475. SeV-Mediated Gene Transfer after Three Re-Administrations in Mice and Sheep

Top of pageAbstract Recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane and leads to high, but transient gene expression. The treatment of chronic diseases may require repeated administration of the virus. We have previously shown that three repeat administrations of recombinant Sendai virus (DF/SeV) did not lead to measurable luciferase (lux) expression after the third administration. However, as part of these experiments we noted that even after one administration, lux expression was lower than expected for a given virus dose. Sequence analysis revealed that the lux gene contained sequences that may be recognised by SeV as |[ldquo]|stuttering|[rdquo]| sequences and may interfere with gene expression. In an attempt to improve SeV-mediated lux expression we introduced several silent mutations to modify these putative stuttering sequences. SeV-mediated lux expression in the lung was increased 2.5-fold (n=10, p<0.005), when compared to the unmodified lux. We next repeated the re- administration experiment using both weekly and monthly dosing intervals [dose 1: SeV-GFP, dose 2: SeV-GFP, dose 3: SeV-lux, 108 pfu/mouse/dose (n=20/dose)]. Lux expression was measured two days after SeV-lux administration. Lux expression after three monthly administrations of SeV was significantly reduced compared to single administration, but remained significantly (p<0.005) higher than untransduced controls. Lux expression was similar to expression levels that can be achieved with the non-viral gene transfer agent GL67. To assess whether the expression pattern is similar in a host in which wild type SeV infection is of no apparent significance (as in men), we transduced sheep with DF/SeV-lacZ. We first determined the time-course of b-gal expression after aerosolisation of 2|[times]|1010 pfu/sheep (n=4) using a Trudell AeroProbe catheter. Bronchial biopsies (Bx, n=8/sheep/time-point) were collected 2-28 days after transduction and bgal expression compared to pre-treatment values. Similar to mice, gene expression in sheep was maximal 2 days after gene transfer (UT: 43.7|[plusmn]|4.3; d2: 5705.7|[plusmn]|3708.2 RLU/mg protein, p<0.05) and had returned to pre-treatment values 7 days after transduction. After monthly repeat administration (doses 1+2: DF/ SeV-empty, dose 3: DF/SeV-lacZ, n=4) we also detected low but significant (p<0.05) residual bgal expression (47.5|[plusmn]|7.1 RLU/mg protein, n=20 bx/sheep) compared to sheep receiving 3 doses of DF/SeV-empty (29.5|[plusmn]|1.1 RLU/mg protein, n=4). In conclusion, the expression pattern of SeV is similar in mice and sheep. Expression is transient, but after repeat administration of 3 doses, low but significant gene expression similar to levels seen with non-viral gene transfer agents can be detected.