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. 2019 Dec 27;9(1):19864.
doi: 10.1038/s41598-019-56462-3.

AAV-mediated Expression of HLA-G1/5 Reduces Severity of Experimental Autoimmune Uveitis

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Free PMC article

AAV-mediated Expression of HLA-G1/5 Reduces Severity of Experimental Autoimmune Uveitis

Elizabeth Crabtree et al. Sci Rep. .
Free PMC article

Abstract

Non-infectious uveitis (NIU) is an intractable, recurrent, and painful disease that is a common cause of vision loss. Available treatments of NIU, such as the use of topical corticosteroids, are non-specific and have serious side effects which limits them to short-term use; however, NIU requires long-term treatment to prevent vision loss. Therefore, a single dose therapeutic that mediates long-term immunosuppression with minimal side effects is desirable. In order to develop an effective long-term therapy for NIU, an adeno-associated virus (AAV) gene therapy approach was used to exploit a natural immune tolerance mechanism induced by the human leukocyte antigen G (HLA-G). To mimic the prevention of NIU, naïve Lewis rats received a single intravitreal injection of AAV particles harboring codon-optimized cDNAs encoding HLA-G1 and HLA-G5 isoforms one week prior to the induction of experimental autoimmune uveitis (EAU). AAV-mediated expression of the HLA-G-1 and -5 transgenes in the targeted ocular tissues following a single intravitreal injection of AAV-HLA-G1/5 significantly decreased clinical and histopathological inflammation scores compared to untreated EAU eyes (p < 0.04). Thus, localized ocular gene delivery of AAV-HLA-G1/5 may reduce the off-target risks and establish a long-term immunosuppressive effect that would serve as an effective and novel therapeutic strategy for NIU, with the potential for applications to additional ocular immune-mediated diseases.

Conflict of interest statement

M.H. and B.G. are coinventors on the AAV-HLA-G technology evaluated herein, are listed on a patent (pending) that is owned by North Carolina State University and the University of North Carolina, and they have received associated royalties. The remainder of the authors do not have competing interests.

Figures

Figure 1
Figure 1
AAV8 gene delivery to EAU Iris and Ciliary Body. Representative histological images of GFP immunofluorescence following intravitreal injection of AAV8-GFP. Transgene expression (green) was examined by immunostaining. The left image is of the iris and ciliary body, without primary antibody; the right image is with anti-GFP antibody. To confirm gene delivery to inflamed uvea, rats were injected with either scAAV8-GFP (3 × 1010 viral genomes; 3 μL; n = 6) or balanced salt solution (3 μL; n = 3) and experimental autoimmune uveitis was induced in 3 of the AAV-GFP injected rats and 3 balanced salt solution rats one week following intraocular injections. Animals were euthanized 2 weeks following induction of EAU and tissues were collected for histopathology. The results demonstrate strong GFP staining in cells of inflamed ciliary body and iris following intravitreal injection of AAV8-GFP. The control section (left), without primary antibody, shows several focal areas of green fluorescence, assumed to be autofluorescence of red blood cells based on the anatomic location of the fluorescence.
Figure 2
Figure 2
AAV8-HLA-G1/5 Improves EAU clinical scores. Rats were treated by intravitreal (IVT) injections with scAAV8-HLAG1/5 (n = 6 rats, 12 eyes) or topically treated with dexamethasone (Dex) (n = 5 rats, 10eyes); no treatment experimental autoimmune uveitis (EAU) rat (n = 6 rats, 12 eyes) and healthy rats (untreated, no EAU induction) (n = 3 rats, 6 eyes) were used as controls. One week after IVT injections, EAU was induced by immunization with IRBP and ocular inflammation was examined by slit lamp biomicroscopy. (a) Bar graph of EAU clinical scores revealed that EAU clinical scores peaked at days 12 and 13 after uveitis induction. Mean clinical scores were significantly less in eyes treated with a single intravitreal dose of scAAV8-HLA-G1/5 or topical dexamethasone (applied topically every 6 hours) compared to untreated EAU eyes on each day from 12 through 14 post-induction (P = 0.03 to 0.001); Pairwise Wilcoxon tests). There were no significant differences in mean clinical scores between eyes treated with scAAV8 HLA-G1/5 or dexamethasone at any day. Healthy eyes had inflammatory scores of 0 for each day of the study. (b) Retinal images (top row) taken on day 9 did not reveal any gross retinal damage in any treatment group; topical corticosteroid treatment (A), HLAG1/5 (B), EAU no treatment (C), healthy or control rat (D). Representative images taken on day 12 after EAU induction, demonstrate dilated iris blood vessels, hypopyon, fibrin, posterior synechia and severe miosis in EAU eyes with no treatment (G), whereas normal eyes were observed in eyes treated with topical corticosteroids (E) and HLAG (F) treated rats; Healthy eye (H).
Figure 3
Figure 3
AAV8-HLA-G1/5 Improves EAU histology scores. (a) Representative images of ocular histology demonstrate iris thickening and severe inflammatory cell infiltration in the ciliary body, iris, anterior chamber and vitreous body, as well as moderate vasculitis formation in experimental autoimmune uveitis (EAU) untreated eyes (C,D). Mild infiltration of inflammatory cells was observed in the vitreous and retina in topical dexamethasone and HLA-G1/5 treated EAU eyes (E&F, G&H, respectively). Control healthy eyes (A&B). (hematoxylin & eosin staining, original magnification: 20x and 40x). The histological infiltrative scores (b) and structural scores (c) were significantly decreased in the scAAV8-HLA-G1/5 treated (n = 4 eyes) and dexamethasone (dex) treated EAU eyes (n = 4 eyes) as compared to the non-treated EAU eyes (n = 4 eyes) (p = 0.01, p = 0.02; Wilcoxon test). There was no significant difference between healthy eyes (n = 2 eyes) and either EAU treatment group. Each point is the average score of an individual eye of two blinded observers and mean scores of each treatment group are denoted by the horizontal bars.
Figure 4
Figure 4
AAV8-HLAG1/5 expression and ocular distribution. AAV8-HLAG1/5 expression and ocular distribution. (a) HLA-G abundance examination by qRT-PCR in selected tissues are presented as vector cDNA /host transcript (GAPDH), n = 4 rats, 8 eyes, Mann-Whitney test, **p < 0.01. Results represent experiments done in triplicate with mean value expressed; (b) Vector genome copy number in distinct ocular tissues (cornea, iris/ciliary body, and retina) are shown as vector genome copy number/ug of host genome DNA, n = 4 rats, 8 eyes, Mann-Whitney test, **p < 0.01.

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