title: Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells.
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.
authors.affiliation: CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2TA, UK. tbettinger@brg.bracco.com
authors.forename: T
authors.initials: T
authors.lastname: Bettinger
authors.forename: R C
authors.initials: RC
authors.lastname: Carlisle
authors.forename: M L
authors.initials: ML
authors.lastname: Read
authors.forename: M
authors.initials: M
authors.lastname: Ogris
authors.forename: L W
authors.initials: LW
authors.lastname: Seymour
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008555:Melitten / Q000737:chemistry / Q000235:genetics; D008845:Microinjections; D008938:Mitosis; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000502:physiology*; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D008164:Luminescent Proteins; D009842:Oligopeptides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D008555:Melitten; D012313:RNA; D008156:Luciferases