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Gene Liau, Kollu Nageswara Rao, Victor V Bartsevich, Jeff Smith, Derek Jantz, Matthew Hirsch; Allele-specific gene editing for the treatment of autosomal dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4473.
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Targeted gene knockout is an emerging approach for the treatment of autosomal-dominant disorders such as P23H RHO mediated autosomal dominant retinitis pigmentosa (adRP), a common form of inherited blindness. A single nucleotide distinguishes P23H RHO from the WT copy which collectively present in a heterozygous manner. Previous works have suggested that down-regulation of the mutant allele in presence of the WT copy prevents retinal degeneration. Therefore, a 930nt I-CreI based designer homing endonuclease (HE) to specifically knockout the mutant RHO allele was engineered and validated in a self-complementary AAV vector. The specificity of this HE for P23H RHO was demonstrated in vitro. Preclinical evaluations in a human P23H RHO adRP mouse model were undertaken to determine if this approach demonstrates clinical potential for preventing vision loss in adRP patients.
A proprietary ARCUS platform was employed to generate a HE that can specifically target the human RHO allele carrying the P23H mutation. The targeted 22 nucleotide sequence differs from the wild type allele only by a single base pair. Specificity of the nuclease was first evaluated in CHO cell lines engineered to contain a single strand annealing GFP reporter that harbors either the WT or P23H RHO sequence. Gene editing efficiency was evaluated by flow cytometry and immunoblotting. To evaluate HE activity in vivo, a scAAV5 vector harboring P23H RHO HE cDNA under control of a photoreceptor specific promoter was administered to the human P23H RHO adRP mouse model via sub-retinal injection. scAAV5-GRK-HE cleavage efficiency of the mutant RHO allele was calculated using sequencing and by digital PCR. In vivo retinal function was evaluated by electroretinography and the subcellular localization of rhodopsin in photoreceptors by histology.
We have generated a HE that is highly specific for the mutant P23H RHO allele. Sub-retinal delivery of scAAV5-GRK-HE decreases the amount of rhodopsin in outer nuclear layer in transduced photoreceptors when compared to non-transduced cells.
This is the first study to demonstrate that a single chain HE can efficiently discriminate a one nucleotide difference and specifically target the P23H RHO allele. This result coupled with decreased rhodopsin mislocalization in an adRP model, foreshadows possible use of scAAV HE-based gene editing of P23H RHO to treat adRP patients.
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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