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Liang Li, Qizhao Wang, Haoliang Huang, Yang Sun, Jeffrey L Goldberg, Yang Hu; Neuroprotection of Retinal Ganglion Cells by AAV2-gamma-Synuclein Promoter-Mediated CRISPR/Cas9 Gene Editing. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2253. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma, the leading cause of irreversible blindness, is characterized by progressive death of retinal ganglion cells (RGCs) and optic nerve (ON) degeneration. There is no effective neuroprotective treatment to prevent RGC/ON degeneration, but an increasing number of genes involved in RGC intrinsic signaling has been found to be important for RGC/ON survival and regeneration. The challenge is how to modulate these promising neural repair targets safely and efficiently in vivo. We decided to establish an adeno-associated virus (AAV)-mediated RGC-specific gene therapy strategy by combining an RGC-specific promoter with CRISPR/Cas9 gene editing.
We used the anatomical and technical advantages of mouse RGC/ON model system and AAV-mediated spatially and temporally controlled genetic manipulation to perform low throughput screening of 14 promoters to determine their specificity and potency in mouse RGCs in vivo. Unilateral ON crush severed as a severe ON injury model for testing whether CRISPR/Cas9-mediated knockdown of CHOP and SARM1, two pro-degeneration genes in RGCs, would provide efficient neuroprotection. We used PERG and OCT for functional and structural analysis of RGC/ON loss in living mice followed by definitive histological quantification of surviving RGC somata and axons post-mortem. Each treatment group contained 8-12 mice.
We found that mouse γ-synuclein (mSncg) promoter drives potent, sustained transgene expression in RGCs specifically. We also observed, for the first time in mouse, that CRISPR/Cas9 driven by AAV2-mSncg promoter significantly knocks down CHOP and SARM1 in RGCs and preserves the morphology and function of acutely injured RGC somata and axons in vivo.
This proof of concept study demonstrates that gene therapy that uses AAV to combine RGC-specific mSncg promoter and CRISPR/Cas9 gene editing to directly modulate endogenous degenerative genes holds great potential in treating devastating optic neuropathies, including glaucoma.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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