HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Liu, J. Articles by Hauswirth, W. W. Search for Related Content PubMed PubMed Citation Articles by Liu, J. Articles by Hauswirth, W. W.

Ribozyme Knockdown of the -Subunit of Rod cGMP Phosphodiesterase Alters the ERG and Retinal Morphology in Wild-Type Mice

¹From the Departments of Ophthalmology and ³Molecular Genetics and Microbiology, and the ⁴Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, Florida.

PURPOSE. To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the -subunit of the rod cGMP phosphodiesterase (PDE) mRNA in the retina of wild-type mice.

METHODS. Two hammerhead ribozymes, HRz35 and HRz42, were designed to target the PDE gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDE gene were monitored by reverse transcription–polymerase chain reaction (RT-PCR) and Western immunoblot assay.

RESULTS. The ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K_cat of 0.48 minute^–1 and a K_m of 980 nM, and HRz42 showed a K_cat of 0.17 minute^–1 and a K_m of 971 nM. Both ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and full-length mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a- and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDE mRNA and protein paralleled the ERG results.

CONCLUSIONS. Ribozyme-mediated somatic knockdown of wild-type PDE mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent.

This article has been cited by other articles:

				V. Justilien, J.-J. Pang, K. Renganathan, X. Zhan, J. W. Crabb, S. R. Kim, J. R. Sparrow, W. W. Hauswirth, and A. S. Lewin SOD2 Knockdown Mouse Model of Early AMD Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4407 - 4420. [Abstract] [Full Text] [PDF]

				P. J. DeMarco Jr, G. L. Yarbrough, C. W. Yee, G. Y. McLean, B. T. Sagdullaev, S. L. Ball, and M. A. McCall Stimulation via a Subretinally Placed Prosthetic Elicits Central Activity and Induces a Trophic Effect on Visual Responses Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 916 - 926. [Abstract] [Full Text] [PDF]