TY - JOUR
T1 - Synthetic Monopartite Peptide That Enables the Nuclear Import of Genes Delivered by the Neurotensin-Polyplex Vector
AU - Lopez-Salas, Francisco E.
AU - Nadella, Rasajna
AU - Maldonado-Berny, Minerva
AU - Escobedo-Sanchez, Maria L.
AU - Fiorentino-Pérez, Rosana
AU - Gatica-García, Bismark
AU - Fernandez-Parrilla, Manuel A.
AU - Mario Gil, Moreno
AU - Reyes-Corona, David
AU - García, Ubaldo
AU - Orozco-Barrios, Carlos E.
AU - Gutierrez-Castillo, Maria E.
AU - Martinez-Fong, Daniel
N1 - Funding Information:
This work was supported by Grant #254686 from Conacyt (DMF).
Funding Information:
The authors thank Rafael Leyva, Ricardo Gaxiola, and René Pánfilo Morales who are appointed to the Unit for Production and Experimentation of Laboratory Animals of the Center for Research and Advanced Studies. F.E.L.-S., B.G.G., and M.A.F.-P. were recipients of doctoral fellowships from the Consejo Nacional de Ciencia y Tecnología (Conacyt) de México. The authors also thank Dr. José Luis Reyes Sánchez and Ma. del Carmen Namorado Tonix for allowing the use of the spectrofluorimeter. The authors gratefully acknowledge the financial support from Conacyt Grant #254686 (D.M.-F.).
Publisher Copyright:
©
Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2020/12/7
Y1 - 2020/12/7
N2 - Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/β pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/β pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.
AB - Neurotensin (NTS)-polyplex is a multicomponent nonviral vector that enables gene delivery via internalization of the neurotensin type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells. An approach to improving its therapeutic safety is replacing the viral karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which performs the nuclear import activity, by a shorter synthetic peptide (KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid DNA translocation through the expression of the green fluorescent protein or red fluorescent protein fused with KPRa and internalization assays and whole-cell patch-clamp configuration experiments in a single cell together with importin α/β pathway blockers. We showed that KPRa electrostatically bound to plasmid DNA increased the transgene expression compared with KPSV40 and enabled nuclear translocation of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation was blocked with ivermectin or mifepristone, suggesting importin α/β pathway mediation. KPRa also enabled NTS-polyplex-mediated expression of reporter or physiological genes such as human mesencephalic-derived neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa is a synthetic monopartite peptide that showed nuclear import activity in NTS-polyplex vector-mediated gene delivery. KPRa could also improve the transfection of other nonviral vectors used in gene therapy.
KW - cancer
KW - fusion proteins
KW - gene therapy
KW - importins
KW - nonviral vectors
KW - Parkinson's disease
UR - http://www.scopus.com/inward/record.url?scp=85095817637&partnerID=8YFLogxK
U2 - 10.1021/acs.molpharmaceut.0c00755
DO - 10.1021/acs.molpharmaceut.0c00755
M3 - Artículo
C2 - 33125243
AN - SCOPUS:85095817637
SN - 1543-8384
VL - 17
SP - 4572
EP - 4588
JO - Molecular Pharmaceutics
JF - Molecular Pharmaceutics
IS - 12
ER -