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

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Therapeutics

Background:

• Efficient oligonucleotide delivery for therapeutic applications remains a significant challenge.
• Peptide nucleic acids (PNAs) are DNA analogues that can be conjugated to cell-penetrating peptides (CPPs) to enhance cellular uptake.
• Conventional CPPs often utilize endosomal pathways, limiting cargo bioavailability.

Purpose of the Study:

• To design and synthesize PNA-peptide conjugates as splice switching oligonucleotides (SSOs) targeting the Mnk2 gene for cancer therapy.
• To investigate the cellular uptake mechanism and therapeutic potential of these conjugates.
• To evaluate the effect of PNA-peptide conjugates on Mnk2 alternative splicing and cancer cell viability.

Main Methods:

• Synthesis of PNA-peptide conjugates, including those with a nuclear localization sequence (NLS) or a cytosol localizing internalization peptide (CLIP6).
• Assessment of cellular uptake and mechanism of entry in human glioblastoma cells (U87) using CLIP6-PNA.
• Analysis of Mnk2 gene splicing modulation and impact on glioblastoma cell death upon incubation with PNA-peptide conjugates.

Main Results:

• CLIP6-PNA conjugates demonstrated effective cellular uptake in glioblastoma cells.
• The CLIP6-PNA conjugate exclusively utilized a non-endosomal mechanism for cellular membrane translocation.
• Incubation with PNA-peptide conjugates led to the up-regulation of the tumor-suppressive Mnk2a isoform, resulting in cancer cell death.

Conclusions:

• PNA-peptide conjugates, particularly CLIP6-PNA, offer an efficient strategy for oligonucleotide delivery.
• The non-endosomal uptake mechanism enhances bioavailability and therapeutic efficacy.
• Targeting Mnk2 splicing with PNA-peptide conjugates presents a promising therapeutic approach for cancer treatment, specifically glioblastoma.