Experimental And Lab Model Of Potential Gene Therapy In Keratoconus

Keratoconus is characterised by progressive stromal weakness and thinning. Levels of collagen crosslinking enzyme lysyl oxidase (LOX) ARE inversely related to the severity of keratoconus. This study explores potential benefits of enhancing LOX expression in keratoconic tissues.

Tertiary eye care hospital and associated Research Hospital, Bengaluru, India

Human donor corneal lenticules were obtained from SMILE surgeries. Recombinant adeno-associated viral(AAV) vectors were constructed expressing wild-type LOX gene. AAV LOX driven by CMV promoter was packaged in AAV9 capsids and purified by sequential isopycnic ultracentrifugation. These vectors transduced ex-vivo donor corneal lenticules in culture media with recombinant AAV expressing GFP as controls. Mouse corneas were transduced in-vivo with 5uL of 1e10vg vectors contralaterally with LOX and GFP expressing AAV, after lightly debriding corneas and applying vector solution as eye-drop. LOX, Collagen I, Collagen IV and MMP9 levels were measured in human ex-vivo tissues  and mouse corneas by immunofluorescence(IF) and mRNA analysis.

AAV transduction of corneal tissues was safe in mouse corneas with no haze/other ocular surface events on periodic examination. Gene therapy vectors effectively transduced corneal fibroblasts in both primary human and mouse corneas. Higher levels of LOX expression correlated with higher expression of collagens and ECM/fibrosis genes like Fibronectin and a-SMA, and lower basal levels of MMP9.

Recombinant AAV-mediated LOX therapy in ectatic corneas is safe and effective. LOX augmentation enhances expression of collagens and reduces MMP9 levels, thus blocking the thinning processes and strengthening the cornea. Reduced MMP9 can alleviate the inflammatory milieu, hence treating keratoconus.