Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Background: Degeneration of optic nerve (ON) axons and loss of retinal ganglion cells (RGCs) are the pathological hallmarks of Primary Open Angle Glaucoma (POAG). Elevation of intraocular pressure (IOP) due to dysfunction of trabecular meshwork (TM) is known to induce neurodegeneration. However, the early pathological events of glaucomatous neurodegeneration are poorly understood due to lack of robust and faithful mouse model that replicates all features of human POAG. Here, we report the generation and characterization of a novel Cre-inducible transgenic mouse model of myocilin (MYOC), the leading known genetic cause of POAG. Using this model, we further explore early pathological events of glaucomatous neurodegeneration due to chronic IOP elevation. Methods: We generated a Cre-inducible transgenic mouse model expressing DsRed-tagged Y437H mutant of human myocilin (Tg.CreMYOCY437H). A single intravitreal injection of helper adenovirus (HAd) 5 expressing empty cassette or Cre was performed in adult Tg.CreMYOCY437H mice, and glaucoma phenotypes including IOP, outflow facility, structural and functional loss of RGCs, ON degeneration, gliosis, and axonal transport deficits were examined at various stages of IOP elevation. Results: An intravitreal injection of HAd5-Cre led to selective MYOC expression in the TM at the level similar to endogenous Myoc. Expression of mutant MYOC induced biochemical and ultrastructural changes in TM leading to reduced outflow facility and significant IOP elevation. Notably, sustained IOP elevation led to significant functional and structural loss of RGCs and progressive ON degeneration. Glaucomatous neurodegeneration was associated with activation of astrocytes and neurodegenerative changes in the optic nerve head (ONH) region. Remarkably, chronic IOP elevation blocked anterograde axonal transport at the ONH prior to axonal degeneration and RGC loss. Interestingly, impaired axonal transport was associated with loss of cytoskeleton proteins including microtubules and neurofilaments resulting into accumulation of mitochondria in the ONH and neuronal dysfunction. Conclusions: Our studies indicate that Cre-inducible Tg.CreMYOCY437H mice recapitulates all glaucoma phenotypes observed in POAG patients. Importantly, sustained IOP elevation impairs axonal transport at ONH leading to glaucomatous neurodegeneration.

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The double whammy of ER-retention and dominant-negative effects in numerous autosomal dominant diseases: significance in disease mechanisms and therapy

Gariballa,

Mohamed,

Badawi

et al. 2024

J Biomed Sci

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The endoplasmic reticulum (ER) employs stringent quality control mechanisms to ensure the integrity of protein folding, allowing only properly folded, processed and assembled proteins to exit the ER and reach their functional destinations. Mutant proteins unable to attain their correct tertiary conformation or form complexes with their partners are retained in the ER and subsequently degraded through ER-associated protein degradation (ERAD) and associated mechanisms. ER retention contributes to a spectrum of monogenic diseases with diverse modes of inheritance and molecular mechanisms. In autosomal dominant diseases, when mutant proteins get retained in the ER, they can interact with their wild-type counterparts. This interaction may lead to the formation of mixed dimers or aberrant complexes, disrupting their normal trafficking and function in a dominant-negative manner. The combination of ER retention and dominant-negative effects has been frequently documented to cause a significant loss of functional proteins, thereby exacerbating disease severity. This review aims to examine existing literature and provide insights into the impact of dominant-negative effects exerted by mutant proteins retained in the ER in a range of autosomal dominant diseases including skeletal and connective tissue disorders, vascular disorders, neurological disorders, eye disorders and serpinopathies. Most crucially, we aim to emphasize the importance of this area of research, offering substantial potential for understanding the factors influencing phenotypic variability associated with genetic variants. Furthermore, we highlight current and prospective therapeutic approaches targeted at ameliorating the effects of mutations exhibiting dominant-negative effects. These approaches encompass experimental studies exploring treatments and their translation into clinical practice.

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Lentiviral mediated delivery of CRISPR/Cas9 reduces intraocular pressure in a mouse model of myocilin glaucoma

Cited by 3 publications

References 93 publications

Subconjunctival Administration of an Adeno-Associated Virus Expressing Stanniocalcin-1 Provides Sustained Intraocular Pressure Reduction in Mice

Subconjunctival Administration of an Adeno-Associated Virus Expressing Stanniocalcin-1 Provides Sustained Intraocular Pressure Reduction in Mice

Ocular hypertension impairs axonal transport in the optic nerve head leading to neurodegeneration in a novel Cre-inducible mouse model of myocilin glaucoma

The double whammy of ER-retention and dominant-negative effects in numerous autosomal dominant diseases: significance in disease mechanisms and therapy

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