Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme

M. Gorbatyuk, Verline Justilien, J. Liu, W. W. Hauswirth, A. S. Lewin

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

To develop an allele independent ribozyme for the treatment of autosomal dominant retinitis pigmentosa (ADRP) associated with mutations in the rhodopsin (RHO) gene, a ribozyme targeting dog, mouse, human but not rat rhodopsin (RHO) mRNA was designed and tested in vitro. Activity of this ribozyme was tested in tissue culture by co-transfection of HEK 293 cells with plasmids expressing opsin mRNA and ribozyme, followed by quantitative RT-PCR to evaluate the level of RHO mRNA. For experiments in vivo, Rz525 driven by the mouse opsin proximal promoter was inserted in plasmids with AAV 2 terminal repeats (TR) and packaged in AAV serotype 5 capsids. AAV-Rz525 was injected subretinally into the right eyes of P23H rat pups. Left eyes were injected with virus expressing GFP from the identical promoter. Animals were analyzed at 4, 8 and 12 weeks post-injection by full field scotopic electroretinography (ERG). After 12 weeks, animals were sacrificed and retinas were dissected, fixed and sectioned. Rz525 had high catalytic activity in vitro and led to a 50% reduction of RHO mRNA in cells. AAV-Rz525 injection into P23H transgenic rats led to significant preservation (about 50%) of scotopic ERG a- and b-wave amplitudes. Histological analysis showed an increased number of ONL nuclei in the central and superior retina of treated eyes relative to control eyes. RT-PCR analysis revealed 46% reduction of transgenic (mouse) RHO mRNA in right eyes relative to left eyes and no change in rat RHO mRNA. AAV5 delivery of Rz525 resulted in a partial rescue of the light response and structural preservation of photoreceptors in transgenic rats. This ribozyme may be a useful component of an RNA replacement gene therapy for ADRP.

Original languageEnglish (US)
Pages (from-to)44-52
Number of pages9
JournalExperimental Eye Research
Volume84
Issue number1
DOIs
StatePublished - Jan 1 2007
Externally publishedYes

Fingerprint

Catalytic RNA
Rhodopsin
Alleles
Messenger RNA
Transgenic Rats
Opsins
Electroretinography
Retinitis Pigmentosa
Retina
Plasmids
Polymerase Chain Reaction
Injections
Raphe Nuclei
Terminal Repeat Sequences
HEK293 Cells
Capsid
Genetic Therapy
Transgenic Mice
Transfection
Dogs

Keywords

  • adeno associated virus
  • autosomal dominant retinitis pigmentosa
  • gene therapy
  • rhodopsin
  • ribozyme

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme. / Gorbatyuk, M.; Justilien, Verline; Liu, J.; Hauswirth, W. W.; Lewin, A. S.

In: Experimental Eye Research, Vol. 84, No. 1, 01.01.2007, p. 44-52.

Research output: Contribution to journalArticle

Gorbatyuk, M. ; Justilien, Verline ; Liu, J. ; Hauswirth, W. W. ; Lewin, A. S. / Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme. In: Experimental Eye Research. 2007 ; Vol. 84, No. 1. pp. 44-52.
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