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

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.

SUBMITTER: Gorbatyuk M

PROVIDER: S-EPMC1778459 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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

Gorbatyuk M M Justilien V V Liu J J Hauswirth W W WW Lewin A S AS

Experimental eye research 20061101 1

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 vi ...[more]

PMID: 17083931

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Project description:To characterize the optical coherence tomography (OCT) appearances of photoreceptor degeneration in the rhodopsin P23H transgenic rat (line 2) in relation to the histological, ultrastructural, and electroretinography (ERG) findings.Homozygous rhodopsin P23H transgenic albino rats (line 2, very-slow degeneration model) were employed. Using OCT (Micron IV®; Phoenix Research Labs, Pleasanton, CA, USA), the natural course of photoreceptor degeneration was recorded from postnatal day (P) 15 to P 287. The OCT images were qualitatively observed by comparing them to histological and ultrastructural findings at P 62 and P 169. In addition, each retinal layer was quantitatively analyzed longitudinally during degeneration, compared it to that observed in wild type Sprague-Dawley (SD) rats. The relationships between the ERG (full-field combined rod-cone response, 3.0 cds/m2 stimulation) findings and OCT images were also analyzed.In the qualitative study, the two layers presumably corresponding to the photoreceptor inner segment ellipsoid zone (EZ) and interdigitation zone (IZ) were identified in the P23H rat until PN day 32. However, the photoreceptor inner and outer segment (IS/OS) layer became diffusely hyperreflective on OCT after P 46, and the EZ and IZ zones could no longer be identified on OCT. In contrast, in the SD rats, the EZ and IZ were clearly distinguished until at least P 247. The ultrastructural study showed partial disarrangements of the photoreceptor outer segment discs in the P23H rats at P 62, although a light-microscopic histological study detected almost no abnormality in the outer segment. In the quantitative study, the outer retinal layer including the outer plexiform layer (OPL) and the outer nuclear layer (ONL) became significantly thinner in the P23H rats than in the SD rats after P 71. The thickness of the IS/OS layer was maintained in the P23H rats until P 130, and it became statistically thinner than in the SD rats at P 237. The longitudinal attenuation in the amplitude of the a- and b-waves of ERG was significantly correlated with the thickness of the combined OPL and ONL but not with that of the IS/OS layer.OCT showed the degenerated photoreceptor IS/OS layer in rhodopsin P23H transgenic rats (line 2) as a diffuse hyperreflective zone, even in the early stage, with the partially disarranged and destabilized OS discs recognizable by ultrastructural assessment but not by a histological study. The amplitude of the a- and b-waves mainly depends on the thickness of the OPL and ONL layer rather than the thickness of the photoreceptor IS/OS layer in P23H rats.

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

Publications

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

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