclic nucleotide gated channel beta 3 (CNGB3) accounts for approximately 50% of this autosomal recessive disease. CNGB3 knock-out (KO) mice have been successfully used as an animal model for gene replacement therapy. However, the mouse retina lacks a cone-dominant macula and is comprised of less than 3% cones, compared to cone rich human macula, making clinical translation difficult for any cone-related retinal degeneration. To address this problem of rod-bias in the mouse, we generated a cone exclusive mouse model lacking CNGB3 by crossing CNGB3 KO mice with mice lacking Neural retina leucine zipper (NRL KO) and characterized the natural history out to four months postnatal.

 

Methods

 

CNGB3/NRL double KO (DKO) mice were generated by crossing NRL KO mice with CNGB3 KO lines. We followed the natural history of CNGB3/NRL DKO mice with photopic electroretinography (ERG) at 0.5, 1, 2, 3 and 4 months of age, respectively. Responses were elicited at four increasing light intensities (1.25 cds/m2, 5cds/m2, 10cds/m2 and 25cds/m2). For comparison, ERG measurements were also recorded from age-matched CNGB3 KO and NRL KO mice (data collection ongoing). Following ERG, one animal from each age group was sacrificed and their retinas collected for histological analysis.

 

Results

 

Photopic recordings at the highest flash intensity revealed that cone amplitudes of CNGB3 KO mice were ~50 µV at 16 days and remained low throughout the following 3 months. In contrast, ERG amplitudes of CNGB3/NRL DKO mice increased from ~50 µV at 16 days to 150 µV at 2 months and remained stable thereafter. There was a significant difference in the CNGB3 and CNGB3/NRL model at 1, 2, and 3 months (p<0.001). We are currently processing retinas from both CNGB3 KO and CNGB3/NRL DKO.

 

Conclusions

 

ERG data with CNGB3 KO mice confirmed previous findings that CNGB3 KO mice do not completely lose cone-mediated ERG function. Residual cone-mediated ERG was further increased when CNGB3 mice were crossed with NRL-/- mice, reaching to 160 µV at 3 month post-natal age. The CNGB3/NRL DKO mouse model may provide insight into the disease progression of ACHM in the cone rich human fovea and perhaps serve as a model for evaluating gene replacement based therapies.

 

 

Average photopic b-wave amplitudes of CNGB3 and CNGB3/NRL mice at 0.5, 1, 2 and 3 months of age (means±SE).

 

Keywords: 538 gene transfer/gene therapy • 539 genetics • 510 electroretinography: non-clinical