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Background: Gene therapy can be viewed as a promising/valuable therapeutic approach directed to cancer treatment, including glioblastoma. Concretely, the combination of gene therapy with chemotherapy could increase its therapeutic index due to a synergistic effect. In this context, bovine serum albumin (BSA)-coated temozolomide (TMZ)-peptide (WRAP5)/p53 gene-based plasmid DNA complexes were developed to promote payload co-delivery. Methods: Design of experiments (DoE) was employed to unravel the BSA-coated TMZ-WRAP5/p53 nanocomplexes with the highest potential by considering the nitrogen to phosphate groups ratio (N/P), and the BSA concentration as inputs and the size, polydispersity index, surface charge and p53-based plasmid complexation capacity (CC) as DoE outputs. Results: The obtained quadratic models were statistically significant (p-value < 0.05) with an adequate coefficient of determination, and the correspondent optimal points were successfully validated. The optimal complex formulation had N/P of 1.03, a BSA concentration of 0.08%, a size of approximately 182 nm, a zeta potential of +9.8 mV, and a pDNA CC of 96.5%. The optimal nanocomplexes are approximately spherical. A cytotoxicity assay showed that these BSA-coated TMZ-WRAP5/p53 complexes did not elicit toxicity in normal brain cells, and a hemolysis study demonstrated the hemocompatibility of the complexes. The complexes were stable in cell culture medium and fetal bovine serum and assured pDNA protection and release. Moreover, the optimal BSA-coated complexes were able of gene transcription and promoted a significant inhibition of glioblastoma cell viability. Conclusions: The reported findings instigate the development of future research to evaluate their potential utility to TMZ/p53 co-delivery. The DoE tool proved to be a powerful approach to explore and tailor the composition of BSA-coated TMZ-WRAP5/p53 complexes, which are expected to contribute to the progress toward a more efficient therapy against cancer and, more specifically, against glioblastoma. [ABSTRACT FROM AUTHOR]

The lack of effective delivery systems has slowed the development of mitochondrial gene therapy. Delivery systems based on cell-penetrating peptides (CPPs) like the WRAP (tryptophan and arginine-rich peptide) family conjugated with a mitochondrial targeting sequence (MTS) have emerged as adequate carriers to mediate gene expression into the mitochondria. In this work, we performed the PEGylation of WRAP/pDNA nanocomplexes and compared them with previously analyzed nanocomplexes such as (KH)9/pDNA and CpMTP/pDNA. All nanocomplexes exhibited nearly homogeneous sizes between 100 and 350 nm in different environments. The developed complexes were biocompatible and hemocompatible to both human astrocytes and lung smooth muscle cells, ensuring in vivo safety. The nanocomplexes displayed mitochondria targeting ability, as through transfection they preferentially accumulate into the mitochondria of astrocytes and muscle cells to the detriment of cytosol and lysosomes. Moreover, the transfection of these cells with MTS–CPP/pDNA complexes produced significant levels of mitochondrial protein ND1, highlighting their efficient role as gene delivery carriers toward mitochondria. The positive obtained data pave the way for in vivo research. Using confocal microscopy, the cellular internalization capacity of these nanocomplexes in the zebrafish embryo model was assessed. The peptide-based nanocomplexes were easily internalized into zebrafish embryos, do not cause harmful or toxic effects, and do not affect zebrafish's normal development and growth. These promising results indicate that MTS–CPP complexes are stable nanosystems capable of internalizing in vivo models and do not present associated toxicity. This work, even at an early stage, offers good prospects for continued in vivo zebrafish research to evaluate the performance of nanocomplexes for mitochondrial gene therapy. [ABSTRACT FROM AUTHOR]

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide–transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression—a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma. [ABSTRACT FROM AUTHOR]

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is mainly caused by an oncogenic mutation in the KIT receptor tyrosine kinase. Targeting KIT using tyrosine kinase inhibitors, such as imatinib and sunitinib, provides substantial benefit; however, in most patients, the disease will eventually progress due to KIT secondary mutations leading to treatment failure. Understanding how GIST cells initially adapt to KIT inhibition should guide the selection of appropriate therapies to overcome the emergence of resistance. Several mechanisms have been broadly implicated in the resistance to imatinib anti-tumoral effects, including the reactivation of MAPK signaling upon KIT/PDGFRA targeted inhibition. This study provides evidence that LImb eXpression 1 (LIX1), a protein we identified as a regulator of the Hippo transducers YAP1 and TAZ, is upregulated upon imatinib or sunitinib treatment. LIX1 silencing in GIST-T1 cells impaired imatinib-induced MAPK signaling reactivation and enhanced imatinib anti-tumor effect. Our findings identified LIX1 as a key regulator of the early adaptative response of GIST cells to targeted therapies. [ABSTRACT FROM AUTHOR]

Mitochondria are membrane-bound cellular organelles of high relevance responsible for the chemical energy production used in most of the biochemical reactions of cells. Mitochondria have their own genome, the mitochondrial DNA (mtDNA). Inherited solely from the mother, this genome is quite susceptible to mutations, mainly due to the absence of an effective repair system. Mutations in mtDNA are associated with endocrine, metabolic, neurodegenerative diseases, and even cancer. Currently, therapeutic approaches are based on the administration of a set of drugs to alleviate the symptoms of patients suffering from mitochondrial pathologies. Mitochondrial gene therapy emerges as a promising strategy as it deeply focuses on the cause of mitochondrial disorder. The development of suitable mtDNA-based delivery systems to target and transfect mammalian mitochondria represents an exciting field of research, leading to progress in the challenging task of restoring mitochondria's normal function. This review gathers relevant knowledge on the composition, targeting performance, or release profile of such nanosystems, offering researchers valuable conceptual approaches to follow in their quest for the most suitable vectors to turn mitochondrial gene therapy clinically feasible. Future studies should consider the optimization of mitochondrial genes' encapsulation, targeting ability, and transfection to mitochondria. Expectedly, this effort will bring bright results, contributing to important hallmarks in mitochondrial gene therapy. [ABSTRACT FROM AUTHOR]

Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood–brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma. [ABSTRACT FROM AUTHOR]

Therapeutic peptides have regained interest as they can address unmet medical needs and can be an excellent complement to pharmaceutic small molecules and other macromolecular therapeutics. Over the past decades, correctors and potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel causing cystic fibrosis (CF) when mutated, were developed to reduce the symptoms of the patients. In this context, we have previously designed a CFTR-stabilizing iCAL36 peptide able to further increase the CFTR amount in epithelial cells, thereby resulting in a higher CFTR activity. In the present study, optimization of the peptidyl inhibitor was performed by coupling five different cell-penetrating peptides (CPP), which are Tat, dTat, TatRI (retro-inverso), MPG, and Penetratin. Screening of the internalization properties of these CPP-iCAL36 peptides under different conditions (with or without serum or endocytosis inhibitors, etc.) was performed to select TatRI as the optimal CPP for iCAL36 delivery. More importantly, using this TatRI-iCAL36 peptide, we were able to reveal for the first time an additive increase in the CFTR amount in the presence of VX-445/VX-809 compared to VX-445/VX-809 treatment alone. This finding is a significant contribution to the development of CFTR-stabilizing peptides in addition to currently used treatments (small-molecule correctors or potentiators) for CF patients. [ABSTRACT FROM AUTHOR]

Together with the nucleus, the mitochondrion has its own genome. Mutations in mitochondrial DNA are responsible for a variety of disorders, including neurodegenerative diseases and cancer. Current therapeutic approaches are not effective. In this sense, mitochondrial gene therapy emerges as a valuable and promising therapeutic tool. To accomplish this goal, the design/development of a mitochondrial-specific gene delivery system is imperative. In this work, we explored the ability of novel polymer- and peptide-based systems for mitochondrial targeting, gene delivery, and protein expression, performing a comparison between them to reveal the most adequate system for mitochondrial gene therapy. Therefore, we synthesized a novel mitochondria-targeting polymer (polyethylenimine–dequalinium) to load and complex a mitochondrial-gene-based plasmid. The polymeric complexes exhibited physicochemical properties and cytotoxic profiles dependent on the nitrogen-to-phosphate-group ratio (N/P). A fluorescence confocal microscopy study revealed the mitochondrial targeting specificity of polymeric complexes. Moreover, transfection mediated by polymer and peptide delivery systems led to gene expression in mitochondria. Additionally, the mitochondrial protein was produced. A comparative study between polymeric and peptide/plasmid DNA complexes showed the great capacity of peptides to complex pDNA at lower N/P ratios, forming smaller particles bearing a positive charge, with repercussions on their capacity for cellular transfection, mitochondria targeting and, ultimately, gene delivery and protein expression. This report is a significant contribution to the implementation of mitochondrial gene therapy, instigating further research on the development of peptide-based delivery systems towards clinical translation. [ABSTRACT FROM AUTHOR]

Aims Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction. Methods and results An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Conclusions Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application. [ABSTRACT FROM AUTHOR]

Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVD) including acute myocardial infarction (AMI) rank first in worldwide mortality and according to the World Health Organization (WHO), they will stay at this rank until 2030. Prompt revascularization of the occluded artery to reperfuse the myocardium is the only recommended treatment (by angioplasty or thrombolysis) to decrease infarct size (IS). However, despite beneficial effects on ischemic lesions, reperfusion leads to ischemia-reperfusion (IR) injury related mainly to apoptosis. Improvement of revascularization techniques and patient care has decreased myocardial infarction (MI) mortality however heart failure (HF) morbidity is increasing, contributing to the cost-intense worldwide HF epidemic. Currently, there is no treatment for reperfusion injury despite promising results in animal models. There is now an obvious need to develop new cardioprotective strategies to decrease morbidity/mortality of CVD, which is increasing due to the aging of the population and the rising prevalence rates of diabetes and obesity. In this review, we will summarize the different therapeutic peptides developed or used focused on the treatment of myocardial IR injury (MIRI). Therapeutic peptides will be presented depending on their interacting mechanisms (apoptosis, necroptosis, and inflammation) reported as playing an important role in reperfusion injury following myocardial ischemia. The search and development of therapeutic peptides have become very active, with increasing numbers of candidates entering clinical trials. Their optimization and their potential application in the treatment of patients with AMI will be discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fernandez Rico, Konate, Josse, Nargeot, Barrère-Lemaire and Boisguérin.)

Cell-penetrating peptide (CPP)-based approaches are excellent method for delivering cell-impermeable compounds/therapeutics such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, and drugs, as covalently or noncovalently conjugated cargo into cells. Nowadays, it is generally accepted that cellular internalization of these CPP-cargoes or CPP-nanoparticles occur via endocytosis-dependent mechanisms or by direct cell translocation.Here, we describe a subset of biophysical and biological methods which can be used to dissect the internalization mechanism of CPPs. Presented protocols and results were shown for the recently developed siRNA-loaded WRAP-based nanoparticles. The rapid and efficient cell delivery of WRAP encapsulated siRNA could be attributed to the main direct cellular translocation of the nanoparticles even if, to some extent, endocytosis-dependent internalization occurred.Deciphering the internalization mechanism is still an important requirement to understand and to optimize the action mode of CPPs or CPP-based nanoparticles as transfection reagents., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.