Using Lipid Nanoparticles for the Delivery of Chemically Modified mRNA into Mammalian Cells

Summary

The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.

Abstract

In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.

Introduction

As a therapeutic molecule, mRNA offers several advantages due to its non-integrative nature and its ability to transfect non-mitotic cells when compared to plasmid DNA (pDNA)¹. Although mRNA delivery was demonstrated in the early 1990s, therapeutic applications were limited due to its lack of stability, its lack of immune activation, and poor translational efficiency². Recently identified chemical modifications, such as pseudouridine 5'-triphosphate (Ψ-UTP) and methyl pseudouridine 5'-triphosphate (me1Ψ-UTP) on mRNA, helped to overcome these limitations, revolutionized mRNA research, and in turn, made mRNA a promising tool in both basic and applied research. The range of applications covers the generation of iPSCs to vaccination and gene therapy^3,4.

In parallel to advancement in mRNA technology, significant advances in non-viral delivery systems made the delivery of mRNA effective, making this technology feasible for multiple therapeutic applications⁵. Among the non-viral vectors, lipid nanoparticles have been found to be effective in delivering nucleic acids^6,7. Recently, Alnylam has received FDA approval of lipid-based siRNA drugs for treating liver diseases, including Patisiran for hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) and Givosiran for acute hepatic porphyrias (AHP)⁸. During the COVID19 pandemic, lipid encapsulated mRNA based vaccines from Pfizer-BioNtech and Moderna demonstrated their efficacy and received FDA approvals^9,10. Thus, lipid enabled mRNA delivery has a great therapeutic potential.

Here, we describe a detailed protocol for the production of chemically modified, in vitro transcribed eGFP mRNA, cationic liposome preparation, mRNA-lipid complex optimization and transfections into mammalian cells (Figure 1).

Protocol

1. Production of me1 Ψ-UTP modified mRNA In vitro transcription (IVT) DNA template preparation NOTE: For IVT DNA template (T7 promoter- open reading frame (ORF) of the gene) preparation, design a gene-specific primer set for the gene of interest. Add the T7 promoter (5'-NNNNNNTAATACGACTCACTATAGGGNNNNNN-3') sequence before gene-specific forward primer. Prepare PCR reaction mixture as described in Table 1. NOTE: Run at least four PCR reactions to increase…

Representative Results

We optimized the protocol for me1Ψ-UTP modified mRNA production, liposome preparation, and mRNA transfection experiments with cationic liposomes into multiple mammalian cells (Figure 1). To synthesize mRNA, the mammalian codon-optimized eGFP IVT template was amplified from the mEGFP-N1 mammalian expression vector and purified by organic extraction/ethanol precipitation method (Figure 2). Later, me1Ψ-UTP modified RNA and mRNA were produced by the IVT pr…

Discussion

Therapeutic applications of unmodified mRNAs have been limited due to their shorter half-life and their ability to activate intracellular innate immune responses, which in turn lead to poor protein expression in transfected cells¹¹. Katalin et al. demonstrated that RNA containing modified nucleosides such as m5C, m6A, ΨU, and me1Ψ-UTP could avoid TLR activation¹². More importantly, incorporation of ΨU or me1Ψ-UTP in IVT mRNA showed superior translational…

Materials

Agarose	Lonza	50004
Bath sonicator	DNMANM Industries	USC-100
Cationic lipid	Synthesized in the lab
Chlorofrom	MP biomedicals	67-66-3	"Caution"
Cholesterol	Himedia	GRM335
DEPC water	SRL BioLit	66886
DMEM	Lonza	12-604F
DNA Ladder	GeneDireX	DM010-R50C
DOPE	TCI	D4251
EDTA sodium salt	MP biomedicals	194822
Ethanol	Hayman	F204325	"Caution"
Fetal bovine serum	Gibco	10270
Flow cytometry	BD	FACS Celesta
Fluroscence Microscope	Leica	MI6000B
Gel documentation system	Cell Biosciences	Flurochem E
Glacial acetic acid	Fisher Scientific	85801	"Caution"
mEGFP-N1, Mammalian expression vector	Addgene	54767
N1-Methylpseudo-UTP	Jena Bioscience	NU-890
Phenol:chloroform:isoamyl alchol (25:24:1), pH 8.0	SRL BioLit	136112-00-0	"Caution"
Phosphate Buffer Saline (PBS), pH 7.4	CellClone	CC3041
Probe sonicator	Sonics Vibra Cells	VCX130
RNA ladder	NEB	N0362S
RNase inhibitor	Thermo Scientific	N8080119
SafeView dye	abm	G108
Sodium acetate	Sigma	S7545
Thermocycler	Applied biosystems	4375786
Thermomixer	Eppendrof	22331
Tris buffer	SRL BioLit	71033
Trypsin	Gibco	25200056