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PepFect14 Signaling and Transfection
Luca Falato1, Maxime Gestin2, Ülo Langel2,3
1Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden. luca.falato92@gmail.com.
Methods in Molecular Biology (Clifton, N.J.)
|November 12, 2021
Summary
PepFect14, a modified cell-penetrating peptide (CPP), effectively delivers cargo like splice-correcting oligonucleotides and proteins into cells. Its unique structure enhances stability and facilitates cellular uptake via endocytosis, with potential for chaperone-mediated autophagy modulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- PepFect14 is a novel cell-penetrating peptide (CPP) derived from stearylated transportan-10.
- It features ornithine substitutions for enhanced protease resistance.
- Its positive charge enables complex formation with various negatively charged cargos.
Purpose of the Study:
- To characterize PepFect14 as a delivery vehicle for therapeutic oligonucleotides, DNA, and proteins.
- To investigate the cellular uptake mechanisms of PepFect14/cargo complexes.
- To explore the role of chaperone-mediated autophagy in PepFect14-mediated transfection.
Main Methods:
- Synthesis and characterization of PepFect14.
- Formation and analysis of PepFect14 complexes with splice-correcting oligonucleotides (SCOs), plasmid DNA (pDNA), and proteins.
- Cellular uptake studies using endocytosis inhibitors and receptor analysis (SCARAs).
- Investigation of autophagy pathways and heat shock protein 70 (HSP70) involvement.
Main Results:
- PepFect14 efficiently forms complexes with SCOs, pDNA, and proteins.
- Cellular entry occurs primarily via macropinocytosis and caveolae-mediated endocytosis, involving SCARAs.
- PepFect14 induces chaperone-mediated autophagy, and its inhibition enhances transfection efficacy.
- HSP70 can be delivered into Bomirsky Hamster Melanoma cells using PepFect14.
Conclusions:
- PepFect14 is a versatile and robust CPP for delivering diverse cargos into cells.
- Understanding its interaction with endocytic pathways and autophagy provides insights for optimizing gene/protein delivery.
- PepFect14 offers a promising platform for therapeutic applications requiring efficient intracellular delivery.
