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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Published: February 1st, 2019

DOI:

10.3791/58444

1Department of Thoracic and Cardiovascular Surgery, Clinical Research Laboratory, University Medical Center, 2Atherothrombosis and Vascular Biology, Baker Heart & Diabetes Institute, 3Department of Medicine, Monash University

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.

For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

The use of modified mRNA for therapeutic applications has shown great potential in the last couple of years. In cardiovascular, inflammatory, and monogenetic diseases, as well as in developing vaccines, mRNA is a promising therapeutic agent1.

Protein replacement therapy with mRNA offers several advantages over the classical gene therapy, which is based on DNA transfection into the target cells2. The mRNA function initiates directly in the cytosol. Although the plasmid DNA (pDNA), a construct of double-stranded, circular DNA containing a promoter region and a gene sequence encoding the therapeu....

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1. Generation of Cationic Nanoliposomes (Figure 1)

  1. Dissolve the lipids DC-cholesterol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride) and DOPE (dioleoyl phosphatidylethanolamine), delivered as a powder, in chloroform to achieve a final concentration of 25 mg/mL.
    Note: Store the dissolved lipids at -20 °C.
  2. Work with 25 mg/mL stock solution of both lipids. Mix 40 µL of the dissolved DC-cholesterol and 80 µL of.......

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Using the protocol as described, NLps consisting of the lipids DC-cholesterol and DOPE were prepared using the dry-film method (Figure 1). During the preparation, the nanoliposome solution shows different stages in turbidity (Figure 2).

The encapsulation efficacy of the NLps can then be analyzed after the encapsulation of 1 µg of eGFP-encoding mRNA by analyzing the.......

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The presented protocol describes the generation of NLps with high encapsulation efficacy for synthetically modified mRNA, as well as the reliable transfection of cells in vitro. Moreover, the NLps guarantee the release of mRNA, which in turn, is translated into a functional protein inside the cells. Additionally, the transfections using NLps can be performed in regular cell medium, resulting in high cell viabilities during transfection, and last up to three days after transfection.

To.......

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Name Company Catalog Number Comments
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) AppliChem, Darmstadt, Germany A2231
(3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol) Avanti, Alabama, USA 700001
4 ′,6-diamidino-2-phenylindole (DAPI) Thermo Fisher Scientific, Darmstadt, Germany D1306
BD FACScan system BD Biosciences, Heidelberg, Germany
Cell Fix (10x) BD Biosciences, Heidelberg, Germany 340181
Chloroform Merck, Darmstadt, Germany 102445
Dimethyl sulfoxid (DMSO) Serva Electrophoresis GmbH, Heidelberg, Germany 20385.02
Dioleoyl phosphatidylethanolamine (DOPE) Avanti, Alabama, USA 850725
Fluorescence microscope Zeiss Axio, Oberkochen, Germany
Lipofectamine 2000 Thermo Fisher Scientific, Darmstadt, Germany 11668019
Mini extruder Avanti, Alabama, USA
Nuclease-free water Qiagen, Hilden, Germany 129114
Opti-Mem Thermo Fisher Scientific, Darmstadt, Germany 11058021
PBS buffer (w/o Ca2+/Mg2+) Thermo Fisher Scientific, Darmstadt, Germany 70011044
Quant-iT Ribo Green RNA reagent kit Thermo Fisher Scientific, Darmstadt, Germany Q33140
RPMI (w/o phenol red) Thermo Fisher Scientific, Darmstadt, Germany 11835030
Silica gel Carl Roth, Karlsruhe, Germany P077
Trypsin/EDTA (0.05%) Thermo Fisher Scientific, Darmstadt, Germany 25300054
HotStar HiFidelity Polymerase Kit Qiagen, Hilden, Germany 202602
QIAquick PCR Purification Kit Qiagen, Hilden, Germany 28104

Pseudouridine-5'-Triphosphate (Ψ-UTP)
TriLink Biotechnologies, San Diego, USA N-1019
5-Methylcytidine-5'-Triphosphate (Methyl-CTP) TriLink Biotechnologies, San Diego, USA N-1014
Cyanine 3-CTP PerkinElmer, Baesweiler, Germany NEL580001EA
RNeasy Mini Kit Qiagen, Hilden, Germany 74104
MEGAscript T7 Transcription Kit Thermo Fisher Scientific, Darmstadt, Germany AM1333
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog New England Biolabs, Ipswich, USA S1411L
Antarctic Phosphatase New England Biolabs, Ipswich, USA M0289S
Agarose Thermo Fisher Scientific, Darmstadt, Germany 16500-500
GelRed Biotium, Fremont, USA 41003
peqGOLD DNA ladder mix VWR, Pennsylvania, USA 25-2040
Invitrogen 0.5-10kb RNA ladder Fisher Scientific, Göteborg,
Sweden
11528766

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