Skip to main content
SpringerLink
Log in
Book cover

Synthetic Messenger RNA and Cell Metabolism Modulation pp 73–88Cite as

Nonviral, Cationic Lipid-Mediated Delivery of mRNA

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 969))

Abstract

Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells is often of low efficiency, due to the difficulty of DNA translocation to the nucleus. Rapid lipid-mediated delivery of RNA is preferable to nonviral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we describe techniques for cationic lipid-mediated delivery of RNA encoding reporter genes in a variety of in vitro cell lines and in vivo. We describe optimized formulations and transfection procedures that we have previously assessed by flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in nondividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5–7 h after transfection. These results are consistent with our in vivo delivery results, techniques for which are shown as well. Longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require long term transient gene expression. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression, and greater efficiency, particularly in nondividing cells.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Flotte TR (2007) Gene therapy: the first two decades and the current state-of-the-art. J Cell Physiology 213:301–305

    Article  CAS  Google Scholar 

  2. Li SD, Huang L (2006) Gene therapy progress and prospects: non-viral gene therapy by systemic delivery. Gene Ther 13:1313–1319

    Article  PubMed  CAS  Google Scholar 

  3. Gao X, Kim KS, Liu D (2007) Nonviral gene delivery: what we know and what is next. AAPS J 9:E92–E104

    Article  PubMed  CAS  Google Scholar 

  4. Felgner PL, Tsai YJ, Sukhu L, Wheeler CJ, Manthorpe M, Marshall J, Cheng SH (1995) Improved cationic lipid formulations for in vivo gene therapy. Ann NY Acad Sci 772:1126–1139

    Article  Google Scholar 

  5. Luo D, Saltzman WM (2000) Synthetic DNA delivery systems. Nat Biotechnol 18:33–37

    Article  PubMed  CAS  Google Scholar 

  6. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci 84:7413–7417

    Article  PubMed  CAS  Google Scholar 

  7. Byk G, Scherman D (2000) Genetic chemistry: tools for gene therapy coming from unexpected directions. Drug Dev Res 50:566–572

    Article  CAS  Google Scholar 

  8. Niedzinski EJ, Fujii SK, Lizarzaburu ME, Hecker JG, Nantz MH (2002) A versatile linker for non-toxic polyamine-mediated DNA transfection. Mol Ther 6:279–286

    Article  PubMed  CAS  Google Scholar 

  9. Nantz MH, Dicus CW, Hilliard B, Yellayi S, Scarfo KA, Zou S, Hecker JG (2010) Unsymmetrical hydrophobic domains improve in vivo transfection efficiency. Mol Pharm 7:786–794

    Article  PubMed  CAS  Google Scholar 

  10. Bally MB, Harvie P, Wong FM, Kong S, Wasan EK, Reimer DL (1999) Biological barriers to cellular delivery of lipid-based DNA carriers. Adv Drug Deliv Rev 38:291–315

    Article  PubMed  CAS  Google Scholar 

  11. Girao da Cruz MT, Simoes S, Pires PPC, Nir S, Pedrosa de Lima MC (2001) Kinetic analysis of the initial steps involved in lipoplex-cell interactions: effect of various factors that influence transfection activity. Biochim Biophys Acta 1510:136–151

    Article  CAS  Google Scholar 

  12. Mortimer I, Tam P, MacLachlan I, Graham RW, Saravolac EG, Joshi PB (1999) Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther 6:403–411

    Article  PubMed  CAS  Google Scholar 

  13. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 270:18997–19007

    Article  PubMed  CAS  Google Scholar 

  14. Wangerek LA, Dahl HH, Senden TJ, Carlin JB, Jans DA, Dunstan DE, Ioannou PA, Williamson R, Forrest SM (2001) Atomic force microscopy imaging of DNA-cationic liposome complexes optimized for gene transfection into neuronal cells. J Gene Med 3:72–81

    Article  PubMed  CAS  Google Scholar 

  15. Zou S, Scarfo KA, Nantz MH, Hecker JG (2010) Lipid-mediated delivery of RNA is more efficient that DNA in non-dividing cells. Int J Pharm 389:232–240

    Article  PubMed  CAS  Google Scholar 

  16. Wilke M, Fortunati E, van den Broek M, Hoogeveen AT, Scholte BJ (1996) Efficacy of a peptide- based gene delivery system depends on mitotic activity. Gene Ther 3:1133–1142

    PubMed  CAS  Google Scholar 

  17. Nicolau C, Sene C (1982) Liposome-mediated DNA transfer in eukaryotic cells. Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage. Biochim Biophys Acta 721:185–190

    Article  PubMed  CAS  Google Scholar 

  18. Mattaj IW, Englmeier L (1998) Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem 67:265–306

    Article  PubMed  CAS  Google Scholar 

  19. Wilson GL, Dean BS, Wang G, Dean DA (1995) Nuclear import of plasmid DNA in digitonin-permeabilized cells requires both cytoplasmic factors and specific DNA sequences. J Biol Chem 270:22025–22032

    Google Scholar 

  20. Malone RW, Felgner PL, Verma IM (1989) Cationic liposome-mediated RNA transfection. Proc Natl Acad Sci 86:6077–6081

    Article  PubMed  CAS  Google Scholar 

  21. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468

    Article  PubMed  CAS  Google Scholar 

  22. Brisson B, Tseng W-C, Almonte C, Watkins S, Huang L (1999) Subcellular trafficking of the cytoplasmic expression system. Hum Gene Ther 10:2601–2613

    Article  PubMed  CAS  Google Scholar 

  23. Anderson DM, Hall LL, Ayyalapu AR, Irion VR, Nantz MH, Hecker JG (2003) Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for nonviral mRNA gene delivery to the central nervous system. Hum Gene Ther 14:191–202

    Article  PubMed  CAS  Google Scholar 

  24. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 269:2550–2561

    PubMed  CAS  Google Scholar 

  25. Sambrook D, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 1st, Cold Spring Harbor Lab, Cold Spring Harbor NY

    Google Scholar 

  26. Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070

    Article  PubMed  CAS  Google Scholar 

  27. Anderson DM, Hall LL, Ayyalapu A, Irion VR, Nantz MH, Hecker JG (2003) Stability of mRNA/cationic lipid lipoplexes in human and rat cerebrospinal fluid: methods and evidence for non-viral mRNA gene delivery to the CNS. Hum Gene Ther 14:191–202

    Article  PubMed  CAS  Google Scholar 

  28. Hauck ES, Zou S, Scarfo KA, Nantz MH, Hecker JG (2008) Whole animal in vivo imaging after transient non-viral lipid-mediated gene transfer to the rat central nervous system. Mol Ther 16:1857–1864

    Article  PubMed  CAS  Google Scholar 

  29. Hecker JG, Hall LL, Irion VR (2001) Non-viral gene delivery to the lateral ventricles in rat brain: initial evidence for widespread distribution and expression in the central nervous system. Mol Ther 3:375–384

    Article  PubMed  CAS  Google Scholar 

  30. Balasubramaniam RP, Bennett MJ, Aberle AM, Malone JG, Nantz MH, Malone RW (1996) Structural and functional analysis of cationic transfection lipids: the hydrophobic domain. Gene Ther 3:163–172

    PubMed  CAS  Google Scholar 

  31. Bennett MJ, Nantz MH, Balasubramaniam RP, Gruenert DC, Malone RW (1995) Cholesterol enhances cationic liposome-mediated DNA transfection on human respiratory epithelial cells. Biosci Rep 15:47–53

    Article  PubMed  CAS  Google Scholar 

  32. Bennett MJ, Aberle AM, Balasubramaniam RP, Malone JG, Nantz MH, Malone RW (1996) Considerations for the design of improved cationic amphiphile-based transfection reagents. J Liposome Res 6:545–565

    Article  CAS  Google Scholar 

  33. McKinney JS, Willoughby KA, Liang S, Ellis EF (1996) Stretch-induced injury of cultured neuronal, glial, and endothelial cells. Stroke 27:934–940

    Article  PubMed  CAS  Google Scholar 

  34. Promega Corporation (2012) Transfast transfection reagent. Promega Corporation website, catalog number E2431-

    Google Scholar 

  35. Corish P, Tyler-Smith C (1999) Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng 12:1035–1040

    Article  PubMed  CAS  Google Scholar 

  36. University of Arizona (2008) Southwest animal imaging resource

    Google Scholar 

  37. Ignowski JM, Schaffer DV (2004) Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnol Bioeng 86:827–834

    Article  PubMed  CAS  Google Scholar 

  38. Allen MS, Wilgus JR, Chewning CS, Sayler GS, Simpson ML (2006) A destabilized bacterial luciferase for dynamic gene expression studies. Syst Synth Biol 1:3–9

    Article  Google Scholar 

  39. Bartlett DW, Davis ME (2006) Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging. Nucleic Acids Res 34:322–333

    Article  PubMed  CAS  Google Scholar 

  40. Hecker JG, Langer DJ, Marshall BE, Barnathan ES, Kariko K (1995) Feasibility studies of mRNA transfection for intermediate duration, perioperative modulation of CNS responses accessible via the cerebrospinal fluid. Anesthesia & Analgesia AUA Abstracts 9999: 1–2

    Google Scholar 

  41. Hecker JG, Irion VR, Malone RW (1997) Self-limited gene expression in-vitro in neuronal cell cultures and in-vivo in rat brain using mRNA/cationic lipid complexes. Anesthesia Analgesia 84: S360

    Google Scholar 

  42. Hecker JG, Irion VR (1998) Advances in transient expression of neuroprotective intracellular proteins in-vivo in rat brain. AUA 45th Annual Meeting May 7–9

    Google Scholar 

  43. Hecker JG, Nantz MH (2008) Novel methods and models for rapid, widespread delivery of genetic materials to the CNS using non-viral, cationic lipid-mediated vectors. PCT/US2006/048093

    Google Scholar 

  44. Hecker JG, Irion VR (1998) Expression of protective intracellular proteins in-vivo in rat brain using mRNA/cationic lipid complexes. Am Soc Gene Ther 1st Mtg, May 28–31, 154A

    Google Scholar 

  45. Hecker JG, Irion VR (1998) Advances in self-limited gene expression of protective intracellular proteins in vivo in rat brain using mRNA/cationic lipid complexes. Anesthesia Analgesia 86: S346

    Google Scholar 

  46. Kariko K, Muramatsu H, Ludwig J, Weissman D (2012) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res 2011;39(21):e142

    Google Scholar 

  47. Kariko K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther 16:1833–1840

    Article  PubMed  CAS  Google Scholar 

  48. Kariko K, Megyeri K, Xiao Q, Barnathan ES (1994) Lipofectin-aided delivery of ribozyme targeted to human urokinase receptor mRNA. FEBS Lett 352:41–44

    Article  PubMed  CAS  Google Scholar 

  49. Kariko K, Keller JM, Harris VA, Langer DJ, Welsh FA (2001) In vivo protein expression from mRNA delivered into adult rat brain. J Neurosci Methods 105:77–86

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine, Seattle, WA, USA

    James G. Hecker

Authors
  1. James G. Hecker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James G. Hecker .

Editor information

Editors and Affiliations

  1. The Anlyan Center, Department of Genetics, Yale School of Medicine, Cedar Street 300, New Haven, 06520-8005, Connecticut, USA

    Peter M. Rabinovich

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this protocol

Cite this protocol

Hecker, J.G. (2013). Nonviral, Cationic Lipid-Mediated Delivery of mRNA. In: Rabinovich, P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology, vol 969. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-260-5_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-260-5_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-259-9

  • Online ISBN: 978-1-62703-260-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation