In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor

Background: Recently, we synthesized a nonviral gene vector capable of tran sfecting cell lines taking advantage of neurotensin (NT) internalization. T he vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polypl ex. This hypothesis was tested here using reporter genes encoding green flu orescent protein or chloramphenicol acetyl transferase. Materials and Methods. NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involve d in the propidium iodide-labeled polyplex internalization and reporter gen e expression. Results. Polyplex internalization was observed within dopamine neurons but not within glial cells, and was pre-vented by both hypertonic sucrose solut ion and SR-48692, a selective nonpeptide antagonist of NT receptors. Report er gene expression was observed in dopamine neurons from 48 hr up to 15 day s after NT-polyplex injection, and was prevented by SR-48692. However, no e xpression was seen when the NT-polyplex was injected into the ansiform lobu le of the cerebellum, which contains low- but not high-affinity NT receptor s. Neither internalization nor expression was observed in cultured glial ce lls, despite the NT-polyplex binding to those cells that was prevented by l evocabastine, a low-affinity NT receptor antagonist. Conclusions. These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in v ivo. NT polyfection may be used to transfer genes of physiologic interest t o nigrostriatal dopamine neurons, and to produce transgenic animal models o f dopamine-related diseases.