Codon optimized RPGR leads to improved stability and rescue with AAV8 gene therapy in X-linked retinitis pigmentosa

Abstract

Purpose : Mutations in the gene RPGR^ORF15 cause 70% of X-linked retinitis pigmentosa (XLRP). Sequence instability of RPGR^ORF15 can not only lead to disease but also to unpredictable recombination errors during viral vector cloning. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein.

Methods : Codons of the human RPGR^ORF15 coding sequence (CDS) were optimized and tested for expression efficiency and sequence stability in vitro using immunohistochemistry, flow cytometry, and western blotting. Recombinant RPGR was characterized by western, mass spectrometry and glutamylation assays. Codon optimized RPGR (coRPGR^ORF15) was tested in vivo in two relevant animal models (Rpgr^-/y and C57BL/6J^Rd9/Boc) and in C57BL6/J wild-type mice. Electroretinography, scanning laser ophthalmoscopy and immunohistochemistry were used to assess safety and efficacy of coRPGR^ORF15 based gene therapy.

Results : The human coRPGR^ORF15 CDS shows superior sequence stability and expression levels in vitro. The glutamylation pattern in the RPGR protein derived from coRPGR^ORF15 CDS is indistinguishable from the wild-type variant, implying that codon-optimization does not significantly alter post-translational modification. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr^-/y and C57BL/6J^Rd9/Boc) and shows good safety in C57BL6/J wild-type mice.

Conclusions : Optimizing the CDS of human coRPGR^ORF15 overcomes the inherent sequence instability of therapeutic transgenes developed for XLRP gene therapy. While maintaining post-translational characteristics and increasing expression levels, coRPGR^ORF15 based gene therapy shows excellent safety in C57BL6/J wild-type mice and efficacy in two relevant animal models of human XLRP disease. This work provides the basis for clinical trial development to treat patients with XLRP due to RPGR mutations.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.