title: Knockdown of wild-type mouse rhodopsin using an AAV vectored ribozyme as part of an RNA replacement approach.
abstract: OBJECTIVE
  To develop a hammerhead ribozymes (Rz) that might be exploited in a "digest and replace" gene therapy strategy for autosomal dominant retinitis pigmentosa (ADRP) caused by mutations in the gene for rhodopsin (RHO).
  
  
  METHODS
  A ribozyme (Rz397) was designed to hybridize with an accessible region in rhodopsin mRNA. It was tested in vitro to determine the kinetics of cleavage of a target oligonucleotide. Following transfection of cultured cells, reduction of rhodopsin mRNA in response to Rz397 was measured RT-PCR. The gene for the ribozyme (Rz397) was inserted in an adeno-associated virus (AAV2) vector and packaged in AAV2 capsids. The virus was injected subretinally in the eyes of C57BL/6J (RHO+/+) and rhodopsin knockout hemizygous (RHO+/-) mice at postnatal days 6 (P6) and 30 (P30). Mice were analyzed by full-field electroretinography (ERG). The reduction of opsin protein was measured by western blot analysis and visualized by immunocytochemistry. Reduction of rhodopsin mRNA was assessed using in situ hybridization. Morphometric microscopy of fluorescent antibody-antigen complexes and autoradiography of retinas were used to quantify levels of rhodopsin protein and mRNA, respectively.
  
  
  RESULTS
  Transient co-transfection of HEK 293 cells with a wild-type rhodopsin cDNA and Rz397 resulted in an approximately 60% reduction of RHO mRNA one day after transfection. RHO+/- -mice injected with AAV2-Rz397 at P6 showed a 50% reduction in b-wave amplitudes in injected eyes relative to saline injected contralateral eyes. However, injection of RHO+/- -animals at one month and of RHO+/+-animals at either age had no impact on ERG. Nevertheless, we detected an 80% reduction of opsin protein in ribozyme-injected eyes of hemizygous mice (by western blot) and a 50% reduction in opsin content in RHO+/+ mice (by morphometry). These reductions were confirmed by in situ hybridization.
  
  
  CONCLUSIONS
  AAV2-Rz397 led to significant (greater than or equal to 50%) reduction of rhodopsin mRNA and protein in mice. It affected ERG amplitudes only when injected in hemizygous RHO knockout pups. This RNA inhibitor may prove useful in treating animal models of ADRP as part of an RNA replacement approach.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA. mari653@ufl.edu
authors.forename: M S
authors.initials: MS
authors.lastname: Gorbatyuk
authors.forename: J J
authors.initials: JJ
authors.lastname: Pang
authors.forename: J
authors.initials: J
authors.lastname: Thomas
authors.forename: William W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: Alfred S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D004596:Electroretinography; D015316:Genetic Therapy; D005822:Genetic Vectors; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism; D017299:Rod Opsins / Q000378:metabolism; D014162:Transfection
chemical_list: D016337:RNA, Catalytic; D012333:RNA, Messenger; D017299:Rod Opsins; C427204:hammerhead ribozyme; D012243:Rhodopsin