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Guo-jie Ye, Jilin Liu, Ewa Budzynski, Peter J Sonnentag, T. Michael Nork, Nader Sheibani, Chunjuan Song, Mark Shearman, Jeffrey D Chulay; Product Development for Gene Therapy of XLRP: Evaluation of Promoters, AAV Capsids in Non-human Primates and Rational Design of RPGR ORF15 cDNA. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1180.
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© ARVO (1962-2015); The Authors (2016-present)
To select the preferred adeno-associated virus (AAV) capsid and promoter, and to construct a stable full-length retinitis pigmentosa GTPase regulator (RPGR) cDNA for AAV-based gene therapy of X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene
GRK1 and IRBP are two small photoreceptor specific promoters able to drive RPGR transgene expression in dogs with XLRP disease (Beltran et al, 2012). The ability of IRBP and GRK1 promotors, and AAV2tYF and AAV5 capsids, to target photoreceptors was evaluated in non-human primates (NHPs) by subretinal injection of AAV vectors containing a GFP expression cassette. Cynomolgus macaques receiving bilateral subretinal injection of 0.1 mL of AAV vector at a concentration of 5 x 1011 vg/mL or 2 x1012 vg/mL were followed for up to 12 weeks. GFP expression was evaluated by observation of fluorescence (in-life), and by qRT-PCR and immunohistochemistry (post-mortem). hRPGR cDNA was codon modified based on the cDNA sequence published by Alan Wright (variant C; NM_001034853). The codon modified hRPGR cDNA encodes a full length hRPGR protein whose amino acid sequence is 100% identical to the published sequence. Synthesized hRPGR cDNA was first cloned in pUC57 cloning vector and then in AAV plasmid for AAV production. Stability of hRPGR cDNA sequence was confirmed by DNA sequencing at each step of cloning and also in the AAV preparations
In NHPs, direct comparison between AAV2tYF-GRK1-GFP and AAV5-GRK1-GFP revealed that AAV2tYF-GRK1-GFP achieved an overall better GFP-reporter gene expression in photoreceptors than AAV5-GRK1-GFP at both dose levels. In contrast to the observation from published studies in dogs, minimal or no GFP expression was present in the eyes receiving the AAV5-IRBP-GFP vector. The redesigned full-length RPGR cDNA, cloned in either a conventional cloning vector or in an AAV plasmid, was stable during large scale plasmid production and AAV vector manufacturing.
The GRK1 promoter was significantly more efficient than IRBP promoter at directing expression in primate photoreceptors and AAV2tYF is a preferred capsid. A stable RPGR cDNA that encode full-length, native RPGR protein was obtained. These results should inform the design of an AAV vector for treatment of patients with XLRP.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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