Ocular Instillation of Vitamin A-Coupled Liposomes Containing HSP47 siRNA Ameliorates Dry Eye Syndrome in Chronic Graft-Versus-Host Disease

Abstract

Introduction Chronic graft-versus-host disease (GVHD) is characterized by multiorgan fibrosis and affects the quality of life of transplant survivors. Ocular manifestations are found in 40-60% of GVHD patients, and the most common symptom is dry eye syndrome associated with lacrimal gland fibrosis. We previously demonstrated heat shock protein 47 (HSP47), a collagen specific molecular chaperone, plays a critical role in excessive collagen synthesis in chronic GVHD, and systemic administration of vitamin A-coupled liposomes carrying HSP47 siRNA (VA-lip HSP47) ameliorates skin and salivary gland fibrosis in chronic GVHD (Yamakawa, et al, Blood 2018). In the current study, we evaluated the role of HSP47 in ocular GVHD and tested if ocular instillation of VA-lip HSP47 could ameliorate dry eye syndrome in a mouse model of chronic GVHD. Method BALB/c mice received 6-Gy total body irradiation followed by intravenous injection with 8 × 106 bone marrow cells and 20 × 106 splenocytes from minor histocompatibility antigen-mismatched B10.D2 or syngeneic BALB/c donors on day 0. Instillation of 2µl VA-lip HSP47 eye drops was performed daily after bone marrow transplantation (BMT). Allogeneic control recipients were daily treated with ocular instillation of VA-lip containing scrambled siRNA after BMT. Exocrine function of the lacrimal gland was evaluated as wet cotton length after inserting cotton threads under the lower eyelids for 15 seconds. Results Histological studies demonstrated massive fibrosis and accumulation of HSP47+ activated fibroblasts in the lacrimal glands 21 days after allogeneic BMT. The amounts of collagen deposit were significantly elevated in association with reduced tear production in allogeneic mice compared to syngeneic controls on day +35 (Fig. 1). Next, we tested if HSP47 could be a therapeutic target in ocular GVHD by using VA-lip HSP47 eye drops. First, fluorescent-labeled VA-lip HSP47 eye drops were daily administrated unilaterally from day +29 to +33 after allogeneic BMT to test if VA-lip HSP47 could reach to lacrimal glands after eye instillation. We found VA-lip HSP47 particles were distributed in the lacrimal glands on treated side but not on the contralateral side. Ocular instillation of VA-lip HSP47 daily on days +1 to +34 after allogeneic BMT depleted HSP47+ fibroblasts, reduced the amounts of collagen deposit, and ameliorated fibrosis in the lacrimal glands, in association with preserved lacrimal excretory function (Fig. 1). Finally, we found that ocular instillation of VA-lip HSP47 started on day +21 after allogeneic BMT ameliorated established lacrimal gland fibrosis and dry eye syndrome in chronic GVHD (Fig. 2). Conclusions Ocular instillation of VA-lip HSP47 is a promising prophylactic and therapeutic treatment against lacrimal gland fibrosis and dry eye syndrome in chronic GVHD.