Mouse gamma-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Wang, Q., Zhuang, P. S., Huang, H., Li, L., Liu, L., Webber, H. C., Dalal, R., Siew, L., Fligor, C. M., Chang, K. C., Nahmou, M., Kreymerman, A., Sun, Y., Meyer, J. S., Goldberg, J. L., Hu, Y.
2020
Abstract
Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno associated virus (AAV)-mediated gene transfer to perform a low throughput in vivo screening in both male and female mouse eyes and identified the mouse ?-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with CRISPR/Cas9 gene editing can knockdown pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.Significance Statement:Here we present an RGC-specific promoter, mouse ?-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and CRISPR/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.
View details for DOI 10.1523/JNEUROSCI.0102-20.2020
View details for PubMedID 32300046
Abstract
Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.
View details for DOI 10.3791/60409
View details for PubMedID 31789319
Abstract
Understanding the molecular mechanism of glaucoma and development of neuroprotectants are significantly hindered by the lack of a reliable animal model that accurately recapitulates human glaucoma. Here we sought to develop a mouse model for the secondary glaucoma that is often observed in humans after silicone oil (SO) blocks the pupil or migrates into the anterior chamber following vitreoretinal surgery. We observed significant intraocular pressure (IOP) elevation after intracameral injection of SO, and that SO removal allows IOP to return quickly to normal. This simple, inducible and reversible mouse ocular hypertension model shows dynamic changes of visual function that correlate with progressive RGC loss and axon degeneration. It may be applicable with only minor modifications to a range of animal species in which it will generate stable, robust IOP elevation and significant neurodegeneration that will facilitate selection of neuroprotectants and investigating the pathogenesis of ocular hypertension-induced glaucoma.
View details for PubMedID 31090540
