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B. S. Pawlyk, X. Liu, O. V. Bulgakov, M. A. Sandberg, S. Khani, A. F. Wright, A. J. Smith, R. R. Ali, E. L. Berson, T. Li; Replacement Gene Therapy in a Mouse Model of X-Linked Retinitis Pigmentosa Lacking RPGR. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5356. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The retinitis pigmentosa GTPase regulator (RPGR), localized in the photoreceptor connecting cilium, is essential for the maintenance and survival of rod and cone photoreceptors. Mutations in the gene encoding RPGR account for > 70% of all cases of X-linked RP. We evaluated whether replacement gene therapy with RPGR could restore photoreceptor structure and function in a mouse model of X-linked RP lacking RPGR.
Cohorts of RPGR knockout (RPGR-/-) from 1 to 21 months of age were studied. Mice were given a subretinal injection in one eye of an AAV2/5 vector in which an abbreviated form of murine RPGR ORF15 cDNA was placed under the transcriptional control of a human rhodopsin kinase (hRK) promoter. Fellow eyes received a GFP vector. Mice were evaluated from 2 weeks to 14 months post-injection (up to 25 months of age) by electroretinography (ERG), immunofluorescence, light microscopy, and immunoblotting analysis for evidence of RPGR protein expression, subcellular localization, and photoreceptor rescue.
In both young and aged RPGR-/- mice, RPGR protein expression was detected by immunofluorescence as early as 2 weeks post-injection and was correctly localized to the photoreceptor connecting cilium. By immunoblotting analysis, RPGR was of the anticipated size (~ 190 kD) and at 2-3x the expression level of endogenous RPGR in wildtype mice. There were more cones with better preserved outer segments in the treated eyes than in control eyes. Rod and cone ERG amplitudes were higher in treated eyes. Significant rod photoreceptor loss was still observed in the treated eyes at older ages.
Vector mediated RPGR gene delivery led to robust expression of RPGR at the photoreceptor connecting cilium and to a partial preservation of photoreceptor structure and function in RPGR-/- mice. Fine-tuning of the transgene expression to match the endogenous RPGR expression level is an important future direction to optimize RPGR gene therapy.
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