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Related Experiment Video

Updated: May 29, 2025

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Engineered PepFect14 analog for efficient cellular delivery of oligonucleotides.

Abhijit Biswas1, Kapilraj Periyasamy2, Maria Maloverjan1

  • 1Institute of Technology, University of Tartu, Nooruse 1, Tartu 50411, Estonia.

Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie
|February 1, 2025
PubMed
Summary

Cell-penetrating peptides (CPPs) are crucial for delivering therapeutic oligonucleotides (ONs). Researchers modified PepFect14 (PF14) to create PF14-Lys, a more effective CPP for ON delivery in vitro and in vivo.

Keywords:
Cell-penetrating peptideNucleic acid deliveryPeptide design

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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.