AAV vector-meditated expression of HLA-G reduces injury-induced corneal vascularization, immune cell infiltration, and fibrosis.

Over 1.5 million individuals suffer from cornea vascularization due to genetic and/or environmental factors, compromising visual acuity and often resulting in blindness. Current treatments of corneal vascularization are limited in efficacy and elicit undesirable effects including, ironically, vision loss. To develop a safe and effective therapy for corneal vascularization, adeno-associated virus (AAV) gene therapy, exploiting a natural immune tolerance mechanism induced by human leukocyte antigen G (HLA-G), was investigated. Self-complementary AAV cassettes containing codon optimized HLA-G1 (transmembrane) or HLA-G5 (soluble) isoforms were validated in vitro. Then, following a corneal intrastromal injection, AAV vector transduction kinetics, using a chimeric AAV capsid, were determined in rabbits. One week following corneal trauma, a single intrastromal injection of scAAV8G9-optHLA-G1 + G5 prevented corneal vascularization, inhibited trauma-induced T-lymphocyte infiltration (some of which were CD8+), and dramatically reduced myofibroblast formation compared to control treated eyes. Biodistribution analyses suggested AAV vectors persisted only in the trauma-induced corneas; however, a neutralizing antibody response to the vector capsid was observed inconsistently. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to safely and effectively treat corneal vascularization and inhibit fibrosis while alluding to broader roles in ocular surface immunity and allogenic organ transplantation.

Gelam honey potentiates ex vivo corneal keratocytes proliferation with desirable phenotype expression.

BACKGROUND: This study aimed to evaluate the effects of Gelam honey on corneal keratocytes proliferative capacity and phenotypic characterization via MTT assay, gene expression and immunocytochemistry. METHODS: Corneal keratocytes from New Zealand white rabbits were cultured in basal medium (BM) and serum enriched medium (BMS). Serial dilutions of Gelam honey (GH) were added to both media and cells were cultured until passage 1. MTT assay was performed on corneal keratocytes in both media to ascertain the optimal dose of GH that produced maximum proliferation. RESULTS: Gelam honey at the concentration of 0.0015% in both media showed the highest proliferative capacity with no morphological changes compared to their respective controls. The gene expression of aldehyde dehydrogenase (ALDH), a marker for quiescent keratocytes and vimentin, a marker for fibroblast, were higher in the GH enriched groups. The alpha smooth muscle actin (α-SMA) expression, marker for myofibroblast, was lower in GH treated groups compared to the controls. Immunocytochemistry results were in accordance to the gene expression analyses. CONCLUSION: Gelam honey at a concentration of 0.0015% promotes ex vivo corneal keratocytes proliferation while retaining desirable phenotype expression. The results serve as a basis for the development of Gelam honey as a potential natural product in promoting corneal wound healing.