Literature Interpretation: AAV-DJ-Mediated MYOC Gene Silencing as a Gene Therapy Strategy for Myocilin-Associated Glaucoma
Release time:
2026-04-02
Research Background:
Open-angle glaucoma (OAG) is a leading cause of irreversible blindness worldwide, characterized by progressive degeneration of retinal ganglion cells (RGCs) and subsequent visual field defects. Among the genetic causes of OAG, mutations in the MYOC gene account for approximately 2%–4% of adult-onset primary open-angle glaucoma and up to 10% of juvenile open-angle glaucoma. MYOC-associated glaucoma typically presents with significantly elevated intraocular pressure (IOP). Such mutations lead to myocilin protein misfolding and accumulation within the trabecular meshwork (TM), inducing endoplasmic reticulum (ER) stress, impairing TM function, and ultimately causing elevated IOP — a key driver of glaucoma progression. Conventional glaucoma treatments include medications, laser therapy, and surgery, all aimed at lowering IOP; however, none directly address the underlying molecular defect. Gene therapy represents a promising strategy to target the root cause of MYOC-associated open-angle glaucoma.
This study aimed to evaluate the efficacy of adeno-associated virus serotype DJ (AAV-DJ)-mediated delivery of a short hairpin RNA targeting MYOC (shMYOC) in a transgenic MYOC P370L mouse model (Tg-MYOC P370L) for the treatment of MYOC mutation-associated open-angle glaucoma (OAG).
Research Methods:
Transgenic mice carrying the human MYOC Pro370Leu mutation were generated using the CRISPR/Cas9 system.
Brief procedure: Cas9 messenger RNA (mRNA), single-guide RNA (sgRNA), and donor DNA were co-injected into fertilized mouse eggs. The sgRNA guided Cas9 to create a double-strand break at the mouse H11 locus, enabling insertion of the CAG-3×Flag-hMYOC-P370L-polyA expression vector at that site. The H11 locus, located between the Eif4enif1 and Drg1 genes, is a safe harbor for efficient exogenous promoter-driven gene expression. Founder mice were genotyped using PCR, and the genotype was verified by DNA sequencing.
Post-intervention assessments included myocilin aggregation, expression of ER stress markers, IOP, aqueous humor outflow facility, retinal ganglion cell (RGC) survival, and visual function. Mice were anesthetized with 3% isoflurane for induction and maintained with 2.5% isoflurane. IOP was measured using a Tonolab rebound tonometer (Icare Finland Oy, Helsinki) at 4–6 minutes after anesthesia induction, following established protocols. All longitudinal IOP measurements were performed within the same circadian time period. During measurement, the tonometer was held perpendicular to the floor, with the probe vertically aligned to the center of the cornea. Six consecutive measurements were taken per eye, and the average was automatically calculated as the IOP value. Three rounds of measurements were repeated for each eye, and all data were recorded.
All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Experimental Animal Welfare and Ethics Committee of Capital Medical University (Approval Nos.: AEEI-2017-11, AEEI-2023-037). Our animal facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Research Results:
In this study, we injected AAV-DJ–shMYOC (1×10¹⁰ vg/eye) into the anterior chamber via a transcorneal transiridal approach to specifically target the human MYOC transgene. Unlike gene-editing tools such as CRISPR, shRNA acts at the mRNA level and does not alter the overall gene expression profile of cells that do not express the target protein, thus offering a favorable safety profile.
Given that most MYOC mutations are clustered in the C-terminal region, we designed the shRNA to target the upstream region of the coding sequence, enabling broad-spectrum knockdown of various MYOC mutants. We first validated the knockdown efficiency of candidate shMYOC constructs in vitro and selected the most effective vector for subsequent in vivo experiments.
AAV-DJ–shMYOC injection did not induce significant inflammation in the anterior chamber. Administration of AAV-DJ–shMYOC to 3-month-old mice effectively prevented the age-related IOP elevation observed in control mice (AAV-DJ–shControl).
At two months post-injection, human myocilin expression in the TM tissue was significantly reduced in shMYOC-treated eyes compared to the shControl group.
Furthermore, RNA and protein extracted from "TM ring" samples (including the iris root, cornea, and adjacent sclera) revealed that AAV-DJ–shMYOC transfection significantly reduced the expression of myocilin and ER stress markers, including BiP (Hspa5), CHOP (Ddit3), and ATF4 (Atf4), at both the mRNA and protein levels compared to AAV-DJ–shControl.
These results demonstrate that AAV-DJ–shMYOC efficiently reduces myocilin expression in the trabecular meshwork and alleviates ER stress in adult Tg-MYOCP370L mice.
Research Conclusions:
This study provides strong evidence that AAV-DJ-mediated MYOC gene silencing represents a promising gene therapy strategy for myocilin-associated glaucoma. By directly targeting the genetic cause of the disease, this approach has the potential to achieve long-term stable intraocular pressure control and neuroprotection. Future studies should focus on addressing key issues related to clinical translation, laying the groundwork for eventual clinical application.
Acknowledgments
This study was supported by the Beijing Natural Science Foundation (7254321), the National Natural Science Foundation of China (82130029), and the National Natural Science Foundation of China (82201170).
Publication Information
Authors: Yue Wan; Xiangxiang Liu; Xuejing Yan; Shen Wu; Yufei Teng; Luyi Han; Jingxue Zhang; Ningli Wang
Publisher: IOVS Investigative Ophthalmology & Visual Science, January 2026, Vol.67, 48.
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