Synthetic Monopartite Peptide That Enables the Nuclear Import of Genes Delivered by the Neurotensin-Polyplex Vector

Abstract

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.

Focused ultrasound on the substantia nigra enables safe neurotensin-polyplex nanoparticle-mediated gene delivery to dopaminergic neurons intranasally and by blood circulation

“…The fusogenic peptide translocates the NPs to the cytoplasm, thus avoiding pDNA endosomal degradation [ 3 , 8 ]. A karyophilic peptide, also electrostatically bound to the pDNA, contributes to pDNA compaction and targets it to the cell nucleus through the α/β importin system to express the codifying sequence [ 10 ]. Physical characterization of NTS-polyplex NPs using transmission and scanning electron microscopies and dynamic light scattering shows that their size ranges from 50 to 150 nm and possess an electrical surface charge of 22 ± 7 mV [ 3 , 5 , 7 , 11 , 12 ].…”

“…Evans Blue (EB) dye was used as a positive control of BBB opening [ 52 ]. Immediately after FUS, the same amount of NTS-polyplex NPs harboring the enhanced green fluorescent protein (GFP) plasmid, proven safety in vitro [ 3 , 10 ] and in vivo [ 14 ], was delivered via the internal carotid artery, retro-orbital venous sinus, or nasal mucosa [ 52 ] to demonstrate GFP expression in nigral dopaminergic neurons and axonal projections. The harmful effect of this procedure was evaluated through neuroinflammation cell variables and the condition of the dopaminergic nigrostriatal system over time.…”