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Aerosolized Non-viral Nucleic Acid Delivery in the Vaginal Tract of Pigs

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Abstract

Purpose

The human pathogen Chlamydia trachomatis is worldwide the leading cause of bacterial sexually transmitted disease. Nasal or vaginal nucleic acid vaccination is a promising strategy for controlling genital Chlamydia trachomatis infections. Since naked nucleic acids are generally not efficiently taken up by cells, they are often complexed with carriers that facilitate their intracellular delivery.

Methods

In the current study, we screened a variety of commonly used non-viral gene delivery carriers for their ability to transfect newborn pig tracheal cells. The effect of aerosolization on the physicochemical properties and transfection efficiency of the complexes was also evaluated in vitro. Subsequently, a pilot experiment was performed in which the selected complexes were aerosolized in the vaginal tract of pigs.

Results

Both mRNA and pDNA containing lipofectamine and ADM70 complexes showed promise for protein expression in vitro, before and after aerosolization. In vivo, only lipofectamine/pDNA complexes resulted in high protein expression levels 24 h following aerosolization. This correlates to the unexpected observation that the presence of vaginal mucus increases the efficiency of lipofectamine/pDNA complexes 3-fold, while the efficiency of lipofectamine/mRNA complexes and ADM70/mRNA and ADM70/pDNA complexes decreased.

Conclusions

As aerosolization was an easy and effective method to deliver complexes to the vaginal tract of pigs, we believe this application technique has future potential for both vaginal and perhaps nasal vaccination using non-viral gene delivery vectors.

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Abbreviations

C.:

Chlamydia

DLS:

Dynamic light scattering

DMEM:

Dulbecco’s modified Eagle’s medium

DMPE:

1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine

DMSO:

Dimethylsulfoxide

DOPE:

Dioleoylphosphatidylethanolamine

DOTAP:

1,2-dioleoyl-3-trimethylammonium-propane

FCS:

Fetal calf serum

GFP:

Green fluorescent protein

GL67:

Genzyme lipid 67

Luc:

Luciferase

MTT:

3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide

NPTr:

Newborn pig tracheal

PEG:

Polyethylene glycol

PEI:

Polyethyleneimine

ROI:

Regions of interest

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ACKNOWLEDGMENTS AND DISCLOSURES

Oliwia Andries is a doctoral fellow of the Research Foundation Flanders. Their financial support is acknowledged with gratitude. E. De Clercq has a PhD fellowship from the Special Research Fund of Ghent University. We also acknowledge funding support from the Research Foundation Flanders (FWO grant n° G.0621.10) and the Special Research Fund (BOF) of Ghent University. Dr. Bakr Ahmed is acknowledged for the collection of pig’s cervical mucus. J. M. García Fernández and J. M. Benito acknowledge financial support from the Junta de Andalucía (contract number FQM2012-1467), and the European Regional Development Funds (FEDER).

Compliance with Ethical Standards

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Ghent University).

Conflict of Interest

The authors declare that they have no conflict of interest.

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    Authors and Affiliations

    1. Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, B-9000, Ghent, Belgium

      Katrien Remaut, Koen Rombouts, Ian Vandenbussche & Sarah Van Praet

    2. Laboratory of Immunology and Animal Biotechnology, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, Ghent, B-9000, Belgium

      Evelien De Clercq, Matthias Van Gils & Daisy Vanrompay

    3. Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820, Merelbeke, Belgium

      Oliwia Andries, Laetitia Cicchelero & Niek Sanders

    4. Institute for Chemical Research, CSIC, University of Sevilla, Americo Vespucio 49, Isla de la Cartuja, E-41092, Sevilla, Spain

      Juan Manuel Benito & José Manuel Garcia Fernandéz

    5. Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Coupure links, 653, B-9000, Ghent, Belgium

      Daisy Vanrompay

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    1. Katrien Remaut
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    Correspondence to Daisy Vanrompay.

    Additional information

    Niek Sanders and Daisy Vanrompay contributed equally to this work.

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    Suppl Fig 1

    Cell viability. NPTr cells were treated with different mRNA/carrier complexes as depicted in the x-axis. Blanc represents non-transfected cells and DMSO served as positive control. * Significantly different from non-treated cells (blanc) (P ≤ 0.05). (JPG 1.12 mb)

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    Remaut, K., De Clercq, E., Andries, O. et al. Aerosolized Non-viral Nucleic Acid Delivery in the Vaginal Tract of Pigs. Pharm Res 33, 384–394 (2016). https://doi.org/10.1007/s11095-015-1796-x

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    • DOI: https://doi.org/10.1007/s11095-015-1796-x

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