Original Article
Regulation of human GDNF gene expression in nigral dopaminergic neurons using a new doxycycline-regulated NTS-polyplex nanoparticle system

https://doi.org/10.1016/j.nano.2017.02.006Get rights and content

Abstract

The human glial-cell derived neurotrophic factor (hGDNF) gene transfer by neurotensin (NTS)-polyplex nanoparticles functionally restores the dopamine nigrostriatal system in experimental Parkinson's disease models. However, high levels of sustained expression of GDNF eventually can cause harmful effects. Herein, we report an improved NTS-polyplex nanoparticle system that enables regulation of hGDNF expression within dopaminergic neurons. We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element (TRE). Another bifunctional plasmid contained the enhanced green fluorescent protein (GFP) gene. Transient transfection experiments in N1E-115-Nurr1 cells showed that doxycycline (100 ng/mL) activates hGDNF and GFP expression. Doxycycline (5 mg/kg, i.p.) administration in rats activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific doxycycline-regulated system suitable for nanomedicine-based treatment of Parkinson's disease.

Graphical Abstract

We constructed NTS-polyplex nanoparticles containing a single bifunctional plasmid that codes for the reverse tetracycline-controlled transactivator advanced under the control of NBRE3x promoter, and for hGDNF under the control of tetracycline-response element. Those NPs were injected into the rat substantia nigra. Accordingly, doxycycline administration activated hGDNF expression only in transfected dopaminergic neurons, whereas doxycycline withdrawal silenced transgene expression. Our results offer a specific regulatable system free of hGDNF transcriptional leakage for use in gene therapy for Parkinson's disease.

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Section snippets

Plasmids

pNBRE3x-rtTA-Adv-TRE-hGDNF (5229 bp; bifunctional plasmid) codes for rtTA-Adv under the control of NBRE3x promoter and for hGDNF under the control of TRE (Figure S1). The TRE-hGDNF sequence (1230 bp) was released with XhoI from pTRE-Tight-hGDNF and subcloned into XhoI site of pNBRE3x -Tet-On-Adv.

pNBRE3x-rtTA-Adv-TRE-EGFP (5309 bp; bifunctional plasmid) codes for rtTA-Adv under the control of 3xNBRE promoter and for GFP under the control of TRE (Figure S1). The TRE-EGFP sequence (1310 bp) was

Physical features of NTS-nanoparticles with the bifunctional plasmids

SEM analysis showed that NTS-polyplex NPs harboring the plasmid pNBRE3x-rtTA-Adv-TRE-hGDNF (Figure 1, A and B) or pNBRE3x-rtTA-Adv-TRE-GFP (Figure 1, E and F) have a diameter of 40 to 120 nm. DLS analysis showed a mean diameter of NST-polyplex NPs containing the plasmid pNBRE3x-rtTA-Av-TRE-hGDNF of 185.7 nm and a mean zeta potential of +22.6 mV for NPs (Figure 1, C and D), whereas the NPs with the plasmid pNBRE3x-rtTA-Av-TRE-EGFP had a mean diameter of 179.3 nm and a mean zeta potential of

Discussion

Our results offer an improved NTS-polyplex NP system that enables transgene regulation within nigral DA neurons. The decrease in pDNA size by the inclusion of an hDAT minimum promoter (NBRE3x promoter) allowed the incorporation of the key regulatory components (rtTA-Adv and TRE) of the Tet On advanced system in a single plasmid that also contains the transgene of interest (bifunctional plasmid). The size of NTS-polyplex NPs containing the Dox-regulated plasmid can fit the endosome space that

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    Armando J. Espadas-Alvarez was the recipient of scholarship from CONACYT (210279).

    The work was supported by CONACYT 254686 (DMF) and FINNOVA 224222 (APV).

    Competing Interests: The authors have declared that no competing interests exist. Funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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