Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides

Ulrik L. Rahbek, Kenneth A. Howard, David Oupicky, Devika S. Manickam, Mingdong Dong, Anne F. Nielsen, Thomas B. Hansen, Flemming Basenbacher, Jørgens Kjems

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Background: Small interfering RNAs (siRNAs) can induce specific gene silencing through cytoplasmic mRNA cleavage and nuclear transcriptional silencing, necessitating delivery to different cellular compartments. This study presents a reducible copolypeptide (rCPP) carrier containing different molar ratios of a histidine-rich peptide (HRP) and nuclear localization sequence (NLS) peptide to modulate intracellular trafficking of transfected siRNA and primary RNA transcripts (pri-miRNA). Methods: Polyplex formation using siRNA and rCPP was demonstrated using photon correlation spectroscopy and atomic force microscopy. Confocal and fluorescence microscopy were used to investigate cellular uptake and nuclear trafficking whilst endogenous enhanced green fluorescent protein (EGFP) knockdown in H1299 cells was evaluated using flow cytometry. Transcriptional gene silencing of endogenous EF1A was verified using real-time reverse-transcription polymerase chain reaction (RT-PCR) and pri-miRNA nuclear processing was demonstrated using Northern analysis. Results: rCPP-based polyplexes showed rapid cellular uptake and low cytotoxicity. Labelled components revealed intact polyplexes after 2 h that exhibited directed movements consistent with endosomal trafficking. Polyplex-mediated knockdown of EGFP increased with greater HRP content. The inclusion of NLS promoted nuclear localization of transfected siRNAs and pri-miRNAs to the nuclear compartment allowing for transcriptional silencing of EF1A and Drosha and Dicer dependent expression of mature miRNA, respectively. Conclusion: Our results demonstrate that reducible copolypeptides can be used as carriers for the non-toxic cellular delivery of siRNA and pri-miRNA. The nuclear targeting of rCPPs can be utilized for delivery of siRNAs and pri-miRNAs to the nuclear compartment for transcriptional gene silencing or endogenous processing into mature miRNA, respectively, which could potentially lead to improved therapeutic approaches.

Original languageEnglish (US)
Pages (from-to)81-93
Number of pages13
JournalJournal of Gene Medicine
Volume10
Issue number1
DOIs
StatePublished - Jan 1 2008

Fingerprint

MicroRNAs
Small Interfering RNA
Gene Silencing
Histidine
Peptides
Intranuclear Inclusion Bodies
Atomic Force Microscopy
RNA Precursors
Photons
Fluorescence Microscopy
Confocal Microscopy
Reverse Transcription
Spectrum Analysis
Flow Cytometry
Polymerase Chain Reaction
Messenger RNA

Keywords

  • Copolypeptides
  • Nuclear trafficking
  • Polyplexes
  • pri-miRNA
  • siRNA

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

Cite this

Rahbek, U. L., Howard, K. A., Oupicky, D., Manickam, D. S., Dong, M., Nielsen, A. F., ... Kjems, J. (2008). Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides. Journal of Gene Medicine, 10(1), 81-93. https://doi.org/10.1002/jgm.1120

Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides. / Rahbek, Ulrik L.; Howard, Kenneth A.; Oupicky, David; Manickam, Devika S.; Dong, Mingdong; Nielsen, Anne F.; Hansen, Thomas B.; Basenbacher, Flemming; Kjems, Jørgens.

In: Journal of Gene Medicine, Vol. 10, No. 1, 01.01.2008, p. 81-93.

Research output: Contribution to journalArticle

Rahbek, UL, Howard, KA, Oupicky, D, Manickam, DS, Dong, M, Nielsen, AF, Hansen, TB, Basenbacher, F & Kjems, J 2008, 'Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides', Journal of Gene Medicine, vol. 10, no. 1, pp. 81-93. https://doi.org/10.1002/jgm.1120
Rahbek, Ulrik L. ; Howard, Kenneth A. ; Oupicky, David ; Manickam, Devika S. ; Dong, Mingdong ; Nielsen, Anne F. ; Hansen, Thomas B. ; Basenbacher, Flemming ; Kjems, Jørgens. / Intracellular siRNA and precursor miRNA trafficking using bioresponsive copolypeptides. In: Journal of Gene Medicine. 2008 ; Vol. 10, No. 1. pp. 81-93.
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