Evaluation of alternative splice variant activity in an in vivo model of arthritis
Since angiogenesis plays a key role in RA, we next evaluated the therapeutic potential of ASV in an extensively validated mouse model of arthritis – namely, acute CIA. On the day of disease onset, replication-incompetent alternative splice variant-expressing adenoviruses were administered as a single dose of 1 × 107 plaque-forming units. The severity of arthritis in the mice was consecutively recorded for the following 10 days.
Control adenovirus (LacZ) was without significant effect on disease severity (Table 3 and Figures 5 and 6). In contrast, treatment with either Tie1-751 (Table 3 and Figure 5) or VEGFR1-541 (Table 3 and Figure 6) alternative splice variant adenoviruses significantly reduced disease severity, as evidenced by decreased clinical scores (P < 0.001), reduced paw thickness (P < 0.001), and reduced joint inflammation and destruction (P < 0.01 and P < 0.001 for VEGFR1-541 and Tie1-751, respectively). An example of the joint histology for untreated, LacZ ASV-treated and Tie1-751 ASV-treated mice is shown in Figure 5c, with quantitative analysis of the histology depicted in Table 3.
Table 3  Effect of alternative splice variant-expressing adenoviruses on joint inflammation and destruction  Following onset of arthritis, mice were treated with the alternative splice variant adenovirus indicated. Data presented as P values of mice treated with the indicated recombinant alternative splice variant adenoviruses as compared with untreated mice, and are expressed as the P value of clinical scores, paw swelling, and histological evaluation. For clinical scores and paw swelling, data were analyzed using two-way analysis of variance versus untreated mice. For histological evaluation, H & E and toluidine blue stained sections were scored for pannus formation, synovitis, and bone and cartilage erosion. Data were analyzed using the chi-square test for trend versus untreated mice.
Figure 5  Inhibition of murine collagen-induced arthritis by Tie1-751. On the day of arthritis onset, mice received intravenously 1 × 107 plaque-forming units of adenoviruses expressing either LacZ (○) or Tie1-751 alternative splice variants (ASV) (●), or remained untreated (□) as indicated. (a) Clinical score was recorded daily, and data were analyzed by two-way analysis of variance versus untreated mice. LacZ, not significant (P = 0.3734); Tie1-751, P < 0.001; n = 6 per group. (b) Paw swelling was recorded using calipers daily, and data were analyzed by two-way analysis of variance versus untreated mice. LacZ, not significant (P = 0.5134); Tie1-751, P < 0.001. Data are means of n = 6. (c) Serial sections of mouse hind feet were stained with either H & E (left panels) or toluidine blue (right panels). Figure shows tibia–tarsus joint sections from untreated mice (top panels), from LacZ adenovirus-treated mice (middle panels), and from Tie1-751 ASV adenovirus-treated mice (bottom panels). Sections are shown at 40× magnification; scale bar = 20 μm. (d) Pharmacokinetics of Tie1-751 from the ASV-expressing adenovirus. Sera from untreated mice or mice treated intravenously with 1 × 109 plaque-forming units of Tie1-751 ASV adenovirus were analyzed after the indicated times by western blot, followed by scanning and quantitation using Tie1-751 standard. (e) Effect of recombinant Tie1-751-Fc protein on clinical score. Results are from mice on day 10 of arthritis. Filled bars, untreated mice; empty bars, mice treated with recombinant Tie1-751-Fc 30 mg/kg, three times weekly. Data are means of n = 6. **P < 0.01 for Tie-751-Fc treated mice versus untreated mice.
Figure 6  Differential effects of alternative splice variant-expressing adenoviruses on collagen-induced arthritis mice. On the day of arthritis onset, mice received intravenously 1 × 107 plaque-forming units of the indicated alternative splice variant (ASV)-expressing adenoviruses. Clinical scores ((a), (c), and (e))and paw thickness measured by calipers ((b), (d), and (f))were recorded daily. Data were analyzed by two-way analysis of variance versus untreated mice (Table 3). (a) and (b) Mice received adenoviruses expressing either LacZ (○), VEGFR1-541 (■), VEGFR2-712 (▲) or VEGFR3-765 (●), or remained untreated (□). Data are means of n = 5 per group. (c) and (d) Mice received adenoviruses expressing either LacZ (○), Met-877 (■), Tie1-751 (▲) or FGFR1-320 (●), or remained untreated (□). Data are means of n = 6 per group. (e) and (f) Mice received adenoviruses expressing either LacZ (○), RAGE-387 (■), PDGFRβ-366 (▲), c-Kit-413 (●), or CSF1R-306 (◆), or remained untreated (□). Data are means of n = 6 per group.   The presence of Tie1-751 in mouse sera was confirmed by western blotting (Figure 5d). The effectiveness of Tie1-751 in CIA was confirmed using recombinant Tie1-751-Fc protein (Figure 5e).
A less marked disease-modifying effect was seen with the adenovirus encoding FGFR1-320 (Table 3 and Figure 6), which reduced clinical scores and paw thickness (P < 0.01) but without achieving a statistically significant improvement of joint histological evaluation (P < 0.057). Similarly, VEGFR2-712 reduced the clinical score (P < 0.001) but failed to affect the paw thickness and the histological scores (Table 3 and Figure 6).
Treatment with ASV derived from VEGFR3, RAGE, Met, c-Kit, PDGFRβ, and CSF1R adenoviruses did not generate a significant effect on any of the disease parameters (Table 3 and Figure 6).