July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Mirtron gene therapy for the treatment of rhodopsin-related dominant retinitis pigmentosa
Author Affiliations & Notes
  • Harry Otway Orlans
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, ENGLAND, United Kingdom
    Ophthalmology, Imperial College Healthcare NHS Trust, London, United Kingdom
  • Michelle E McClements
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, ENGLAND, United Kingdom
  • Alun R Barnard
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, ENGLAND, United Kingdom
  • Cristina Martinez-Fernandez dela Camara
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, ENGLAND, United Kingdom
  • Robert E MacLaren
    Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, ENGLAND, United Kingdom
    Oxford Eye Hospital, Oxford, United Kingdom
  • Footnotes
    Commercial Relationships   Harry Orlans, Oxford University Innovation (P); Michelle McClements, Oxford University Innovation (P); Alun Barnard, Oxford University Innovation (P); Cristina Martinez-Fernandez dela Camara, None; Robert MacLaren, Oxford University Innovation (P)
  • Footnotes
    Support  MRC/Fight for Sight grant MR/N00101X/1
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5195. doi:
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      Harry Otway Orlans, Michelle E McClements, Alun R Barnard, Cristina Martinez-Fernandez dela Camara, Robert E MacLaren; Mirtron gene therapy for the treatment of rhodopsin-related dominant retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5195.

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

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Abstract

Purpose : RNA replacement gene therapy has shown promise for mutation-independent treatment of rhodopsin (RHO)-related autosomal dominant retinitis pigmentosa (ADRP). This strategy relies on a careful balance between gene suppression & augmentation. Mirtrons are atypical RNA interference effectors that are spliced from parent transcripts as introns. We have developed artificial mirtrons directed against RHO and packaged these within an adeno-associated virus (AAV)2/8Y733F vector together with a RHO coding sequence (CDS) rendered resistant to mirtrons by codon modification. Control of both mirtrons and transgene by a single RNA Pol-II human RHO promoter allows predictable matching between the two arms of the therapy as well as cell-specific expression of both so as to limit off-target effects.

Methods : Mirtron designs were validated for splicing efficiency when nested within the GFP CDS with accurate splicing determined by fluorescence following HEK293 cell transfection. Human/mouse RHO knockdown was quantified in vitro using the Dual Glo luciferase assay (Promega). The mirtron-RHO knockdown/replacement AAV was tested by subretinal injection in the RhoP23H/+ knock-in mouse model of ADRP. RNA replacement was quantified by RT-qPCR using cDNA derived from injected retinas and the effect of treatment on retinal structure and function determined by optical coherence tomography (OCT) and electroretinography (ERG).

Results : For individual mirtrons in vitro splicing efficiency ranged 0.3-100% whilst RHO knockdown ranged 0-83%. Placement of mirtrons in the 5’ untranslated region of rather than within the transgene increased both splicing (p<0.01) and potency (p<0.0001), and delivery of multiple mirtrons in tandem resulted in additive RHO-suppression (p<0.0001). RHO codon-modification conferred complete resistance to mirtron-mediated knockdown in vitro. When delivered by AAV subretinally in Rho-/- mice, this transgene rescued RHO protein expression and scotopic ERG function. Subretinal injection of mirtron-RHO AAV in the RhoP23H/+ model resulted in a 34.1±5.0% reduction in total retinal mouse Rho expression (p=0.0011) and slowed degeneration as measured by OCT (p=0.0184) and ERG.

Conclusions : This represents the first ever demonstration of use of artificial mirtrons as gene therapy tools in vivo. This novel technology could be adapted for the treatment of a range of dominant and polygenic diseases of the eye and elsewhere.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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