Ocular graft versus host disease (OGvHD) develops in nearly 50% of patients after allogeneic hematopoietic stem cell transplantation (HSCT) and frequently manifests as dry eye disease (DED).  The aim of this study was to evaluate the efficacy of a single subconjunctival (SCJ) dose of adeno-associated virus (AAV) gene therapy encoding human leukocyte antigen G (HLA-G) isoforms 1 and 5 to induce ocular immune tolerance and inhibit development of clinical signs of OGvHD.

 A Major histocompatibility mismatch chronic OGvHD murine model was evaluated. Seven days after HSCT, mice were dosed subconjunctivally with scAAV8-HLA-G1/5 (1e9vg/eye), topical cyclosporine (CSA) (twice daily), or left untreated.  Body weights and tear production (red thread test) were recorded, and eyelid, corneal opacity, and corneal fluorescein retention were scored through day 44 after HSCT.

Body weights were recorded prior to ocular injections and every other day thereafter. Tear production was recorded weekly by using phenol red threads (PRT) (Zone Quick Tread, Oasis Medical, San Dimas, CA, USA) placed into the lateral canthus of the eye for 20 s, and the amount of tear fluid was recorded in mm.  Clinical scores (0-4) of eyelid inflammation, corneal opacity, and corneal fluorescein retention were recorded, following slit lamp biomicroscopy (KOWA SL-17, Torrance, CA), three times per week through day 44 after HSCT with the observer masked to the treatment groups.

The most prominent clinical signs affecting ocular tissue following allogenic HSCT manifested as lid margin, cornea, and corneal fluorescein retention score changes. Individual mice received a score of 0-4 for the grade of each category.   

Each database sheet (Tab) has the same organizational format, with columns of Mouse ID (identification), Cohort, Treatment (CSA-cyclosporine; AAV-HLAG1/5 (subconjunctival) or no treatment. The timepoint is the day following HSCT. and finally, the score assigned (based on the description previously). 

Eyelid Score Tab: Lid margins were evaluated on the severity of edema, skin swelling, and if the eyelids were able to open.

Cornea Scores Tab: The clinical scale of corneal involvement ranged from normal and clear to variable stages of keratopathy to corneal ulcerations.

Fluorescein Retention Scores Tab: Corneal fluorescein retention scores were evaluated based on geographic coverage and density of the stain.

PRT Scores Tab: Phenol red thread tear evaluation.  This a measurement of tear production using the PRT thread.  The values are read in mm of wetting over 20 seconds. 

Weights % Tab: body weights and percentage of baseline of the mice over time

Histology Scores Tab: Histology scores: Additionally, histologic images of the cornea and limbus were collected and analyzed using ImageJ software to measure epithelial, stromal, and total corneal thickness.   Epithelial thickness is the thickness in uM of the corneal epithelium, corneal thickness is the total corneal thickness in uM, and ratio is the ratio of the epithelial thickness to total corneal thickness.  The #cells at the limbus is the number of mononuclear cells at the superior limbus quantitated in an area measuring 20x20um from the limbus extending posteriorly.

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