Abstract
X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRORF15). Despite successful RPGRORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGRORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. 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. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. 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/6JRd9/Boc) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations.
Highlights
Than Wild-Type retinitis pigmentosa GTPase regulator (RPGR) The nucleotide sequence of RPGRORF15 coding sequence (CDS) was refined by reducing the frequency of low-abundance codons from 10% in the wild-type RPGRORF15 to 1%, while increasing major codon usage from 32% to 56% (Figure S1)
The fact that it is still a goal that has not translated into a clinical trial is mainly due to the fact that RPGR is a complex gene with high propensity for mutational changes.[12,25]
Even with regulatory approval for a phase I trial, production of clinical grade AAV for RPGR gene therapy is associated with significant risks because the RPGR transgene might spontaneously mutate in the course of vector production according to good manufacturing practice (GMP) guidelines
Summary
1854 Molecular Therapy Vol 25 No 8 August 2017 a 2017 The Authors. Www.moleculartherapy.org to improve efficiency of transduction without the need to increase the MOI. This is crucial as therapeutic transgene expression levels are achieved in the target tissue with minimal required number of viral vector particles, thereby reducing the risk of, e.g., capsid-related immunogenicity.[15]. AAV8 was chosen as viral vector because of its transduction characteristics of primate photoreceptors following subretinal injection and its proven clinical safety profile.[18,19]
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