101. Rational Design and Cloning of a Stable RPGR ORF15 cDNA Encoding the Full-Length Native RPGR Protein

Introduction: The retinitis pigmentosa GTPase regulator (RPGR) cDNA contains a long purine-rich repetitive sequence in ORF15 exon that is unstable during recombinant DNA manipulation. This complicates efforts to develop AAV-based vectors for gene therapy of X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene. We reasoned that the stability of RPGR-ORF15 cDNA could be significantly improved by rational design of the cDNA sequence through codon modification without changing the amino acid sequence. Methods: A human RPGR cDNA was designed based on Genbank reference mRNA sequence NM_001034853 that encodes hRPGR isoform C. The 3459 bp coding sequence was codon-optimized based on human codon usage and further modified to reduce tandem repeats, and adjust G/C content, where possible. The codon-modified cDNA (hRPGRco), which encodes a full-length hRPGR protein of 1152 amino acids that is 100% identical to the published sequence, was synthesized and cloned into conventional cloning vector pUC57 and sub-cloned into various vectors, including an AAV plasmid (used for AAV production by transfection) and an HSV shuttle plasmid (used to make an HSV helper for AAV production using AGTC's proprietary HSV-based AAV manufacturing system). Stability of the hRPGRco cDNA sequence was verified by DNA sequencing at multiple steps: after sub-cloning, large scale plasmid production, production of recombinant HSV helper virus clones that carry the hRPGRco expression cassette and production of AAV vector. Stability of plasmids carrying the hRPGRco expression cassette was also analyzed after serial passage of bacterial clones cultured at various temperatures and during multiple passages. Results: Plasmids carrying the redesigned hRPGRco cDNA were grown stably in bacteria at 37°C. The hRPGRco cDNA was 100% correct in AAVplasmids, in shuttle vectors used to produce HSV helpers, and in the rHSV helper virus passed multiple rounds in mammalian cells. The sequence was also confirmed 100% correct in AAV vectors produced by plasmid transfection for animal efficacy studies. Conclusions: A stable RPGR cDNA that encodes full-length, native RPGR protein was obtained and will allow large scale production of an AAV vector for treatment of patients with XLRP.