Improved 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-poly-L-lysine (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+/-1.5%, and in vivo, 5+/-4%. 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+/-5.96% of internalization and 20.35+/-0.82% of expression in N1E-115 cells, whereas karyophilic-NT-polyplex yielded 13.75+/-3.88% and 10.94+/-2.04%, 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.
Navarro-Quiroga
Iván
I
Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional de México, Apartado postal 14-740, 07000 México DF, Mexico.
Antonio González-Barrios
Juan
J
Barron-Moreno
Fernando
F
González-Bernal
Víctor
V
Martinez-Arguelles
Daniel B
DB
Martinez-Fong
Daniel
D
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics*; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D004298:Dopamine / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006389:Hemagglutinins, Viral / Q000235:genetics*; D046911:Macromolecular Substances; D008297:Male; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009496:Neurotensin / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D018028:Receptors, Neurotensin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D013378:Substantia Nigra / Q000166:cytology / Q000378:metabolism
D036022:Capsid Proteins; D006389:Hemagglutinins, Viral; D046911:Macromolecular Substances; D018028:Receptors, Neurotensin; D011993:Recombinant Fusion Proteins; C062481:VP1 protein, polyomavirus; C452559:hemagglutinin HA-2 fusogenic peptide, Influenza virus; D009496:Neurotensin; D004247:DNA; D004298:Dopamine