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 therapy3,4.
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 acids6,7. 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 approvals9,10. 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
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
MS thanks the Department of Biotechnology, India, for the financial support (BT/PR25841/GET/119/162/2017), Dr Alok Srivastava, Head, CSCR, Vellore, for his support and Dr Sandhya, CSCR core facilities for imaging and FACS experiments. We thank R. Harikrishna Reddy and Rajkumar Banerjee, Applied Biology Division, CSIR-Indian Institute of Chemical Technology Uppal Road, Tarnaka, Hyderabad, 500 007, TS, India, for their help in analyzing physico-chemical data of the liposomes. Vigneshwaran V, and Joshua A, CSCR for their help in video making.
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 |
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