Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase

AIM: To evaluate the potential of two trabecular meshwork (TM)-specific promoters, Chitinase 3-like 1 (Ch3L1) and matrix gla protein (MGP), for improving specificity safety in glaucoma gene therapy based on self-complementary AAV2 (scAAV2) vector technologies. METHODS: An scAAV2 with C3 transferase (C3) as reporter (scAAV2-C3) was selected. The scAAV2-C3 vectors were driven by Ch3L1 (scAAV2-Ch3L1-C3), MGP (scAAV2-MGP-C3), enhanced (scAAV2-eMGP-C3) cytomegalovirus (scAAV2-CMV-C3), respectively. cultured primary human TM cells treated each at different multiplicities infections. Changes cell morphology observed phase contrast microscopy. Actin stress fibers Rho GTPases/Rho-associated kinase pathway-related molecules assessed immunofluorescence staining, real-time quantitative polymerase chain reaction Western blot. Each injected intracamerally into one eye rat low high doses In vivo green fluorescence visualized a Micron III Retinal Imaging Microscope. Intraocular pressure (IOP) monitored using rebound tonometer. Ocular responses evaluated slit-lamp histopathology analysis examined hematoxylin eosin staining. RESULTS: culture studies, vector-mediated expression induced morphologic changes, disruption actin cytoskeleton reduction fibronectin inhibiting signaling pathway. At same dose, these changes significant scAAV2-CMV-C3 or scAAV2-Ch3L1-C3, but not scAAV2-eMGP-C3 scAAV2-MGP-C3. low-injected IOP significantly decreased scAAV2-Ch3L1-C3-injected eyes scAAV2-MGP-C3-injected scAAV2-eMGP-C3-injected eyes. high-injected Similar to scAAV2-CMV-C3, scAAV2-Ch3L1-C3 showed efficient transduction both corneal endothelium. anterior segment tissues eyes, no obvious morphological found except TM. Inflammation absent. CONCLUSION: scAAV2-transduced cells, promoter-driven efficiency is close that cytomegalovirus, obviously higher than MGP. chamber eye, transgene pattern presumably affected promoter, promoter. These findings would provide useful reference improvement vector.