Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity.

AVI BioPharma, Inc., Corvallis, OR 97333, USA.
Nucleic Acids Research (Impact Factor: 8.81). 07/2007; 35(15):5182-91. DOI: 10.1093/nar/gkm478
Source: PubMed

ABSTRACT Arginine-rich cell-penetrating peptides (CPPs) are promising transporters for intracellular delivery of antisense morpholino oligomers (PMO). Here, we determined the effect of L-arginine, D-arginine and non-alpha amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24 CPP-PMOs. Insertion of 6-aminohexanoic acid (X) or beta-alanine (B) residues into oligoarginine R8 decreased the cellular uptake but increased the splice-correction activity of the resulting compound, with a greater increase for the sequences containing more X residues. Cellular toxicity was not observed for any of the conjugates up to 10 microM. Up to 60 microM, only the conjugates with > or = 5 Xs exhibited time- and concentration-dependent toxicity. Substitution of L-arginine with D-arginine did not increase uptake or splice-correction activity. High concentration of serum significantly decreased the uptake and splice-correction activity of oligoarginine conjugates, but had much less effect on the conjugates containing X or B. In summary, incorporation of X/B into oligoarginine enhanced the antisense activity and serum-binding profile of CPP-PMO. Toxicity of X/B-containing conjugates was affected by the number of Xs, treatment time and concentration. More active, stable and less toxic CPPs can be designed by optimizing the position and number of R, D-R, X and B residues.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Antisense oligonucleotide (AO) - mediated splice correction therapy for Duchenne muscular dystrophy (DMD) has shown huge promise from recent phase IIb clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide (MSP) and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide-AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to morpholino oligomers (PMOs), approximately 25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas less than 2% in corresponding tissues treated with either MSP-PMO or unmodified PMO under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-PMO can be an alternative option to current AOs.Molecular Therapy (2014); doi:10.1038/mt.2014.63.
    Molecular Therapy 04/2014; · 7.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
    PLoS ONE 01/2014; 9(8):e104999. · 3.53 Impact Factor
  • Source

Full-text (2 Sources)

Available from
Jun 1, 2014