PepFect14 Signaling and Transfection

PepFect14 is a cell-penetrating peptide (CPP) derived from stearylated transportan-10 (strearil-TP10) with which it shares the stearic acid residue on C′ terminus and the amino acid sequence except for lysines that in PepFect14 are substituted with ornithines. Being non-proteinogenic amino acids, ornithines make PepFect14 less sensitive to serum proteases and due to its positive charges the CPP can form complexes with negatively charged cargos, such as splice correcting oligonucleotides (SCOs), plasmid DNA (pDNA), and proteins. It has been reported that PepFect14/SCO complexes enter the cells mainly through endocytosis, in particular: macopinocitosys and caveolae-mediated endocytosis through the interaction with two receptors of the scavenger receptors class A family (SCARAs). PepFect14 and its complexes trigger the chaperone-mediated autophagy response involving the heat shock protein family (HSP70) whose inhibition leads to an increase of PepFect14 transfection efficacy. Exploiting the interaction between HSP70 and PepFect14 and their ability to form nanoparticle. HSP70 has been delivered in Bomirsky Hamster Melanoma cells (BHM) using PepFect14 of which a protocol is described at the end of this chapter.