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.

CRISPR/Cas9 Plasmid Delivery Through the CPP: PepFect14.

Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The first one can be used to silence a specific gene or a portion of it and the second one to insert new DNA, in presence of a donor template, in a targeted position in the genome. In order to exploit one of these two mechanisms, three major targeted nucleases have been developed: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein). The last one seems to be the most promising tool among the others for gene editing. By using the properties and versatility of the Cell Penetrating Peptide (CPP) PepFect14, we developed a protocol to deliver a plasmid encoding for CRISPR-Cas9 and Green Fluorescent Protein (GFP) in BHM cell line expressing luciferase (Bomirsky Hamster Melanoma pLuc). Aiming to knocking down the luciferase gene in the cell line and to expressing GFP. Having two fast and easy read-outs of the plasmid's activity at the same time. Furthermore, by labeling the CRISPR plasmid with Cy5 it is possible to have a visual confirmation of the cellular uptake of the pDNA/CPP complex, via fluorescent microscopy, as described.

Cell-Penetrating Peptides Delivering siRNAs: An Overview.

Cell-Penetrating Peptides (CPP) are valuable tools capable of crossing the plasma membrane to deliver therapeutic cargo inside cells. Small interfering RNAs (siRNA) are double-stranded RNA molecules capable of silencing the expression of a specific protein triggering the RNA interference (RNAi) pathway, but they are unable to cross the plasma membrane and have a short half-life in the bloodstream. In this overview, we assessed the many different approaches used and developed in the last two decades to deliver siRNA through the plasma membrane through different CPPs sorted according to three different loading strategies: covalent conjugation, complex formation, and CPP-decorated (functionalized) nanocomplexes. Each of these strategies has pros and cons, but it appears the latter two are the most commonly reported and emerging as the most promising strategies due to their simplicity of synthesis, use, and versatility. Recent progress with siRNA delivered by CPPs seems to focus on targeted delivery to reduce side effects and amount of drugs used, and it appears to be among the most promising use for CPPs in future clinical applications.

Effect of small molecule signaling in PepFect14 transfection.

Cell-penetrating peptides can be used to deliver oligonucleotide-based cargoes into cells. Previous studies have shown that the use of small molecule drugs could be an efficient method to increase the efficacy of delivery of oligonucleotides by cell-penetrating peptides either as targeting agents that can be used in formulation with the cell-penetrating peptide and its cargo or as cell signaling modulators that facilitates the cellular uptake of the treatment. This study presents two aims. The first aim is the identification of small molecule drugs that would induce a synergic effect on the transfection of splice correcting oligonucleotides assisted by PepFect14. The second aim is to identify the mechanisms behind the effect of small molecule drugs modulation of cell-penetrating peptide assisted transfection of oligonucleotides. Through an optimized, high-throughput luciferase assay for short oligonucleotide delivery using cell-penetrating peptides, and the simultaneous addition of a small molecule drug library, we show that three small molecule drugs (MPEP, VU0357121 and Ciproxifan) induced an increase in the transfection efficacy of PepFect14 in complex with a short single-stranded oligonucleotide in HeLa pLuc705 cells. These three drugs are described in the literature to be highly specific for their respective target receptors. However, none of those receptors are expressed in our cell line, indicating a yet non-described pathway of action for these small molecules. We show that the indicated small molecules, without interfering with the particles formed by PepFect14 and the oligonucleotide, interfere via still unidentified interactions in cell signaling, leading to an up-regulation of endocytosis and a higher efficacy in the delivery of short splice correcting oligonucleotides in complex with PepFect14.

The hypoxic expression of the glucose transporter RAG1 reveals the role of the bHLH transcription factor Sck1 as a novel hypoxic modulator in Kluyveromyces lactis.

Glucose is the preferred nutrient for most living cells and is also a signaling molecule that modulates several cellular processes. Glucose regulates the expression of glucose permease genes in yeasts through signaling pathways dependent on plasma membrane glucose sensors. In the yeast Kluyveromyces lactis, sufficient levels of glucose induction of the low-affinity glucose transporter RAG1 gene also depends on a functional glycolysis, suggesting additional intracellular signaling. We have found that the expression of RAG1 gene is also induced by hypoxia in the presence of glucose, indicating that glucose and oxygen signaling pathways are interconnected. In this study we investigated the molecular mechanisms underlying this crosstalk. By analyzing RAG1 expression in various K. lactis mutants, we found that the bHLH transcriptional activator Sck1 is required for the hypoxic induction of RAG1 gene. The RAG1 promoter region essential for its hypoxic induction was identified by promoter deletion experiments. Taken together, these results show that the RAG1 glucose permease gene is synergistically induced by hypoxia and glucose and highlighted a novel role for the transcriptional activator Sck1 as a key mediator in this mechanism.