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

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.