title: RPE65 gene delivery restores isomerohydrolase activity and prevents early cone loss in Rpe65-/- mice.
abstract: OBJECTIVE
  Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65-/-) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65-/- mice.
  
  
  METHODS
  Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65-/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina.
  
  
  RESULTS
  High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65-/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65-/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin alpha subunit in Rpe65-/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65-/- mice.
  
  
  CONCLUSIONS
  RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65-/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.
authors.affiliation: Department of Cell Biology, The University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Gennadiy
authors.initials: G
authors.lastname: Moiseyev
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Jian-Xing
authors.initials: JX
authors.lastname: Ma
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins; D002851:Chromatography, High Pressure Liquid; D003623:Dark Adaptation; D005136:Eye Proteins / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D015316:Genetic Therapy; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D012162:Retinal Degeneration / Q000201:enzymology / Q000517:prevention & control*; D012172:Retinaldehyde / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000378:metabolism; D015293:Transducin / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism; D019745:cis-trans-Isomerases / Q000096:biosynthesis* / Q000235:genetics; D000099178:Retinoid Isomerohydrolase
chemical_list: D002352:Carrier Proteins; D005136:Eye Proteins; D012172:Retinaldehyde; D017299:Rod Opsins; D015293:Transducin; D014801:Vitamin A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase