Advancements in Gene Therapy for Glaucoma: Targeting the Actin Cytoskeleton and Oxidative Stress

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Glaucoma, a leading cause of global visual loss, affects approximately 80 million individuals worldwide [1]. The disease involves progressive damage to the optic nerve head, primarily attributed to high intraocular pressure (IOP) [2,3].

The most prevalent form, primary open-angle glaucoma (POAG), affects around 70 million people, while steroid-induced glaucoma (SIG) arises from long-term glucocorticoid use [2,4,5,6,7]. Current treatments primarily focus on lowering IOP, but there is a pressing need for interventions addressing neuroprotection as well.

Actin Cytoskeleton Targeting Therapies

Recently, the Food and Drug Administration approved a novel class of glaucoma therapies targeting the actin cytoskeleton [11]. The actin cytoskeleton, crucial for maintaining trabecular meshwork (TM) homeostasis, is altered in glaucoma [12,13]. Fibronectin and alpha-smooth muscle actin (alpha-SMA) play pivotal roles in TM remodeling [14,15,16].

A breakthrough in this field involves Exoenzyme C3 transferase (C3), derived from Clostridium botulinum, which inhibits Rho GTPases, key regulators of TM contractility [17,18,19,20,21,22,23]. Recent studies demonstrated that C3 protects retinal ganglion cells (RGCs) by inhibiting RhoA signaling and reducing IOP [25]. This inhibition of Rho GTPase using C3 emerges as a promising avenue for glaucoma therapy.

Oxidative Stress and Thioredoxins

Oxidative stress is implicated in glaucoma pathogenesis, contributing to RGC loss, increased IOP, neuroinflammation, and apoptosis [26,27,28]. Thioredoxins (Trxs), a family of redox proteins, play vital roles in cellular redox homeostasis and protect against oxidative stress [29]. Trx2, a member of this family, has been shown to support RGC survival by reducing oxidative stress and apoptosis [30,31]. Targeting oxidative stress pathways, particularly through Trx2, presents a potential therapeutic strategy for glaucoma.

Fusion Proteins and Gene Therapy

The use of fusion proteins in gene therapy offers a novel approach by delivering multiple genes through a single vector [32]. This strategy, exemplified by the regulatory-approved Aflibercept and Vabysmo, demonstrates enhanced efficacy in ophthalmology [34,35,36]. In this study, a self-complementary adeno-associated virus 2 (scAAV2) vector carrying a fusion gene of C3 and Trx2 was developed. scAAV2, with its self-complementary genome, ensures faster and more efficient transgene expression [37,38,39].

Advantages of Gene Therapy for Glaucoma

Gene therapy for glaucoma presents advantages over conventional treatments, providing a long-lasting effect with a single administration, addressing issues of patient compliance and adverse effects [40]. The success of AAV-based gene therapy in ophthalmic conditions, such as Luxturna for Leber congenital amaurosis, underscores its potential for glaucoma treatment [41,42].

Experimental Findings

The recombinant scAAV-Trx2-C3 vector efficiently transduced endothelial cells in vitro and in vivo. Intracameral delivery of Trx2-C3 via AAV2 effectively decreased IOP and prevented RGC cell death in a DEX-induced glaucoma model. Moreover, scAAV-Trx2-C3 reduced fibronectin and alpha-SMA expression in glaucoma mouse models. These findings collectively suggest that scAAV-Trx2-C3 holds promise as a gene therapy vector for glaucoma treatment.

Discussion

Glaucoma, characterized by progressive optic nerve head damage and visual field loss, is a multifaceted disease influenced by various factors, with intraocular pressure (IOP) being a significant risk factor [2,3]. Despite efforts to reduce IOP, the degeneration of retinal ganglion cells (RGCs) persists, indicating the need for comprehensive therapeutic strategies addressing mechanical and biological aspects of the disease [28,44,45,46].

This study introduces a novel approach using a self-complementary adeno-associated virus 2 (scAAV2) vector carrying a fusion gene of the C3 and Trx2 proteins to target both IOP regulation and neuroprotection [49,50].

The fusion protein Trx2-C3 aims to address multiple aspects of glaucoma pathogenesis. C3, derived from Clostridium botulinum, inhibits RhoA activity, a crucial regulator of TM contractility, ultimately affecting IOP [17,22,23,25]. On the other hand, Trx2, a potent antioxidant, safeguards cells from oxidative stress, a known contributor to glaucoma [26,27,28,30,31]. The fusion construct’s design mirrors established methods in ophthalmology, exemplified by aflibercept and faricimab-svoa [35,51,52].

In glaucoma pathophysiology, TM fibrosis, particularly in open-angle glaucoma (OAG), plays a pivotal role in increased IOP [13,15,53]. Traditional treatments target the Rho/ROCK pathway, regulating cytoskeletal dynamics, but dual-target therapies, such as scAAV2-Trx2-C3, offer a promising alternative. By inhibiting RhoA activity and reducing fibronectin deposits, scAAV2-Trx2-C3 showcases its potential in regulating TM cells and mitigating fibrosis [56,57].

The DEX-induced glaucoma model used in this study mimics steroid-induced glaucoma, providing insights into molecular and cellular alterations in TM and potential therapeutic strategies [54,55]. The administration of scAAV2-Trx2-C3 through intracameral delivery significantly lowered IOP in the DEX-induced mouse model, demonstrating its efficacy in mitigating glaucoma-associated IOP elevation.

Although the study’s short-term nature limits the observation period, the sustained reduction in IOP for up to 4 weeks post-injection is a positive outcome [56].

Despite IOP reduction being a crucial aspect, the study extends its focus to neuroprotection, addressing the need for therapies preventing RGC damage. Glaucoma patients may experience progression despite IOP management, emphasizing the importance of neuroprotective strategies [58,59,60].

Trx2’s role in regulating oxidative stress on RGCs has been well-documented [64], and the study shows scAAV2-Trx2-C3 effectively reduces reactive oxygen species (ROS) levels in DEX-treated cells in vitro. Furthermore, intracameral delivery of scAAV2-Trx2-C3 prevents apoptotic cell death of RGCs in the DEX-induced glaucoma mouse model, affirming its potential neuroprotective effects.

Moreover, scAAV2-Trx2-C3 exhibits promising anti-fibrotic effects, reducing fibronectin and alpha-SMA expression in DEX-induced glaucoma mouse models. This suggests a broader impact on the pathological processes underlying TM fibrosis.

In conclusion, the presented study introduces a novel gene therapy approach, utilizing scAAV2-Trx2-C3, to address both IOP regulation and neuroprotection in glaucoma. The fusion construct demonstrates efficacy in lowering IOP, preventing RGC apoptosis, and reducing fibrotic markers.

While further research and long-term studies are warranted, the study lays the foundation for a potentially transformative gene therapy for glaucoma, providing a comprehensive strategy to combat this debilitating eye disease.


reference link : https://www.mdpi.com/1422-0067/24/22/16253

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