Dose-ranging and further therapeutic evaluation of a bicistronic humanized TrkB-BDNF gene therapy for glaucoma in rodents.

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. Supplementary Information: The online version contains supplementary material available at 10.1186/s44477-025-00003-y.