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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)
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.
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).
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.
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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