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M. Natkunarajah, A.J. Smith, Y. Duran, K. Balaggan, B. Pawlyk, A. Wright, R. Ali; AAV Mediated Gene Replacement Therapy in the RPGR Knockout Mouse– A Model of X–Linked Retinitis Pigmentosa . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5224.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mutations in RPGR–ORF15 account for the majority of cases of XLRP. The gene encodes the default RPGR protein, and a splice variant known as RPGR/ORF15. Both are located in the connecting cilia of photoreceptors, where they appear to be involved in the trafficking of rhodopsin and/or other phototransduction proteins between the inner and outer segment of the photoreceptor. The RPGR knockout mouse ( RPGR–/–) has been developed as a model of XLRP, and has allowed us to investigate the potential of somatic default RPGR/ORF15 gene replacement. Methods: Four RPGR expression constructs were cloned and packaged into AAV2 delivery vectors. Two constructs contained the full–length murine RPGR cDNA (exon 1–19); CBA–default RPGR and CMV–default RPGR, and two contained splice variants; CMV–ORF14/15 and CMV–ORF15. These were injected into mice (postnatal age 4 weeks) using a subretinal technique in the right eye, over two time points one week apart. The left eye was uninjected and used as an internal control. ERGs were performed prior to injection and then every month thereafter. Immunohistochemistry was carried out on retinal sections of sacrificed mice to check for construct expression at a variety of time points ( 2 months –7 months post injection). Results: We found expression of all injected constructs in the RPGR knockout mouse. Immunostaining using antibodies to RPGR showed correct localisation of all RPGR variants to the connecting cilia of photoreceptors. ERG analysis at the latest time point (11 months for the CBA–default RPGR) shows a trend towards slower photoreceptor degeneration in the treated eyes. Conclusions: We have shown correct transgene product localisation to the connecting cilia from both default RPGR constructs and two splice variants ORF14/15 and ORF15. The RPGR knockout mouse shows an inherently slow natural rate of photoreceptor degeneration, but preliminary ERG data in the treated eyes shows a trend towards retarding this process, and suggest that functional rescue of the mouse model for XLRP may be achieved.
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