Volume 12, Issue 2, Pages 369-373 (August 2005) Local Administration of an Adeno-associated Viral Vector Expressing IL-10 Reduces Monocyte Infiltration and Subsequent Photoreceptor Damage during Experimental Autoimmune Uveitis  Cathryn A. Broderick, Alexander J. Smith, Kam S. Balaggan, Anastasios Georgarias, Prateek K. Buch, Peter C. Trittibach, Susie E. Barker, Gian-Marco Sarra, Adrian J. Thrasher, Andrew D. Dick, Robin R. Ali  Molecular Therapy  Volume 12, Issue 2, Pages 369-373 (August 2005) DOI: 10.1016/j.ymthe.2005.03.018 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Murine IL-10 cDNA was purchased from Invivogen (UK) and subcloned between the CMV promoter and the IRES region of the AAV.cmv.CNTF.ires.eGFP construct following removal of the CNTF cDNA to generate the construct pd10.cmv.IL-10.ires.eGFP. The constructs were used to generate recombinant rAAV as previously described [24,25]. Antigen-specific splenocytes were isolated from immunized mice 14 days post-subcutaneous administration and rechallenged in vitro for 48 h. Test wells contained AAV.IL-10 conditioned medium. The biological function of the IL-10 transgene was confirmed by the ability of conditioned medium to decrease (A) antigen-specific lymphocyte proliferation as determined by[3H]thymidine uptake and (B) CD11b+ myeloid cell expression of MHC class II and CD86 costimulatory molecules as detected by flow cytometry. These decreases were negated by an IL-10 neutralizing antibody and are not observed in control vector conditioned medium-treated cells (unpaired Student’s t test, mean ± SD). Molecular Therapy 2005 12, 369-373DOI: (10.1016/j.ymthe.2005.03.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Subretinal administration of vectors was performed in anesthetized 3- to 4-week-old female C57BL/6 mice (n = 15, Harlan UK Ltd.), under direct retinoscopy through an operating microscope as previously described [26]. A 1.5-cm 34-gauge hypodermic needle (Hamilton, UK) was used to inject 2 μl of virus suspension (genomic titer of 1010 particles/ml) into the subretinal space to produce a bullous retinal detachment in the superior hemisphere and a second in the inferior hemisphere. Where required, left eyes were injected with PBS or titer-matched AAV.GFP as internal controls. Retinal vessels remained patent during and following the injections. All animals received chloramphenicol 1% eye ointment to the cornea. EAU was induced in the AAV-injected group (n = 15) and a control group of 7- to 8-week-old female C57BL/6 mice (n = 6) by injection of 500 μg of IRBP peptide 1–20 (SigmaGenosys, UK) v/v CFA with 2.5 mg/ml M. tub (Sigma, UK) and 1.5 μg pertussis toxin (Sigma) intraperitoneally per mouse in total volumes of 100 μl, as previously described [27]. At 23 days post-EAU, eyes were graded for clinical severity [28]. (A) Normal retinal section grade 0. (B) Disease of grade 2 with vasculitis and retinal infiltration but no photoreceptor loss. (C) Severe disease grade 5 with typical features of granuloma (*), retinal folds (small arrow), loss of photoreceptor layer (arrowhead), and infiltration throughout the retinal layers; notice the retinal thickening compared to (B). H&E sections from AAV.GFP-injected eyes showing (D) extensive retinal infiltration (bar) and (F) vasculitis (*) and choroiditis (arrowhead). (E and G) H&E sections from AAV.IL-10-injected eyes with reduced EAU features. Within the AAV.IL-10 group, there was little photoreceptor layer infiltration or damage. (H) CD45-positive cells are found throughout the AAV.GFP retinal layers (small arrows). (I) In AAV.IL-10-treated eyes CD45-positive cells are rarely detected within the photoreceptor layer. Acetone-fixed retinal sections from AAV.IL-10-treated and AAV.GFP control eyes were stained for expression of nitrotyrosine (1:100, rabbit anti-nitrotyrosine; Upstate Ltd., UK). Primary antibody was detected with goat anti-rabbit: Alexa 594 (Molecular Probes, UK) and sections were counterstained with DAPI (Molecular Probes). Intense levels of positivity were detected (J) within the photoreceptor layer (arrow), (L) in areas of vasculitis, and (N) on cells present in the vitreous (small arrows) within eyes injected with AAV.GFP as expected during EAU. Much reduced levels of nitrotyrosine were detected in corresponding areas of infiltration of AAV.IL-10-injected retina: (K) photoreceptor layer with some infiltrate, (M) no nitrotyrosine by area of vasculitis, some photoreceptor positivity, and (O) very low expression on rare intravitreal cells (small arrows). Original magnifications: A, ×40; B and C, ×20; D–I, ×10; J and K, ×20; M, ×10; L, N, and O, ×40. Molecular Therapy 2005 12, 369-373DOI: (10.1016/j.ymthe.2005.03.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions