Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Optic neuropathies such as glaucoma occur when retinal ganglion cells (RGCs) in the eye are injured. Strong evidence suggests mesenchymal stem cells (MSCs) could be a potential therapy to protect RGCs; however, little is known regarding their effect on the human retina. We, therefore, investigated if human MSCs (hMSCs), or platelet-derived growth factor (PDGF) as produced by hMSC, could delay RGC death in a human retinal explant model of optic nerve injury. Our results showed hMSCs and the secreted growth factor PDGF-AB could substantially reduce human RGC loss and apoptosis following axotomy. The neuroprotective pathways AKT, ERK, and STAT3 were activated in the retina shortly after treatments with labeling seen in the RGC layer. A dose dependent protective effect of PDGF-AB was observed in human retinal explants but protection was not as substantial as that achieved by culturing hMSCs on the retina surface which resulted in RGC cell counts similar to those immediately post dissection. These results demonstrate that hMSCs and PDGF have strong neuroprotective action on human RGCs and may offer a translatable, therapeutic strategy to reduce degenerative visual loss.

Background: Glaucoma is a leading cause of irreversible blindness, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Activation of the Tropomyosin receptor kinase B (TrkB) pathway by mature brain-derived neurotrophic factor (mBDNF) has emerged as a promising neuroprotective strategy, given its critical role in promoting RGC survival in preclinical models. Methods: We advanced the development of a bicistronic adeno-associated viral (AAV) gene therapy vector engineered to co-express human TrkB and mBDNF. The vector was optimized with fully human transgene sequences and evaluated for functional expression and dose scalability to support clinical translation. Expression tracking and efficiency were enhanced by incorporating a self-cleaving 2A peptide sequence. Results: In a mouse model of optic nerve crush, intravitreal administration of 1.52E8 to 7.60E8 genome copies (GC)/eye significantly mitigated RGC damage. In a rat model of laser-induced ocular hypertension, doses ranging from 3.80E8 to 1.90E9 GC/eye preserved both visual function and RGC survival. The 2A peptide facilitated efficient co-expression of TrkB and mBDNF while minimizing interference from endogenous protein pathways. Conclusions: These findings demonstrate that co-expression of human TrkB and mBDNF via a bicistronic AAV vector yields robust, dose-dependent neuroprotection and sustained transgene expression in two distinct models of glaucomatous injury. This gene therapy represents a promising first-in-class candidate for the treatment of glaucoma.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), that starts with atrophic lesions in the outer retina that expand to cover the macula and fovea, leading to severe vision loss over time. Pigment Epithelium-Derived Factor (PEDF) has a diverse-range of properties, including its ability to promote cell survival, reduce inflammation, inhibit angiogenesis, combat oxidative stress, regulate autophagy, and stimulate anti-apoptotic pathways, making it a promising therapeutic candidate for GA. However, the relatively short half-life of PEDF protein has precluded its potential as a clinical therapy for GA since it would require frequent injections. Therefore, we describe administration of a PEDF gene, comparing and contrasting delivery routes, viral and non-viral vectors, and consider the critical challenges for PEDF as a neuroprotectant for GA.