The X-linked RP3 gene codes for the ciliary protein RPGR and accounts for over 10% of inherited retinal degenerations. The critical RPGR-ORF15 splice variant contains a highly repetitive purine-rich linker region that renders it unstable and difficult to adapt for gene therapy. To test the hypothesis that the precise length of the linker region is not critical for function, we evaluated whether adeno-associated virus-mediated replacement gene therapy with a human ORF15 variant containing in-frame shortening of the linker region could reconstitute RPGR function in vivo. We delivered human RPGR-ORF15 replacement genes with deletion of most (314 codons, ‘short form’) or 1/3 (126 codons, ‘long form’) of the linker region to Rpgr null mice. Human RPGR-ORF15 expression was detected post treatment with both forms of ORF15 transgenes. However, only the long form correctly localized to the connecting cilia and led to significant functional and morphological rescue of rods and cones. Thus the highly repetitive region of RPGR is functionally important but that moderate shortening of its length, which confers the advantage of added stability, preserves its function. These findings provide a theoretical basis for optimizing replacement gene design in clinical trials for X-linked RP3.
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We thank Dr Jeng-Shin Lee at the Research Vector Core at Harvard Medical School for AAV vector packaging, and Drs Peter Colosi and Zhijian Wu at the National Eye Institute for helpful discussions. This work was supported by National Eye Institute grant EY10581, NEI core grant for Vision Research (5P30EY14104), the Foundation Fighting Blindness, the Foundation for Retina Research, the Massachusetts Lions Eye Research Fund and by grants from the European Union (AAVEYE), the UK Department of Health, National Institute of Health Research BMRC for Ophthalmology and UK Fight for Sight.
RRA and AJS are co-founders of Athena Vision Limited with a commercial interest in gene therapy for X-linked RP. The remaining authors declare no conflict of interest.
Note added in proof
While this manuscript was under review, the ORF15 cDNA used to treat the canine model of RPGR was found to have undergone multiple deletions and other types of changes, supporting the view that the original 'full-length' RPGR-ORF15 is prone to spontaneous mutations (Deng WT et al. Stability and safety of an AAV vector for treating RPGR-ORF15 X-linked retinitis pigmentosa. Hum Gene Ther 2015; 26: 593-602).
Supplementary Information accompanies this paper on Gene Therapy website
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Pawlyk, B., Bulgakov, O., Sun, X. et al. Photoreceptor rescue by an abbreviated human RPGR gene in a murine model of X-linked retinitis pigmentosa. Gene Ther 23, 196–204 (2016). https://doi.org/10.1038/gt.2015.93
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