In Silico Studies to Support Vaccine Development.

The progress that has been made in computer science positioned in silico studies as an important and well-recognized methodology in the drug discovery and development process. It has numerous advantages in terms of costs and also plays a huge impact on the way the research is conducted since it can limit the use of animal models leading to more sustainable research. Currently, human trials are already being partly replaced by in silico trials. EMA and FDA are both endorsing these studies and have been providing webinars and guidance to support them. For instance, PBPK modeling studies are being used to gather data on drug interactions with other drugs and are also being used to support clinical and regulatory requirements for the pediatric population, pregnant women, and personalized medicine. This trend evokes the need to understand the role of in silico studies in vaccines, considering the importance that these products achieved during the pandemic and their promising hope in oncology. Vaccines are safer than other current oncology treatments. There is a huge variety of strategies for developing a cancer vaccine, and some of the points that should be considered when designing the vaccine technology are the following: delivery platforms (peptides, lipid-based carriers, polymers, dendritic cells, viral vectors, etc.), adjuvants (to boost and promote inflammation at the delivery site, facilitating immune cell recruitment and activation), choice of the targeted antigen, the timing of vaccination, the manipulation of the tumor environment, and the combination with other treatments that might cause additive or even synergistic anti-tumor effects. These and many other points should be put together to outline the best vaccine design. The aim of this article is to perform a review and comprehensive analysis of the role of in silico studies to support the development of and design of vaccines in the field of oncology and infectious diseases. The authors intend to perform a literature review of all the studies that have been conducted so far in preparing in silico models and methods to support the development of vaccines. From this point, it was possible to conclude that there are few in silico studies on vaccines. Despite this, an overview of how the existing work could support the design of vaccines is described.

A Second Life for MAP, a Model Amphipathic Peptide.

Cell-penetrating peptides (CPP) have been shown to be efficient in the transport of cargoes into the cells, namely siRNA and DNA, proteins and peptides, and in some cases, small therapeutics. These peptides have emerged as a solution to increase drug concentrations in different tissues and various cell types, therefore having a relevant therapeutic relevance which led to clinical trials. One of them, MAP, is a model amphipathic peptide with an α-helical conformation and both hydrophilic and hydrophobic residues in opposite sides of the helix. It is composed of a mixture of alanines, leucines, and lysines (KLALKLALKALKAALKLA). The CPP MAP has the ability to translocate oligonucleotides, peptides and small proteins. However, taking advantage of its unique properties, in recent years innovative concepts were developed, such as in silico studies of modelling with receptors, coupling and repurposing drugs in the central nervous system and oncology, or involving the construction of dual-drug delivery systems using nanoparticles. In addition to designs of MAP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy, this review will be focused on unique molecular structure and how it determines its cellular activity, and also intends to address the most recent and frankly motivating issues for the future.

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.

Mitochondrial Targeting Probes, Drug Conjugates, and Gene Therapeutics.

Mitochondria represent an important drug target for many phatology, including neurodegeneration, metabolic disease, heart failure, ischemia-reperfusion injury, and cancer. Mitochondrial dysfunctions are caused by mutation in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins. Cell-penetrating peptides (CPPs) have been employed to overcome biological barriers, target this organelle, and therapeuticaly restore mitochondrial functions. Here, we describe recent methods used to deliver oligonucleotides targeting mitochondrial protein by using mitochondrial penetrating peptides. In particular, we highlight recent advances of formulated peptides/oligonucleotides nanocomplexes as a proof-of-principle for pharmaceutical form of peptide-based therapeutics.

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 in protein mimicry and cancer therapeutics.

Extensive research has been undertaken in the pursuit of anticancer therapeutics. Many anticancer drugs require specificity of delivery to cancer cells, whilst sparing healthy tissue. Cell-penetrating peptides (CPPs), now well established as facilitators of intracellular delivery, have in recent years advanced to incorporate target specificity and thus possess great potential for the targeted delivery of anticancer cargoes. Though none have yet been approved for clinical use, this novel technology has already entered clinical trials. In this review we present CPPs, discuss their classification, mechanisms of cargo internalization and highlight strategies for conjugation to anticancer moieties including their incorporation into therapeutic proteins. As the mainstay of this review, strategies to build specificity into tumor targeting CPP constructs through exploitation of the tumor microenvironment and the use of tumor homing peptides are discussed, whilst acknowledging the extensive contribution made by CPP constructs to target specific protein-protein interactions integral to intracellular signaling pathways associated with tumor cell survival and progression. Finally, antibody/antigen CPP conjugates and their potential roles in cancer immunotherapy and diagnostics are considered. In summary, this review aims to harness the potential of CPP-aided drug delivery for future cancer therapies and diagnostics whilst highlighting some of the most recent achievements in selective delivery of anticancer drugs, including cytostatic drugs, to a range of tumor cells both in vitro and in vivo.

Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models.

Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.

Design and Synthesis of a Peptide-Based Glioma-Targeted Drug Delivery Vector gHope2.

In the current chapter, we are detailing the synthesis path of a tumor-targeted, CPP-functionalized chemotherapeutic drug, as well as in vitro validation of the targeting and cell penetrating functionalities of the construct. The design of targeted drug delivery vehicle is based on a new glioma-specific homing peptide that has been conjugated to doxorubicin. Further functionalization with an 18-amino acid cell penetrating peptide pVEC was achieved, a CPP that was chosen because of its high cell penetrating efficacy and low toxicity. The three elements were combined into one drug delivery construct gHope2, and its tumor-homing and cell penetrating activity was demonstrated in human glioma cell line U87.

Astrocytes promote ethanol-induced enhancement of intracellular Ca2+ signals through intercellular communication with neurons.

Ethanol (EtOH) abuse induces significant mortality and morbidity worldwide because of detrimental effects on brain function. Defining the contribution of astrocytes to this malfunction is imperative to understanding the overall EtOH effects due to their role in homeostasis and EtOH-seeking behaviors. Using a highly controllable in vitro system, we identify chemical signaling mechanisms through which acute EtOH exposure induces a modulatory feedback loop between neurons and astrocytes. Neuronally-derived purinergic signaling primed a subpopulation of astrocytes to respond to subsequent acute EtOH exposures (SEastrocytes: signal enhanced astrocytes) with greater calcium signal strength. Generation of SEastrocytes arose from astrocytic hemichannel-derived ATP and accumulation of its metabolite adenosine within the astrocyte microenvironment to modulate adenylyl cyclase and phospholipase C activity. These results highlight an important role of astrocytes in shaping the overall physiological responsiveness to EtOH and emphasize the unique plasticity of astrocytes to adapt to single and multiple exposures of EtOH.

Status update in the use of cell-penetrating peptides for the delivery of macromolecular therapeutics.

INTRODUCTION: In this review, recent developments and applications with cell-penetrating peptides (CPP) are discussed. CPPs are widely used tools for the delivery of various macromolecular therapeutics, such as proteins and nucleic acids. AREAS COVERED: The current review focuses on recent important advances and reports that demonstrate high clinical and translational potential. Most important clinical developments have occurred with the CPP-drug conjugate approaches that target various protein-protein interactions, and these have been highlighted subsequently. Most of the applications are targeting cancer, but recently, noteworthy advances have taken place in the field of antisense oligonucleotides and muscular dystrophies, lung targeting, and trans-BBB targeting. EXPERT OPINION: Successful applications and clinical development with the drug conjugate approaches are discussed. On the other hand, the reasons of why the nanoparticle approaches are not as far in development are analyzed.

Intracellular delivery of therapeutic antisense oligonucleotides targeting mRNA coding mitochondrial proteins by cell-penetrating peptides.

Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nano-complexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.

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 future of peptides in cancer treatment.

Peptides hold great potential for the cancer therapy and diagnostics. In the current review, the main and most influential areas of peptide cancer therapeutics are overviewed. These include the development of anticancer peptides, use of peptides for drug delivery, and cancer targeting.

Tumor gene therapy by systemic delivery of plasmid DNA with cell-penetrating peptides.

Gene therapy is a prospective strategy for treating cancer. However, finding efficient and tumor-specific gene delivery vectors remains an issue. Tumor responsive cell-penetrating peptide (CPP) PepFect144 (PF144) has previously been shown to deliver reporter gene encoding plasmid DNA specifically into tumors upon systemic administration, but its capability to reduce tumor growth has not yet been evaluated. Here, we study the potential of PF144-based anti-angiogenic gene delivery to inhibit tumor growth by silencing vascular endothelial growth factor (VEGF) expression in tumors. This approach led to the inhibition of tumor growth in both the HT1080 fibrosarcoma model and orthotopic 4T1 breast tumor model. We additionally saw that the addition of αvß3 integrin targeting did not further improve the tumor sensitive CPPs. Our results suggest that activatable cell-penetrating peptide PF144 is a promising nonviral plasmid DNA delivery vector for cancer treatment.

Chitosan enhances gene delivery of oligonucleotide complexes with magnetic nanoparticles-cell-penetrating peptide.

Gene-based therapies, including the delivery of oligonucleotides, offer promising methods for the treatment of cancer cells. However, they have various limitations including low efficiency. Herein, cell-penetrating peptides (CPPs)-conjugated chitosan-modified iron oxide magnetic nanoparticles (CPPs-CTS@MNPs) with high biocompatibility as well as high efficiency were tested for the delivery of oligonucleotides such as plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA. A biocompatible nanocomposite, in which CTS@MNPs was incorporated in non-covalent complex with CPPs-oligonucleotide, is developed. Modifying the surface of magnetic nanoparticles with cationic chitosan-modified iron oxide improved the performance of magnetic nanoparticles-CPPs for oligonucleotide delivery. CPPs-CTS@MNPs complexes enhance oligonucleotide transfection compared to CPPs@MNPs or CPPs. The hydrophilic character of CTS@MNPs improves complexation with plasmid pGL3, splice correction oligonucleotides, and small-interfering RNA payload, which consequently resulted in not only strengthening the colloidal stability of the constructed complex but also improving their biocompatibility. Transfection using PF14-splice correction oligonucleotides-CTS@MNPs showed sixfold increase of the transfection compared to splice correction oligonucleotides-PF14 that showed higher transfection than the commercially available lipid-based vector Lipofectamine™ 2000. Nanoscaled CPPs-CTS@MNPs comprise a new family of biomaterials that can circumvent some of the limitations of CPPs or magnetic nanoparticles.

A precision oncology approach to the pharmacological targeting of mechanistic dependencies in neuroendocrine tumors.

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

Cell-penetrating peptides for siRNA delivery to glioblastomas.

Delivery of small interfering RNA (siRNA) to suppress glioblastoma growth is a hurdle due to the critical obstacles of the blood-brain barrier and the siRNA properties of such as high negative charges and instability in serum. Therefore, the passage of siRNA to targeted cells is limited. Several siRNA carriers have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells. In this study, two CPPs, PepFect 14 (PF14) and the amphipathic peptide PepFect 28 (PF28), were modified with targeting peptides by covalent conjugation and non-covalent complex formation to improve glioma-targeted specificity and gene-silencing efficiency. In conclusion, we have established an efficient non-covalently complexed carrier (PF14:TG1) for siRNA delivery to human glioblastoma cells (U87), showing a significant two-fold increase in gene-silencing efficiency compared to the parent peptide PF14 and also improved specificity to U87 cells compared to non-glioma targeted cells.

Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.

Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings.

BACKGROUND: A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. METHODS: Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. RESULTS: The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. CONCLUSION: These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

Comparison of Peptide- and Lipid-Based Delivery of miR-34a-5p Mimic into PPC-1 Cells.

The microRNA (miRNA) microRNA-34a (miR-34a) regulates a number of genes involved in cell cycle control and is therefore considered to have a high therapeutic potential. MiR-34a expression is often downregulated in cancer cells and its restoration has been shown to exert a tumor-suppressive effect. However, effective and safe delivery of synthetic miRNA analogs into cancer cells remains a challenge. The aim of this study was to evaluate cell-penetrating peptide PepFect (PF)14 as a carrier for delivery of miR-34a-5p into human primary prostate carcinoma-1 (PPC-1) cells. Using microarray expression analysis, we identified a total of 3,283 (1,744 upregulated and 1,539 downregulated) differentially expressed genes in PF14:miR-34a-5p-transfected cells. In comparison, miR-34a-5p delivery with the commercially available lipid-based reagent siPORT-NeoFX (siPORT) had less robust effects on differential expression and affected fewer genes significantly (90 upregulated and 91 downregulated genes). Functional annotation revealed significant enrichment for downregulated genes in processes and pathways associated with the cell cycle and proliferation regulation in PF14:miR-34a-5p-transfected cells. Five genes (ARHGAP1, AXL, CDC25A, FOSL1, and PDGFRA) were identified and validated as relevant quantitative real-time polymerase chain reaction-based markers of miR-34a-5p transfection efficiency. Our experiments revealed novel potential miR-34a-5p targets and demonstrate that PF14 is a reliable transfection reagent for miRNA mimics characterized by high efficiency and low toxicity relative to lipid-based reagents.