Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells

Nucleic Acids Res. 2001 Sep 15;29(18):3882-91. doi: 10.1093/nar/29.18.3882.

Abstract

Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cell-Free System / metabolism
  • Gene Expression
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Proteins / genetics
  • Melitten / chemistry
  • Melitten / genetics
  • Microinjections
  • Mitosis
  • Molecular Sequence Data
  • Oligopeptides / genetics
  • Oligopeptides / physiology*
  • Protein Biosynthesis
  • RNA / genetics
  • RNA / metabolism*
  • RNA, Messenger / administration & dosage
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rabbits
  • Reticulocytes / chemistry
  • Reticulocytes / metabolism
  • Time Factors
  • Transcription, Genetic
  • Transfection / methods*
  • Tumor Cells, Cultured

Substances

  • Luminescent Proteins
  • Oligopeptides
  • RNA, Messenger
  • Green Fluorescent Proteins
  • Melitten
  • RNA
  • Luciferases