Generation and analysis of a transgenic mouse model with a modified human rhodopsin replacement gene with therapeutic potential.

Abstract

Abstract: : Purpose: To generate and analyse an animal model to study the efficacy of the gene replacement element of a therapeutic strategy for autosomal dominant retinitis pigmentosa adRP. Methods: A transgenic mouse was generated that carries a human rhodopsin gene under the control of the mouse rhodopsin promoter. This rhodopsin gene encodes an identical amino acid sequence to the wild type human rhodopsin gene but has a number of nucleotide differences. These differences are designed, using the degeneracy of the genetic code, to allow transcripts from this modified rhodopsin gene when transcribed to escape cleavage by ribozyme or siRNA molecules that target that particular site. The transgenic mice were bred onto a mouse rhodopsin knockout (rho –/–)background. These mice were analysed using electroretinography and histology, comparing retinal function and thickness to that of wild type mice. Results: The modified human rhodopsin gene rescues disease pathology in a rho–/– mouse. Suppression agents such as ribozymes and siRNA molecules that suppress a wild type human rhodopsin transcript at a target site should not suppress the replacement gene transcript. Notably, transcripts from the replacement gene were resistant to siRNA–based suppression in cell culture while wild type transcripts were suppressed efficiently. Conclusions: This model supports the idea of a mutation independent suppression and replacement gene therapeutic strategy for adRP, and the general strategy is also applicable to other dominantly inherited diseases. The suppression element of the strategy involves downregulation of gene expression in a mutation independent manner ensuring its application to patients with any mutation in a given gene. This work demonstrates that the replacement element of the strategy works well in an animal model.

Keywords: retinitis • gene transfer/gene therapy • transgenics/knock–outs