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Simultaneous Targeting of Two Master Regulators of Apoptosis with Dual-Action PNA- and DNA-Peptide Conjugates.

ABSTRACT: Conjugation of peptides with oligonucleotides offers opportunities for combining the strengths of both biopolymer classes. Herein, we show that the combination of a peptide-based module with an antisense oligonucleotide module provides for enhancements of potency and a widened scope of cell delivery options. The peptide unit comprises a Smac mimetic compound (SMCs) which antagonizes the action of inhibitor of apoptosis proteins (IAPs) frequently overexpressed in cancer cells. To counteract SMC resistance, the antisense module downregulates the cellular FLICE-like protein (c-FLIP), a master regulator of the extrinsic apoptosis pathway. We compared c-FLIP antisense units based on oligophosphorothioate (PSO) and peptide nucleic acid (PNA) architectures. Owing to the ease of synthesis, PNA conjugates combined with a cell penetrating peptide (CPP) offer a seemingly ideal solution for cell delivery of dual activity agents. However, our investigations revealed that such congeners have to be handled with care to avoid off-target effects. By contrast, PSO conjugates provided a more robust and specific activity for inducing death of SMC-resistant A549 cells due to a simultaneous activation of caspases and c-FLIP knockdown. We show that lipofection is a convenient approach for delivery of peptide-PSO conjugates into cells. The results highlight that the combination of the peptide and the DNA world confers properties inaccessible by the unconjugated components.

SUBMITTER: Altrichter Y

PROVIDER: S-EPMC7583637 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA length (to adjust binding affinity). In general, the carrier CPP-PNA constructs including the ones with decanoyl modification provided significant increase of the activity of unmodified antisense PNA as well as of antisense octaarginine-PNA conjugates. Antisense activity, and by inference cellular delivery, of unmodified antisense PNA was enhanced at least 20-fold at 6 ?M upon the complexation with an equimolar amount of nonamer carrier decanoyl-CPP-PNA (Deca-cPNA1(9)-(D-Arg)8). The antisense activity of a CPP-PNA ((D-Arg)8-asPNA) (at 2 ?M) was improved 6-fold and 8-fold by a heptamer carrier CPP-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP-PNA carriers may be used as effective cellular delivery vectors for different types of antisense oligomers and also allows use of combinations of (at least two) different CPP ligands.

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Simultaneous Targeting of Two Master Regulators of Apoptosis with Dual-Action PNA- and DNA-Peptide Conjugates.

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