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Maureen A McCall, Archana Jalligampala, gobinda Pangeni, Maha Jabbar, Henry J Kaplan, Wei Wang, Bhubanananda Sahu, Jon E Chatterton, Jeff Smith, Victor Bartsevich, Kristi Viles; Allele-specific Gene Editing Induces Rod Function in a Transgenic Swine Model of Autosomal Dominant Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4229. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The P23H mutation in the rhodopsin (RHO) gene represents the most common form of autosomal dominant retinitis pigmentosa (adRP) in the US (~10%). We used a human P23H (hP23H) transgenic pig model of adRP to evaluate an allele-specific knock out strategy of the hP23H mutant sequence and determined if its inactivation (targeted gene knockout) could be a therapeutic intervention for adRP.
A 930 nt I-CreI based homing endonuclease (HE) designed to knockout the hP23H RHO allele was packaged in a self-complementary AAV vector (scAAV). We evaluated the specificity of the HE for P23H RHO in a transgenic pig model expressing hP23H RHO (TgP23H RHO). scAAV-HE and scAAV-GFP (scAAV-HE/GFP) were coinjected subretinally (~40ul) into TgP23H RHO and wild-type (WT) littermates between postnatal days (P) 3 to 7. In ongoing experiments (50 eyes), we are screening a range of scAAV-HE/GFP titers, as well as controls at regular post injection intervals (≥14 weeks post injection (wpi)). Ocular exams, Optical Coherence Tomography (OCT) and fluorescence fundus imaging assess retinal health, bleb size and position and laminar structure. Full field ERGs assess retinal function.
Untreated TgP23H pigs have no scotopic b-wave at or after P3 (intensity = 0.001cd/m2). Titers between 6e9 and 2e10 vg (scAAV-HE/GFP (1:1)), produce only focal retinal damage around the retinotomy and minor inflammatory response inTgP23H eyes (n=9). At ~ 4 wpi, 7/9 scAAV-HE/GFP injected TgP23H eyes have a small, but significant scotopic ERG b-wave (~5-30μV) compared to baseline, and the b-wave amplitudes in the same scAAV-HE/GFP injected TgP23H eyes doubled at ~9wpi. OCT b-scans of scAAV-HE/GFP injected TgP23H eyes, indicate that outer retinal thickness remains similar to baseline (2 wpi). Fellow control (untreated, vehicle or scAAV-GFP) injected TgP23H eyes (n = 7, 2, 4) show no b-wave response and outer retinal thickness declines compared to 2wpi. A titer of 6e9 vg produces little change in WT retinal structure or function (n=3), although higher titers produce an inflammatory response (n=24).
In a Tg P23H pig model of human adRP, an AAV-930nt I-CreI based designer HE delivered into the subretinal space induces rod function, compared to controls. The results suggest that this scAAV HE-based gene editing of P23H RHO should be applicable to treat adRP patients with the same P23H mutation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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