Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.
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.
AVI BioPharma Inc., Corvallis, Oregon 97333, USA.
N B
NB
Marshall
S K
SK
Oda
C A
CA
London
H M
HM
Moulton
P L
PL
Iversen
N I
NI
Kerkvliet
D V
DV
Mourich
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009025:Morpholines / Q000008:administration & dosage / Q000737:chemistry / Q000493:pharmacokinetics; D060172:Morpholinos; D010446:Peptide Fragments / Q000737:chemistry*; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects*; D016372:RNA, Antisense / Q000008:administration & dosage* / Q000737:chemistry / Q000493:pharmacokinetics; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D000943:Antigens, Differentiation; D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D007376:Interleukin-2; D008070:Lipopolysaccharides; D009025:Morpholines; D060172:Morpholinos; C406562:OX40Ig; D010446:Peptide Fragments; D012322:RNA Precursors; D016372:RNA, Antisense; D001120:Arginine; C414409:penetratin; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse