Figure 3.

Application of the MultiBac system in gene therapy. rAAV particles are produced by co-infection with three different baculoviruses 48, 49. Bac-Rep harbors two expression cassettes that contain genes for the major AAV replication enzyme, Rep78, and an N-terminal truncation of Rep78, Rep52Δ. rAAV-Bac contains AAV inverted terminal repeat (ITR) elements that are required for rescue, replication and packaging of transgene sequences, together with rat leptin cDNA under the control of a chicken β-actin promoter, which is inserted into the rAAV-Bac baculoviral genome by Cre–LoxP-mediated fusion of a specifically tailored donor plasmid [52]. Leptin is a hormone that acts in the brain to reduce food intake and stimulate energy expenditure. Bac-VP produces the AAV virion coat proteins. Complete rAAV virions containing the leptin gene are produced in triply co-infected insect cells and purified 50, 52, and then administered to diet-induced obese rats. An obese rat is shown compared to a normal rat for illustration (bottom). Diet-induced obesity renders laboratory rats (and presumably other species) resistant to leptin treatment. Therefore, it is close to impossible to curtail diet-induced weight gain. This could be overcome by circumventing leptin resistance or restoring leptin actions in obese animals. The surprising outcome of the study involving baculovirus-produced leptin rAAV as a gene therapy vector was that exercise, in this study wheel-running, was required to prevent completely weight gain when combined with the leptin gene therapy intervention, leading to the conclusion that work-out, in tandem with leptin gene delivery, may actually develop into a potential antiobesity treatment [52]. The baculovirus schematic drawing is adapted from an image kindly supplied by K. J. Airenne (University of Kuopio, Finland). The rAAV particles shown are based on PDB entry 1LP3 [59].