Wu RP, Youngblood DS, Hassinger JN, Lovejoy CE, Nelson MH, Iversen PL et al.. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res 35: 5182-5191

AVI BioPharma, Inc., Corvallis, OR 97333, USA.
Nucleic Acids Research (Impact Factor: 9.11). 07/2007; 35(15):5182-91. DOI: 10.1093/nar/gkm478
Source: PubMed


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-α amino acids on cellular uptake, splice-correction activity, cellular toxicity and serum binding for 24
CPP−PMOs. Insertion of 6-aminohexanoic acid (X) or β-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 μM. Up to 60 μM, only the conjugates with ⩾ 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.

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    • "Other examples include Penetratin, a 16 amino acid domain from the Antennapedia protein of Drosophila, a flock house virus (FHV) coat peptide (sequence 35–49) and oligoarginines [96]. Subsequently, conjugates of PMOs with arginine-rich CPPs, termed peptide-PMOs (PPMOs), were found to possess enhanced potency in vitro [117]. Conjugates coupled to R6-Penetratin (R6-Pen) improved serum stability and biological activity profile giving rise to a new class of PMO/PNA internalization peptides called Pips [49]. "
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    ABSTRACT: Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. Copyright © 2015. Published by Elsevier Inc.
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    • "Disruption/destabilization of ligand- 9LR:siRNA by late endosomal proteases could be requisite to promote CPP-membrane interactions for continued release of siRNA. In keeping with our data, substitution of R-CPPs and oligo-L-arginines with D-amino acids, at the terminal positions, obstructed degradation by endosomal proteases, reducing cytoplasmic localization of CPP, bioactivity of attached cargo, and transfection efficiencies (Abes et al., 2008; Fischer et al., 2010; Mason et al., 2007; Verdurmen et al., 2011; Wu et al., 2007). The association of RISC machinery components with intraluminal vesicles and membranes of the late endosome (Lee et al., 2009; Saleh et al., 2006; Siomi and Siomi, 2009) also raises the possibility that ligand-9LR instability, in combination with ionic competition for nucleic acid binding at lower pH, may displace siRNA for direct loading onto the endosomal RISC, enhancing the dynamics of RNA silencing in the cytoplasm. "
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    ABSTRACT: Cell-penetrating peptides (CPPs), such as nona-arginine (9R), poorly translocate siRNA into cells. Our studies demonstrate that attaching 9R to ligands that bind cell surface receptors quantitatively increases siRNA uptake and importantly, allows functional delivery of complexed siRNA. The mechanism involved accumulation of ligand-9R:siRNA microparticles on the cell membrane, which induced transient membrane inversion at the site of ligand-9R binding and rapid siRNA translocation into the cytoplasm. siRNA release also occurred late after endocytosis when the ligand was attached to the L isoform of 9R, but not the protease-resistant 9DR, prolonging mRNA knockdown. This critically depended on endosomal proteolytic activity, implying that partial CPP degradation is required for endosome-to-cytosol translocation. The data demonstrate that ligand attachment renders simple polycationic CPPs effective for siRNA delivery by restoring their intrinsic property of translocation. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Dec 2014 · Chemistry & Biology
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    • "A variety of methods have been developed to improve PNA uptake into cells, and the currently favored approach involves conjugation to cell-penetrating peptides (CPPs) [4]–[6]. Many groups have attempted to understand what makes a CPP a good PNA carrier, but there are discrepancies in the results most likely resulting from the diversity of the cell systems used, location of target transcript (nuclear or not), types of conjugates (linkers, linker localization, peptides), PNA length, and the methods used to evaluate CPP efficacy (assays for biological function versus fluorescent labels that indicate localization) [7]–[12]. The mechanistic details of how the CPP-cargo conjugates enter the cell remains unclear, although recent data suggest that entry occurs through an energy-dependent endocytic pathway [13]. "
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    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.
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