A Novel Fully Humanized RHO adRP Mouse Model

Abstract

Purpose: : To develop a novel mouse model that expresses only human (not mouse) transgenic rod opsin (RHO) mRNAs in mouse rod photoreceptors.

Methods: : The normal human RHOtransgene (NHR-E, Olsson et al., 1992) and the P347S human RHOtransgene (C1, Li et al., 1996) were independently bred to homozygosity onto a mouse RHOknockout background (Humphries et al, 1997; Lem et al., 1999). Genotyping and test breedings of mice containing human RHOtransgenes and mouse RHOknockouts were performed to identify animals of both sexes that were homozygous for the human transgenes and the knockout. In vivo retinal imaging was used to assess the state of retina health. Retinal structure was assessed histologically (outer nuclear layer (ONL) thickness) and ultrastructurally, while function was assessed by electroretinography (ERG).

Results: : A single copy of the P347S human RHOtransgene on the mouse WT RHObackground promoted a slow, progressive retinal degeneration whereas the homozygous C1 line has early onset diffuse panretinal degeneration. The homozygous WT human RHO model exhibited a normal fundus appearance, normal histological features and a normal scotopic ERG pattern (Naka Rushton analysis). The recently achieved fully "humanized" mouse adRP model is being characterized.

Conclusions: : We have developed two novel mouse models on a mouse RHO knockout background: one that expresses two doses of normal human RHO mRNA and another that expresses two doses of P347S mutant human RHO mRNA. The first model can be used to assess the toxicity of therapeutic gene silencing agents (e.g. ribozymes, shRNA). Cross-breeding these two models will result in all neonates expressing single doses of human normal and mutant RHO mRNAs without any mouse RHO mRNA (important for preclinical therapeutic efficacy testing). This adRP model will be used to test gene silencing rescue strategies when the target mRNAs are only precise copies of those that would exist in a human clinical trial. This is a first-of-kind animal model system that mimics human retinal degenerative disease.

Keywords: retinal degenerations: hereditary • transgenics/knock-outs • gene transfer/gene therapy