Figure 1

Improved IT gene transfer by pseudotyping sc-rAAV vectors with the serotype 1 capsid. (A.) sc-rAAV vectors packaged with capsids of the serotypes 1, 2, and 5 were evaluated. Four months after IT vector application (3 × l0⁹ particles/animal), animals were sacrificed, the lower half of the SC including the CE and the surrounding meninges was harvested, frozen at -80°C, pulverized, and subjected to Western blotting (20 μg protein loaded per lane). A representative Western blot demonstrating EGFP expression in rats injected with vectors of the serotypes 1 and 5 is shown. No expression was seen with serotype 2 or in negative control rats injected with PBS (marked as '-'). Actin served as a loading control. (B.) One month after IT vector administration expression with serotype 1 was demonstrable in five out of six rats. The failure to detect EGFP expression in one rat may have been related to IT bleeding at the time of catheter placement and/or to inattention in harvesting the nerve roots of the CE, which were included with the lumbar segments of the SC in this experiment. '(+)' indicates expression in the SC of an EGFP-transgenic mouse, which served as positive control. Equal loading of lanes was confirmed by protein staining of the membranes (not shown). (C.) qPCR demonstrated significantly higher IT expression with serotype 1 (●) compared with serotype 2 (■) (p < 0.01). The scale of the ordinate is inverted indicating the threshold cycle T_c with lower cycle numbers corresponding to higher expression. No expression was seen in PBS controls (▲) as indicated by T_c > 40. The T_c for the house keeping gene ubiquitin varied little and was not different between the groups.