Glaucoma is a multifactorial disease characterized by the gradual apoptosis and degeneration of retinal ganglion cells (RGCs), leading to corresponding vision loss and being one of the leading causes of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is considered the only modifiable risk factor for glaucoma, and lowering IOP is the only clinically proven method to halt or slow its progression.
Fundus photographs of healthy and glaucomatous optic nerve heads
Figure 1: Fundus photographs of healthy and glaucomatous optic nerve heads (right eye)
(A) In a healthy optic nerve head, the neuroretinal rim maintains its normal shape, with the broadest part located in the inferior area, followed by the superior, nasal, and temporal areas (named according to the ISNT rule); (B) In a glaucomatous optic nerve head, the neuroretinal rim is significantly thinner than in a healthy optic nerve head, with a corresponding larger and deeper optic cup.

In pharmacological treatments for glaucoma, the primary mechanisms for lowering IOP include inhibiting aqueous humor secretion or facilitating aqueous humor drainage. Carbonic anhydrase inhibitors (CAIs) are among the oldest IOP-lowering drugs, reducing aqueous humor production by inhibiting the interconversion of carbon dioxide and bicarbonate in the ciliary body. However, topical drug treatments can be complicated by poor patient compliance, low bioavailability, and side effects. Given that glaucoma requires lifelong management as a chronic disease, daily medication and combination therapy with multiple drugs are common in clinical practice. Thus, developing a convenient, effective, and durable treatment method is urgently needed for the long-term management of glaucoma patients. Due to the eye’s accessible nature and immune-privileged characteristics, gene therapy is considered a promising new approach for glaucoma treatment.

On April 25, 2024, the team of Xiulan Zhang from the Zhongshan Ophthalmic Center at Sun Yat-sen University, and the team of Patrick Yu Wai Man from the University of Cambridge jointly published a research paper titled “CRISPR-Cas9-mediated deletion of carbonic anhydrase 2 in the ciliary body to treat glaucoma” in “Cell Reports Medicine” (IF 14.3). Dr. Jiaxuan Jiang and Dr. Kangjie Kong are the co-first authors of this paper. The study used the AAV ShH10 to deliver the CRISPR-Cas9 system, precisely targeting the Car2 gene in the ciliary body. This approach effectively reduced IOP in an experimental glaucoma mouse model and provided long-term mitigation or even prevention of glaucoma damage caused by sustained high IOP, offering a potential new treatment strategy for glaucoma patients.

Graphical abstract of the paper
Figure 2: Graphical abstract of the paper

*PackGene Contributed the vector construction and AAV packaging services for this research

In Vitro Experiments

  • Plasmid Vectors: ssAAV.U6.(Sp)sgRNA.CAG.SV40 NLS-EGFP.WPRE, ssAAV.miniCMV.SpCas9
  • Promoters: U6, miniCMV, CAG
  • Cell Type: Neuro-2a (N2a) cells
  • Transfection Method: Lipofectamine 3000 transfection
  • Detection Methods: Sanger sequencing, T7E1 assay, GUIDE-seq

In Vivo Experiments

  • Viral Vectors: AAV shH10-SpCas9, AAV shH10-sgRNA
  • Promoters: U6, miniCMV, CAG
  • Injection Sites: DBA/2J mice, intravitreal injection
  • Injection Dose: 1 x 10^13 gc/mL, 2μL
  • Detection Time: 1 week post-injection
  • Detection Methods: Immunofluorescence imaging, Capillary-based immunoassay, qPCR, etc.
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