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Shaomei Wang, Benjamin Bakondi, Bin Lu, Augustus Mercado, Yueqin zhou, Sergey Girman; Long term safety and efficacy of gene editing by CRISPR/Cas9 in autosomal dominant mutation model for RP. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2925.
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© ARVO (1962-2015); The Authors (2016-present)
There are diverse genetic causes of Retinitis pigmentosa (RP) including up to 200 different mutations with dominant, recessive and sex-linked inheritance patterns. Gene replacement therapy using adeno-associated virus (AAV) has been applied successfully to an autosomal recessive form of arRP known as Leber Congenital Amaurosis (LCA) resulting from mutations in the RPE65 gene. As proof of principle, others and we have demonstrated the feasibility of using CRISPR/Cas9 in vivoas gene therapy to rescue photoreceptors in rodent models for autosomal dominant RP (adRP). Here we investigate long-term safety and efficacy after AAV8-CRISPR/Cas9 gene editing in a P23H rat model for adRP.
A Staphylococcus aureus Cas9 (SaCas9) and its single guide RNA expression cassette into a single AAV vector (AAV2/8) (AAV2/8-SaCas9/gRNA) (Vector Biolabs) was injected into the subretinal space of P23H rats at early- and late-stages of retinal degeneration. Visual function was tested at several times for this long-term and safety study (one year). At the end of experiments, retinal cryostat sections were examined for general retinal lamination, photoreceptor rescue, retinal specific markers and immune cell markers. DNA was extracted for specific On-and off–target next generation sequencing.
At the early time after AAV2/8-SaCas9/gRNA injection, there was no rescued effect as revealed by electroretinography (ERG) over the untreated fellow eye. However, with time as degeneration progresses, vector treated eyes showed significant better ERG value over control, and this effect lasts for long term (one year). Visual function deterioration with time was also clearly evidence. Histological examination shows there are four layers of rescued photoreceptors in vector treated eye covering large area, while in untreated fellow eyes, there is no photoreceptor remaining. Next generation sequencing of On-and Off-target effect is ongoing.
This long-term study demonstrated that long-term efficacy after a single subretinal delivery of AAV2/8-SaCas9/gRNA into the P23H rat model for adRP was possible. If safety can be achieved, this approach can be translated into human clinics for treating adRP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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