Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.

TitleArginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.
Publication TypeJournal Article
Year of Publication2007
AuthorsMarshall NB, Oda SK, London CA, Moulton HM, Iversen PL, Kerkvliet NI, Mourich DV
JournalJournal of immunological methods
Volume325
Issue1-2
Pagination114-26
Date Published2007 Aug 31
ISSN0022-1759
KeywordsAmino Acid Sequence, Animals, Antigens, CD45, Antigens, Differentiation, Arginine, Base Sequence, Carrier Proteins, Cell Survival, Female, Flow Cytometry, Forkhead Transcription Factors, Gene Expression, Interleukin-2, Leukocytes, Mononuclear, Lipopolysaccharides, Macrophages, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Morpholines, Morpholinos, Peptide Fragments, RNA Precursors, RNA Splicing, RNA, Antisense, T-Lymphocytes
Abstract

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

DOI10.1016/j.jim.2007.06.009
Alternate JournalJ. Immunol. Methods