Scheme of the AAV2 vector construct.

<p>The transgene is under the control of a CMV promoter and followed by BGH poly (A). The expression cassette is flanked by ITRs. CMV promoter, human cytomegalovirus immediate early promoter; hIFN-α, human interferon-alpha; BGH poly (A), BGH poly-adenylation signal; ITR, AAV2 inverted terminal repeats.</p

Intraocular cytokines immunoassay on day 12 and day 21.

<p>Normal mice and mice receiving a subretinal injection and a subsequent IRBP immunization were sacrificed on day 12 and day 21 post-immunization. The ocular cytokine levels were assayed by ELISA. IFN-γ (A), IL-17 (B) and IL-10 (C) levels in rAAV2-CMV-mIL-27p28 treated eyes compared with rAAV2-CMV-GFP treated eyes (p values 0.009 and 0.772 were obtained by a paired T test. The p value 0.024 was calculated using the Mann-Whitney U test). Results are presented as mean ± standard deviation. ND = not detected. (n = 5).</p

Histological examinations on day 14 of EAU.

<p>Images of histological analysis show severe intraocular inflammation in PBS (<b>a,b</b>) and AAV2.GFP injected eyes (<b>c,d</b>) compared with AAV2.IFN-α treated (<b>e,f</b>), AAV2.IL-4 treated (<b>g,h</b>), and AAV2.IFN-α combined with AAV2.IL-4 treated eyes (<b>i,j</b>). (haematoxylin eosin staining, original magnification×100). EAU was significantly reduced in AAV2.IL-4 treated group, AAV2.IL-4 combined with AAV2.IFN-α treated group as compared with controls (<b>k</b>) (p<0.0001, Mann-Whitney U test). The AAV2.IFN-α treated group also shows a significantly decreased uveitis (p = 0.005). Each point is the score of an individual eye. The mean scores of each group are denoted by the horizontal bars.</p

The expression of transgenes following subretinal injection

<p>. In the AAV2.IFN-α injected eyes, the level of IFN-α increases from 14 days (the first time point tested) to three months after injection. For the eyes receiving AAV2.IFN-α combined with AAV2.IL-4 injection, IFN-α level reaches a peak on day 42 and remains at a moderate level until three months after injection (<b>a</b>). IL-4 expression is similar in eyes receiving an injection of AAV2.IL-4 alone as compared to eyes receiving AAV2.IL-4 combined with AAV2.IFN-α (<b>b</b>). Results are expressed as the mean±standard deviation.</p

Clinical evaluation of EAU activity.

<p>EAU was induced in groups of mice receiving subretinal vector or PBS injection. Severe uveitis is observed in the PBS (<b>a</b>) and AAV2.GFP injected eyes (<b>b</b>) as compared to the AAV vectors treated eyes (<b>c–e</b>). Three AAV vector treated groups show significantly attenuated EAU over time as compared with controls (<b>f</b>). Data are presented as mean±standard deviation. The clinical score shows that compared with controls, the AAV2.IFN-α treated group (p = 0.019, Mann-Whitney U test), the AAV2.IL-4 treated group (p<0.0001) and the combined treated group (p<0.0001) developed a significantly reduced EAU (<b>g</b>). Each point represents an individual eye. The average scores of each group are denoted by the horizontal bars.</p

Systemic IRBP-specific immune responses in each group.

<p>DTH responses were elicited on day 19 of EAU and evaluated on day 21. Data show no significant difference of ear swelling among all the tested groups (p>0.05) (<b>a</b>). IRBP-specific lymphocyte proliferation (<b>b</b>) and IL-17 production <i>in vitro</i> (<b>c</b>) show no significant difference among the five tested groups (P>0.05). Results are presented as mean±standard deviation. 5–6 animals per group were used and each experiment was performed three times.</p

The expression of transgene following subretinal injection of rAAV2-CMV-mIL-27p28.

<p>The intraocular IL-27p28 levels at various time points show that IL-27p28 expression is stable over 90 days, its expression reaches a peak on day 150 and remains detectable until 9 months. Results are expressed as the mean ± standard deviation. (n = 3).</p

Histological examination on day 14 of EAU.

<p>Images of histological analysis show severe retinal folding, destruction, damage of the photoreceptor layer and massive inflammatory cell infiltration in the retina and the choroid, as well as intensive vasculitis in PBS injected eyes (A), moderate infiltration, moderate photoreceptor cell damage and medium-sized granulomas in rAAV2-CMV-GFP injected eyes (B). However, a minor infiltration of cells was observed in the choroid in rAAV2-CMV-mIL-27p28 treated eye (C). (hematoxylin eosin staining, magnification ×200) Comparing PBS injected and rAAV2-CMV-GFP injected eyes with rAAV2-CMV-mIL-27p28 treated eyes showed a reduced EAU histological grade (D, p = 0.006, p = 0.003, respectively). The scores of the three chosen eye section are 4, 3 and 0.5, respectively. Each point is the score of an individual eye, the mean scores of each group are denoted by the horizontal lines.</p

Clinical evaluation of EAU activity.

<p>rAAV2-CMV-mIL-27p28 was subretinally injected into the eye, PBS and rAAV2-CMV-GFP were used as controls. Three weeks after injection, mice were immunized to induce EAU with IRBP_161–180 peptide and slit lamp microscopy was used to examine ocular inflammation. Images show significantly more severe inflammation in the PBS (A) and rAAV2-CMV-GFP injected eyes (B) as compared with the rAAV2-CMV-mIL-27p28 treated eye (C). Clinical scoring on day 12 after immunization (D) showed that the PBS injected eyes had a score of 1.86 (±0.8) and the rAAV2-CMV-GFP injected eyes reached a mean clinical score of 2.25 (±0.59), whereas the score of rAAV2-CMV-mIL-27p28 treated eyes was 1 (±0.24, p = 0.024, p<0.001, respectively). The scores of three chosen eye pictures are 3, 3 and 0.5, respectively. Each point represents an individual eye, the average scores of each group are denoted by the horizontal lines.</p

Systemic immune responses following subretinal IL-27p28 gene transfer.

<p>In vivo DTH (A), in vitro IRBP-specific lymphocyte proliferation (B) and IFN-γ and IL-17 (C, D) production showed no significant difference among PBS treated mice, rAAV2-CMV-mIL-27p28 and rAAV2-CMV-GFP treated mice (p>0.05). Results are presented as mean ± standard deviation. (n = 5).</p