论文信息 - I mproved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal

I mproved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal

Recently we reported that neurotensin-SPDP-polyL-l sine (NT-vector) is able to bind plasmid DNA (NT-polyplex) and polyfect cells expressing the high-affinity neurotensin receptor (NTRH) although with low transfecting efficiency: in vitro, 6.5 61.5%, and in vivo, 564%. In this work, we attempted to increase the transfecting efficiency by integrating the hemagglutinin HA2 fusogenic peptide and the Vp1 nuclear localization signal of SV40 to the NT-polyplex (fusogenic-karyophilic-NT-polyplex). Confocal microscopy and flow cytometry analysis showed that the fusogenic-karyophilic-NT-polyplex produced mostly nuclear localization of the plasmid DNA in NTRH-bearing N1E-115 cells. About 50% of N1E-115 cells internalized and expressed the reporter gene when the plasmid DNA was transferred by the fusogenic-karyophilic-NT-polyplex. Although to a less extent, the addition of each viral peptide separately to NT-polyplex (fusogenic-NT-polyplex or karyophilic-NT-polyplex) improved polyfection. Fusogenic-NT-polyplex produced 22.41 65.96% of internalization and 20.35 60.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75 6 .88% and 10.9462.04%, respectively. Basal internalization and expression were detected in N1E-115 cells in the presence of 100 nM SR-48692 and in NTRH-lacking cells. The fusogenic-karyophilic-NT-polyplex was microinjected into the substantia nigra to test its ability for gene transfer in vivo. Fusogenic-karyophilic-NT-polyplex internalization was observed within dopamine neurons only. Reporter gene expression was observed in a high proportion of dopamine neurons up to 60 days after NT-polyfection. Both internalization and expression were prevented by SR-48692. Our results show that the fusogenic-karyophilic-NT-polyplex is a highly efficient and specific gene vector and encourage its use to transfer gene of physiological interest to NTRH-bearing neurons.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Uptake and transporters

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