Abstract
Abstract: :
Purpose: More than a hundred point mutations in the rhodopsin gene are known to cause autosomal dominant retinitis pigmentosa (ADRP), a progressive rod–cone dystrophy. In this study, we have made and analyzed a line of human P23H transgenic mice to determine the line’s usefulness as an in vivo model of ADRP. Methods: A single line of transgenic P23H rhodopsin mice was generated by pronuclear injection of the entire transcriptional unit of the human P23H rhodopsin gene (generous gift from T. Dryja). RT–PCR was performed on total retinal RNA to confirm the expression of the human P23H rhodopsin transgene. A breeding protocol was implemented to yield the transgene on each of three genetic backgrounds with respect to endogenous mouse rhodopsin, homozygous wild type, hemizygous, or null. Full field simultaneous electroretinography (ERG) was performed on dark–adapted mice to establish the rate of retinal degeneration for each of the three genetic backgrounds. Therapeutic ribozymes were cloned and packaged into recombinant AAV virus particles and are currently being tested in vivo. Results:The rate of retinal degeneration observed for transgenic human P23H rhodopsin mice was dependent on the amount of endogenous mouse rhodopsin. On the null background (–/–), electroretinography shows that both transgenic and non–transgenic mice have no rod–mediated ERG signal at 1 month of age. When the human P23H rhodopsin transgene was expressed on the wild type (+/+) mouse rhodopsin background, the rate of retinal degeneration was very slow, with only a 10–15 % reduction in the maximum dark–adapted b–wave amplitude at 5 ½ months of age. When the human P23H rhodopsin transgene was expressed on the hemizygous (+/–) mouse rhodopsin background there was a rapid retinal degeneration, with 80–90% loss of ERG at 3 months of age. Histological analysis of these retinas is in progress. Conclusions: Since rapid retinal degeneration occurs in this line on a hemizygous (+/–) background but not on a wild type (+/+) background, pathology is almost certainly attributable to expression of the P23H allele rather than to rhodopsin over expression. Preliminary results suggest that rAAV–delivered therapeutic hammerhead ribozymes delay the rate of retinal degeneration in human P23H transgenic mice.
Keywords: transgenics/knock–outs • gene transfer/gene therapy • electroretinography: non–clinical