June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Codon optimized RPGR leads to improved stability and rescue with AAV8 gene therapy in X-linked retinitis pigmentosa
Author Affiliations & Notes
  • M Dominik Fischer
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
    Centre for Ophthalmology, University of Tubingen, Tubingen, Germany
  • Michelle E. McClements
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Cristina Martinez-Fernandez de la Camar
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Julia-Sophia Bellingrath
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
    Centre for Ophthalmology, University of Tubingen, Tubingen, Germany
  • Daniyar Dauletbekov
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
    Centre for Ophthalmology, University of Tubingen, Tubingen, Germany
  • Simon C. Ramsden
    Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre, Manchester, United Kingdom
  • Doron G. Hickey
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Alun R Barnard
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
  • Robert E MacLaren
    Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom
    Oxford Eye Hospital, Oxford University Hospitals NHS Trust, The John Radcliffe Hospital, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   M Dominik Fischer, Isis Innovation Ltd. (P), NightStaRx Ltd. (C), NightStaRx Ltd. (F); Michelle McClements, None; Cristina Martinez-Fernandez de la Camar, None; Julia-Sophia Bellingrath, None; Daniyar Dauletbekov, None; Simon Ramsden, None; Doron Hickey, None; Alun Barnard, NightStaRx Ltd. (C); Robert MacLaren, Isis Innovation Ltd. (P), NightStaRx Ltd. (C), NightStaRx Ltd. (F)
  • Footnotes
    Support  UK Medical Research Council clinical research training fellowship (HMRXDS0).
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1573. doi:
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      M Dominik Fischer, Michelle E. McClements, Cristina Martinez-Fernandez de la Camar, Julia-Sophia Bellingrath, Daniyar Dauletbekov, Simon C. Ramsden, Doron G. Hickey, Alun R Barnard, Robert E MacLaren; Codon optimized RPGR leads to improved stability and rescue with AAV8 gene therapy in X-linked retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1573.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Mutations in the gene RPGRORF15 cause 70% of X-linked retinitis pigmentosa (XLRP). Sequence instability of RPGRORF15 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 RPGRORF15 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 (coRPGRORF15) was tested in vivo in two relevant animal models (Rpgr-/y and C57BL/6JRd9/Boc) and in C57BL6/J wild-type mice. Electroretinography, scanning laser ophthalmoscopy and immunohistochemistry were used to assess safety and efficacy of coRPGRORF15 based gene therapy.

Results : The human coRPGRORF15 CDS shows superior sequence stability and expression levels in vitro. The glutamylation pattern in the RPGR protein derived from coRPGRORF15 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/6JRd9/Boc) and shows good safety in C57BL6/J wild-type mice.

Conclusions : Optimizing the CDS of human coRPGRORF15 overcomes the inherent sequence instability of therapeutic transgenes developed for XLRP gene therapy. While maintaining post-translational characteristics and increasing expression levels, coRPGRORF15 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.

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