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CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides.

Abstract

Efficient delivery of oligonucleotides still remains a challenge in the field of oligonucleotide based therapy. Peptide nucleic acid (PNA), a DNA analogue that is typically synthesized by solid phase peptide chemistry, has been conjugated to a variety of cell penetrating peptides (CPP) as a means of improving its cellular uptake. These CPPs typically deliver their cargoes into cells by an endosomal-dependent mechanism resulting in lower bioavailability of the cargo. Herein, we designed and synthesized PNA-peptide conjugates as splice switching oligonucleotides (SSO) targeting the Mnk2 gene, a therapeutic target in cancer. In humans, the MKNK2 gene, is alternatively spliced, generating isoforms with opposite biological activities: Mnk2a and Mnk2b. It was found that the Mnk2a isoform is down-regulated in breast, lung, brain, and colon tumors and is a tumor suppressor, whereas MnK2b is oncogenic. We have designed and synthesized PNAs that were conjugated to either of the following peptides: a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6). CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a nonendosomal mechanism to cross the cellular membranes of glioblastoma cells (U87). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up-regulates the Mnk2a isoform leading to cancer cell death.

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  • Authors+Show Affiliations

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    The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem , Hadassah Ein-Kerem, Jerusalem 91120, Israel.

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    The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem , Hadassah Ein-Kerem, Jerusalem 91120, Israel.

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    The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem , Hadassah Ein-Kerem, Jerusalem 91120, Israel.

    The Institute for Drug Research, The School of Pharmacy and ‡Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, The Faculty of Medicine, The Hebrew University of Jerusalem , Hadassah Ein-Kerem, Jerusalem 91120, Israel.

    Source

    Bioconjugate chemistry 28:12 2017 Dec 20 pg 3036-3042

    MeSH

    Base Sequence
    Cell Line, Tumor
    Cell-Penetrating Peptides
    Drug Carriers
    HeLa Cells
    Humans
    Hydrogen-Ion Concentration
    Oligonucleotides
    Peptide Nucleic Acids

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    29211451