Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal
Por:
Navarro-Quiroga I., Antonio González-Barrios J., Barron-Moreno F., González-Bernal V., Martinez-Arguelles D.B., Martinez-Fong D.
Publicada:
1 ene 2002
Resumen:
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. © 2002 Elsevier Science B.V. All rights reserved.
Filiaciones:
Navarro-Quiroga 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 D.F., Mexico
Programa de Doctorado en Ciencias Biomédicas, Facultad de Medicina, Universidad Nacional Autónoma de México, Apdo. Postal 70-250, 04510 México D.F., Mexico
Antonio González-Barrios J.:
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 D.F., Mexico
Barron-Moreno F.:
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 D.F., Mexico
González-Bernal V.:
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 D.F., Mexico
Martinez-Arguelles D.B.:
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 D.F., Mexico
Martinez-Fong D.:
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 D.F., Mexico
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