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Brian C Gilger, Laura Conatser, Sara Smith, Jacklyn Salmon, Matthew Hirsch; Intrastromal AAV-HLA-G gene therapy to re-establish corneal immune tolerance. Invest. Ophthalmol. Vis. Sci. 2017;58(8):969.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate AAV-HLA-G (human leukocyte antigen G) for reducing corneal vascularization and inducing immune tolerance after injury.
Self-complementary AAV plasmid vectors harboring a codon optimized HLA-G1 or HLA-G5 transcriptional cassette were validated in cell culture prior to in vivo experiments. Seven days after unilateral corneal wounding by NaOH, both eyes of NZW rabbits were injected intrastromally with scAAV8G9 encoding GFP, HLA-G (1+5), or BSS (n=3/group). Vascularization and inflammation were monitored by slit-lamp (Hackett-Macdonald) and photography. Fifty-six days after injury, rabbits were euthanized, eyes removed, fixed, stained, and evaluated for cornea vasculature (CD31) and cellular response (CD8). Histologic scores were recorded for cellular infiltrate, fibrosis, and vascularization. Serum was analyzed for neutralizing antibodies generated to the AAV capsid and liver, brain, kidney, and heart were collected for vector biodistribution assays (Q-PCR).
Corneal edema from intrastromal injection cleared within 24 hours and injections were well tolerated. No difference in inflammatory scores was observed until day 21 after injection, where a decrease in ocular inflammation occurred in AAV-HLA-G injected eyes compared to eyes injected with the GFP vector. Corneal vascularization developed in NaOH injured eyes beginning at approximately 7 days after injury. Eyes injected with HLA-G had significantly less area of vascularization than GFP injected eyes on days 35-54 after injury (Pi[HML1] >0.004). Histology of corneas injected with AAV-HLA-G following wounding appeared similar to non-injured corneas. Cumulative histologic scores were not significantly different in HLA-G or BSS injected non-injured corneas and significantly lower than in corneas injected with GFP (P=0.043). Most infiltrating immune cells, including CD8+ T cells were reduced in AAV-HLA-G injected corneas. Vector genome biodistribution indicated that AAV vectors were restricted to the cornea, however, 50% of subjects elicited a neutralizing antibody response to the vector capsid following a single intrastromal administration.
Evaluation of AAV-HLA-G ocular injection in rabbits is under continued investigation for clinical translation. Furthermore, these data warrant further evaluation of broader roles of AAV-HLA-G in ocular surface immunity and cornea transplantation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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