CLIP6-PNA-Peptide Conjugates: Non-Endosomal Delivery of Splice Switching Oligonucleotides (SSO)

Terese Soudah teresesoudah@gmail.com 1 Maxim Mogilevsky 2 Rotem Karni 2 Eylon Yavin 1
1The School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel
2Department of Biochemistry and Molecular Biology, The Hebrew University of Jerusalem, Jerusalem, Israel

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 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 cargo1-2.

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 downregulated in breast, lung, brain, and colon tumors and is a tumor suppressor whereas MnK2b is oncogenic3. We have designed and synthesized PNAs that were conjugated to either one of the following peptides: a nuclear localization sequence (NLS) and a cytosol localizing internalization peptide (CLIP6)4.

CLIP6-PNA demonstrates effective cellular uptake and exclusively employs a non-endosomal mechanism to cross the cellular membranes of glioblastoma cells (U87-MG). Simple incubation of PNA-peptide conjugates in human glioblastoma cells up regulates the Mnk2a isoform leading to cancer cell death.

Acknowledgment: This research was supported by the Israel Science Foundation (grant No. 480/13). E.Y. acknowledges the David R. Bloom Center for Pharmacy for financial support.

References:

1Oh, S. Y., Ju, Y., Kim, S., and Park, H. (2010) PNA-Based Antisense Oligonucleotides for MicroRNAs Inhibition in the Absence of a Transfection Reagent, Oligonucleotides 20, 225-230.

2Lebleu, B., Moulton, H. M., Abes, R., Ivanova, G. D., Abes, S., Stein, D. A., Iversen, P. L., Arzumanov, A. A., and Gait, M. J. (2008) Cell penetrating peptide conjugates of steric block oligonucleotides, Adv. Drug Del. Rev. 60, 517-529.

3Maimon A., et al. Cell Reports 2014, 7, 1-13.

4Medina, S. H., Miller, S. E., Keim, A. I., Gorka, A. P., Schnermann, M. J., and Schneider, J. P., Angew. Chem. Int. Ed. 2016, 55, 3369-3372.









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