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Protocol

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Biochemistry

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

Published: June 10th, 2022

DOI:

10.3791/62407

1Centre for Stem Cell Research, Christian Medical College Campus
* These authors contributed equally

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.

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.

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 m....

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1. Production of me1 Ψ-UTP modified mRNA

  1. 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.
    1. Prepare PCR reaction mixture as described in Table 1.
      NOTE: Run at least four PCR reactions to increase.......

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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.......

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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.......

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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.

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Name Company Catalog Number Comments
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

  1. Sahin, U., Kariko, K., Tureci, O. mRNA-based therapeutics--developing a new class of drugs. Nature Reviews Drug Discovery. 13 (10), 759-780 (2014).
  2. Schlake, T., Thess, A., Fotin-Mleczek, M., Kallen, K. J. Developing mRNA-vaccine technologies. RNA Biology. 9 (11), 1319-1330 (2012).
  3. Carlile, T. M., et al. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 515 (7525), 143-146 (2014).
  4. Kariko, K., Buckstein, M., Ni, H., Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 23 (2), 165-175 (2005).
  5. Guan, S., Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Therapy. 24 (3), 133-143 (2017).
  6. Srujan, M., et al. The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles. Biomaterials. 32 (22), 5231-5240 (2011).
  7. Dharmalingam, P., et al. Transfection: Cationic Lipid Nanocarrier System Derivatized from Vegetable Fat, Palmstearin Enhances Nucleic Acid Transfections. ACS Omega. 2 (11), 7892-7903 (2017).
  8. Hoy, S. M. Patisiran: First Global Approval. Drugs. 78 (15), 1625-1631 (2018).
  9. Anderson, E. J., et al. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. New England Journal of Medicine. , (2020).
  10. Polack, F. P., et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine. , (2020).
  11. Schlee, M., Hartmann, G. Discriminating self from non-self in nucleic acid-sensing. Nature Reviews Immunology. 16 (9), 566-580 (2016).
  12. Kariko, K., Buckstein, M., Hi, H., Weissman, D. Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification and evolutionary origin of RNA. Immunity. 23 (2), 165-175 (2005).
  13. Mauger, D. M., et al. mRNA structure regulates protein expression through changes in functional half-life. Proceedings of the National Academy of Sciences of the United States of America. 116 (48), 24075-24083 (2019).
  14. Vaidyanathan, S., et al. Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. Molecular Therapy - Nucleic Acids. 12, 530-542 (2018).

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