Original ArticleRegulation of human GDNF gene expression in nigral dopaminergic neurons using a new doxycycline-regulated NTS-polyplex nanoparticle system
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.
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
References (57)
- et al.
NTS-polyplex: a potential nanocarrier for neurotrophic therapy of Parkinson's disease
Nanomedicine
(2012) - et al.
Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model
Brain Res
(2000) - et al.
Neurotensin polyplex as an efficient carrier for delivering the human GDNF gene into nigral dopamine neurons of hemiparkinsonian rats
Mol Ther
(2006) - et al.
Pharmacologically controlled, discontinuous GDNF gene therapy restores motor function in a rat model of Parkinson's disease
Neurobiol Dis
(2014) Aberrant sprouting and downregulation of tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer
Exp Neurol
(2002)The need for regulatable vectors for gene therapy for Parkinson's disease
Exp Neurol
(2008)- et al.
Tight long-term dynamic doxycycline responsive nigrostriatal GDNF using a single rAAV vector
Mol Ther
(2009) - et al.
Safety of the intravenous administration of neurotensin-polyplex nanoparticles in BALB/c mice
Nanomedicine
(2014) - et al.
Improved neurotensin-vector-mediated gene transfer by the coupling of hemagglutinin HA2 fusogenic peptide and Vp1 SV40 nuclear localization signal
Brain Res Mol Brain Res
(2002) - et al.
Synthesis of a non-viral vector for gene transfer via the high-affinity neurotensin receptor
Brain Res Brain Res Protoc
(2000)
The dopamine transporter: role in neurotoxicity and human disease
Toxicol Appl Pharmacol
Characterization of the 5'-flanking region of the human dopamine transporter gene
Brain Res Mol Brain Res
Developmental regulation of neurotensin receptor expression and function in murine neuroblastoma clone N1E-115
Eur J Pharmacol
Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation
J Biol Chem
Characterization of the lipopolysaccharide induced model of Parkinson's disease: role of oxidative stress and neuroinflammation
Neurochem Int
Endosome maturation, transport and functions
Semin Cell Dev Biol
Effect of genome size on AAV vector packaging
Mol Ther
Brain-derived neurotrophic factor transgenic mice exhibit passive avoidance deficits, increased seizure severity and in vitro hyperexcitability in the hippocampus and entorhinal cortex
Neuroscience
Brain-derived neurotrophic factor (BDNF) overexpression in the forebrain results in learning and memory impairments
Neurobiol Dis
Retroviral integration profiles: their determinants and implications for gene therapy
BMB Rep
Innate immune response induced by gene delivery vectors
Int J Pharm
Brain morphometry and the neurobiology of levodopa-induced dyskinesias: current knowledge and future potential for translational pre-clinical neuroimaging studies
Front Neurol
Treatment of Parkinson's disease in the advanced stage
J Neural Transm
Neuroprotection in a 6-hydroxydopamine-lesioned Parkinson model using lactoferrin-modified nanoparticles
J Gene Med
Angiopep-conjugated nanoparticles for targeted long-term gene therapy of Parkinson's disease
Pharm Res
Transgenic expression of human glial cell line-derived neurotrophic factor from integration-deficient lentiviral vectors is neuroprotective in a rodent model of Parkinson's disease
Hum Gene Ther
Overexpression of glial cell line-derived neurotrophic factor using a lentiviral vector induces time- and dose-dependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system
J Neurosci
Long-term glial cell line-derived neurotrophic factor overexpression in the intact nigrostriatal system in rats leads to a decrease of dopamine and increase of tetrahydrobiopterin production
J Neurochem
Cited by (13)
The potential use of tetracyclines in neurodegenerative diseases and the role of nano-based drug delivery systems
2022, European Journal of Pharmaceutical SciencesCitation Excerpt :The in vitro study conducted in LPS-activated murine BV-2 microglial cells demonstrated that D-mino markedly reduced oxidative stress by suppressing NO production and inflammatory cytokine TNF-α production. In Espadas-Alvarez et al. (2017) developed Neurotensin (NTS)-polyplex NPs containing a single bifunctional doxycycline-regulated plasmid in order to obtain an adequate alternative for viral vectors and destined to regulate human GDNF (hGDNF) gene expression. The formulation was assayed in a PD model induced in male Wistar rats.
Nanomaterials as novel agents for amelioration of Parkinson's disease
2021, Nano TodayCitation Excerpt :As described above, measures to transport therapeutic nanomaterials to desired sites include receptor-mediated binding and guidance by physical forces. For example, neurotensin, a 13-amino acid peptide, is considered a suitable targeting molecule for the delivery of NPs to the central nervous system (CNS) neurons due to its high affinity for neurotensin receptors expressed on the plasma membranes of dopaminergic neurons [202]. This ligand-receptor binding ensures a precise delivery of the drug without side effects on other cells.
Polyplexes for gene and nucleic acid delivery: Progress and bottlenecks
2020, European Journal of Pharmaceutical SciencesCitation Excerpt :The authors presented evidence to demonstrate that only the polyplex nanoparticles that selectively targeted the cholecystokinin-B (CCK-B) receptor (thus delivering gastrin siRNA into the tumor) were efficacious in reducing metastasis and the primary tumor size (Burks et al., 2018). In another interesting study, Espadas-Alvarez and coworkers investigated human GDNF gene expression regulation in nigral dopaminergic neurons by utilizing novel doxycycline-regulated neurotensin-polyplex nanoparticles (Espadas-Alvarez et al., 2017). The authors prepared neurotensin-polyplex nanoparticles and incorporated into the delivery system a single bifunctional plasmid that codes for the human glial-cell derived neurotrophic factor under the control of the tetracycline-response element (TRE) promoter and also codes for the reverse tetracycline-controlled transactivator advanced (rtTA-Adv) (also known as rtTA2S-M2) (Liu et al., 2013) under the control of NBRE3x promoter (Espadas-Alvarez et al., 2017).
Inducible Expression of GDNF in Transplanted iPSC-Derived Neural Progenitor Cells
2018, Stem Cell ReportsCitation Excerpt :Overall, there is a clear rationale for growth factor regulation in these types of clinical trial. External factors such as mifepristone, rapamycin, or tetracycline ([Tet], and its analog doxycycline [dox]) can be used to regulate gene expression (Akhtar et al., 2015; Espadas-Alvarez et al., 2017; Tereshchenko et al., 2014). GDNF delivered by direct gene transfer in the rodent has been regulated (Chtarto et al., 2016), but this has yet to be accomplished for engrafted human neural cells engineered to release GDNF.
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.