Abstract
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.
Highlights
The normal cornea is considered to be an immune privileged site that has evolved tolerance to ocular surface flora and environmental irritants in an attempt to prevent overt ocular inflammation that can result in blindness
The results demonstrated decreased immune cell infiltration of the cornea, which included cytotoxic T lymphocytes, and decreased smooth muscle actin staining in the scAAV8G9-optHLA-G treated corneas
The optHLA-G1 transmembrane domain was deleted to generate optHLA-G5 cDNA, and both optHLA-G1 and optHLA-G5 were cloned into a self-complementary associated virus (AAV) plasmid context (Fig. 1A)
Summary
The normal cornea is considered to be an immune privileged site that has evolved tolerance to ocular surface flora and environmental irritants in an attempt to prevent overt ocular inflammation that can result in blindness. These reports provide an important proof of concept for using AAV gene therapy as a prophylactic for cornea vascularization In these examples the effects were incomplete, evaluated only over a short term (2 weeks), and did not demonstrate efficacy in the perceived clinical situation, administration following a cornea trauma. The purpose of this study was to engineer and validate a therapeutic, based on a natural mechanism of immune suppression, for the treatment of corneal vascularization following an insult Towards this end, HLA-G cDNA was enhanced by codon optimization and validated in a self-complementary AAV vector context in vitro[19,20]. The collective data demonstrate the clinical potential of scAAV8G9-optHLA-G to eliminate vascularization of the cornea while alluding to even broader roles in fibrosis, ocular surface immunity, and allogenic organ transplantation
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