Effect of MTX on transgene expression and bioactivity in RA FLS.

<p>In the presence of MTX hIFN-β transgene expression (A) and bioactivity shown by change in IL-1ra (B) and IL-8 (C) remained unaltered in RA FLS. All samples were stimulated with TNF-α. Control cells were stimulated but not transduced with ART-I02. Data shown are mean + SEM (hIFN-β) and mean + SD (cytokines), N = 3–6 experiments, * indicates P<0.05, ** P<0.01 and *** P<0.001.</p

Luminescence of rAAV5-CMV-Fluc detected during longitudinal follow up after intra-articular administration.

<p>Expression was monitored for a total of 7 weeks. Expression is visible as early as the first imaging moment (day 3) and remained stable up till day 49 (A). Images over time of 2 representative animals are shown for day 3, 14 and 49 (end of experiment)(B). Data are shown as mean + SEM, N = 5.</p

Human IFN-β expression levels in RA, rodent, rabbit and NHP FLS.

<p>Comparison of hIFN-β expression levels showed that DOX addition 4 hours post-transduction (‘Dox post’) improved expression in RA FLS more than 4-fold compared to DOX treatment 24 hours pre-transduction (‘Dox pre’) and almost 20-fold compared to medium only. In all three conditions, cells were stimulated with TNF-α (A). Transduction with ART-I02 resulted in increased levels of hIFN-β after stimulation of RA FLS. Without addition of ART-I02 or without stimulation (TNF-α and/or IL1-β), no hIFN-β was detected (B). Double stimulation with TNF-α and IL-1β did not increase the level of transgene expression compared to TNF-α stimulation alone (B). Human IFN-β production was detectable in culture supernatants of FLS of all species (C). For panel B and C, in all conditions doxorubicin was used. Data shown are mean + SEM, N = 2–3 experiments.</p

Vector biodistribution of ART-I02 in arthritic animals.

<p>Vectors were injected intra-articularly (IA) on day 14 after arthritis induction and vector DNA biodistribution was determined 1 and 4 weeks after administration by RT-PCR of a number of tissues (injected joint (right), non-injected joint (left), draining lymphnode, liver, lung, heart, testis, kidney, brain, spleen and blood) (N = 6 per group). Minimal vector spreading outside the joints was detected 1 and 4 weeks after intra-articular administration in animals with (A and B respectively) and without arthritis (C and D respectively). A different pattern was observed after intravenous (IV) administration of the vector, with almost 40% detected in the lungs (E). Data are shown as percentages of the total amount of vector retrieved. CIA, collagen induced arthritis.</p

Vector copies of ART-I02 detected per tissue after intra-articular or intravenous administration.

<p>Besides in the intra-articularly injected right joint, high copy numbers were observed in draining lymph nodes and lung tissue. Intermediate numbers of vector copies were detected in spleen, kidney and blood, low numbers in liver, blood and brain. Levels in heart and testis were very low or below the detection limit of the assay. Data shown are mean + SD, graphs are shown with logarithmic scale. The 100 vg copies detection limit is depicted with a gray dashed line. N = 6 per group. GFP, green fluorescent protein; IA, intra-articular; IV, intravenous; week 1 or 4, number of weeks after vector administration; AA, adjuvant arthritis model.</p

Bioactivity of ART-I02 in human and NHP FLS.

<p>In the presence of hIFN-β the anti-inflammatory cytokine IL-1ra was up regulated in RA FLS (A). Levels of pro-inflammatory cytokines (IL-6, IL-8) as well as MMP-3 were downregulated in RA FLS (A). A quantitative gene reporter bioassay confirmed the presence of bioactive hIFN-β after transduction of ART-I02 in combination with stimulation both in RA FLS as well as NHP FLS (B). In NHP FLS (C), IL-8 was also downregulated significantly, although IL-6 in NHP FLS showed only a trend towards downregulation. All samples were stimulated with TNF-α, except for the human IL-6 data, where samples were stimulated with TNF-α and IL-1β. Control cells were stimulated but not transduced with ART-I02. Data shown are mean + SD, N = 2–6 experiments, * indicates P<0.05, ** P<0.01 and *** P<0.001.</p

NCAP Reduces Histological Measures of Joint Damage.

<p>(A–B) Ankle joints were harvested on study day 16, stained with Toluidine Blue, and scored on a scale of 0-5 for inflammation, pannus formation, cartilage damage and bone resorption (A), with a composite summated score of 0–20 (B). Data are shown as mean+SE score. *p≤0.05 t-test versus CIA/Sham NCAP. (C–D) Representative 50X photomicrographs of ankle joints are shown which have the approximate mean summed score as that of the entire treatment group. Ankle from CIA/Sham NCAP group (C) demonstrates marked inflammation and synovitis (<b>S</b>) and mild cartilage damage (large arrow) and bone resorption (small arrow). Ankle from CIA/NCAP group (D) demonstrates mild inflammation and synovitis (S) and minimal cartilage damage (large arrow) and minimal bone resorption (small arrow).</p

NCAP Effect on Circulating Cytokines.

<p>Study day 16 serum was assayed for IL-1α, IL-1β, IL-2, IL-6, IFN-γ and TNF. Data are shown as mean+SE level, t-test p = NS for all individual cytokine comparisons between CIA/NCAP and CIA/Sham NCAP.</p

Histopathology Scoring Criteria.^*

<p>*Adapted from: Bendele, A., et al., <i>Effects of interleukin-1 receptor antagonist in a slow-release hylan vehicle on rat type II collagen arthritis.</i> Pharm Res, 1998. <b>15</b>(10): p. 1557–61.</p

Treatment Groups.

<p>Treatment Groups.</p