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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 the dissolved DOPE in a glass flask.
   Note: The total lipid amount is 3 mg. To avoid fast evaporation of the chloroform, place the lipids on ic....

Results

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 free.......

Discussion

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.......

Materials

List of materials used in this article

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