Engineered PepFect14 analog for efficient cellular delivery of oligonucleotides

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Delivery

Background:

• Oligonucleotide (ON) therapeutics show great promise but face delivery challenges.
• Cell-penetrating peptides (CPPs) are effective carriers for nucleic acid delivery.
• PepFect14 (PF14) is a widely used CPP for ON delivery.

Purpose of the Study:

• To investigate structural requirements for high efficacy of the CPP PepFect14 (PF14).
• To develop and evaluate novel PF14 analogs for enhanced oligonucleotide delivery.
• To assess the in vitro and in vivo performance of modified CPPs for therapeutic applications.

Main Methods:

• Point mutations were introduced into the PF14 sequence to create analogs.
• Biophysical methods were used to analyze CPP structure and ON condensation into nanoparticles (NPs).
• In vitro assays in reporter cell lines and primary keratinocytes, and in vivo studies in a mouse model were performed.

Main Results:

• The α-helical structure of PF14 is essential for efficient ON delivery; disruption abolishes NP formation and cellular uptake.
• PF14-Lys, a lysine-rich analog, showed superior performance compared to PF14 in delivering splicing-switching oligonucleotides (SSOs) and small interfering RNA (siRNA).
• PF14-Lys efficiently delivered microRNA (miRNA) in keratinocytes and suppressed inflammation in vivo.

Conclusions:

• The α-helical structure and specific charge/hydrophobic face are critical for CPP efficacy.
• PF14-Lys represents a highly promising and versatile delivery vector for various oligonucleotides.
• PF14-Lys demonstrates significant potential for both in vitro and in vivo therapeutic applications.