Multiple disease-specific considerations have led to interest in the potential of gene therapy to permanently correct elevated intraocular pressure (IOP), the main causal risk factor for primary open angle glaucoma (POAG). Since IOP elevation results from abnormal resistance to aqueous humor outflow from the eye through the trabecular meshwork (TM), a means to genetically modify this specialized outflow organ permanently and safely is a prioritized goal. Here we tested different lentiviral vector designs and doses for long-term transgene expression in a large animal model, and investigated whether exogenously introduced myocilin proteins influenced IOP. The anterior chambers of 18 domestic cats (36 eyes) were injected with dual-gene feline immunodeficiency virus (FIV) vectors. Substantial, well-tolerated green fluorescent protein (GFP) expression was achieved in TM and monitored non-invasively in vivo for 1.2-2.3 years, using both 5' cap-translation and internal ribosome entry site (IRES)-translation. In all 36 eyes, post-mortem examination revealed substantial TM transgene expression (which often greatly exceeded that observable non-invasively during life). However, co-expression with enhanced GFP of myocilin or a juvenile glaucoma-associated mutant myocilin did not elevate IOP. These results demonstrate a safe, long-term single and dual gene expression in TM and establish an experimental system for testing candidate therapeutic transgenes for POAG.