Sébastien Deshayes

Peptide‐based nanoparticles (PBN) have emerged as a promising alternative to lipid nanoparticles (LNP) for nucleic acid delivery and efficient cellular uptake. In this study, we evaluated the formulation of WRAP5 (W‐ and R‐rich amphipathic peptide 5)–based PBN using a microfluidic device and assessed the impact of key process parameters, flow rate ratio (FRR), total flow rate (TFR), and mixing channel design, on nanoparticle characteristics. Across 72 formulations encapsulating small interfering RNA (siRNA) or plasmid DNA (pDNA), dynamic light scattering revealed consistent mean sizes ranging from 50 to 70 nm, with a low polydispersity index (PdI < 0.22), independent of FRR, TFR, or mixer type. Stability studies demonstrated that siRNA‐loaded PBN exhibited moderate size increases during storage at 4°C, whereas pDNA‐loaded PBN remained highly stable for up to 70 days. Biological assays confirmed robust activity: WRAP5:siRNA PBN achieved approximately 50% CDK4 silencing in GIST‐T1 cells, and WRAP5:pDNA PBN mediated efficient mCHERRY expression in HeLa cells, regardless of formulation method or storage duration. These findings highlight the robustness and scalability of WRAP5‐based PBN, contrasting with LNP systems that require stringent control of FRR and TFR, and partially underscore their potential for nucleic acid delivery applications.

Background: Gastrointestinal stromal tumors (GIST) are mainly caused by gain-of-function mutations in KIT or PDGFRA genes and constitute the most common malignant neoplasm of mesenchymal origin. Dysregulation of the Hippo pathway and its downstream effectors YAP1 and TAZ has been implicated in GIST progression, yet their individual contributions remain unclear. Given emerging evidence of non‑redundant YAP1/TAZ functions in cancer, we sought to dissect their respective roles in GIST tumorigenic properties. Methods: We developed sequence‑specific siRNAs targeting YAP1 or TAZ, individually or in combination, and delivered them into GIST-T1, GIST‑430, and GIST‑882 cells using WRAP5 peptide‑based nanoparticles. Silencing efficiency and specificity were confirmed by Western blot, RT-qPCR, and immunofluorescence. Functional assays, including wound healing, Transwell migration, MTT metabolic activity, and cell counting, were used to evaluate proliferation and migration. Downstream transcriptional targets (CYR61, CTGF) and signaling proteins (KIT/AKT/ERK pathways) were quantified. Clinical relevance was assessed using progression‑free survival and metastasis data from the ATGsarc GIST cohort. Results: WRAP5 nanoparticles enabled specific and efficient knockdown of YAP1, TAZ, or both, with no cytotoxicity. Across GIST cell lines, TAZ silencing consistently reduced migration and proliferation, whereas YAP1 knockdown had minimal or cell-line-dependent effects. TAZ depletion markedly downregulated CYR61 and CTGF expression, while YAP1 silencing showed limited impact. CYR61 knockdown impaired proliferation and migration in all models, identifying it as a key mediator of TAZ-driven oncogenicity. Clinical analysis confirmed that high TAZ and high CYR61 expression were associated with shorter disease-free survival and metastasis. Conclusion: TAZ, more than YAP1, acts as a major regulator of GIST proliferation and migration through transcriptional control of CYR61. These findings highlight TAZ as a promising therapeutic target and demonstrate the utility of WRAP5-based nanoparticles for selective gene silencing in GIST.

Background: Gastrointestinal stromal tumors (GIST) are mainly caused by gain-of-function mutations in KIT or PDGFRA genes and are the most common neoplasms of the digestive tract. Imatinib (IM), a tyrosine kinase inhibitor (TKI) targeting these oncogenic drivers, has considerably improved patient outcomes, although resistance remains a major challenge. The transcriptional co-activators YAP1 and TAZ, downstream effectors of the Hippo pathway, have emerged as potential oncogenic drivers in various cancers, including GIST. However, their specific roles in KIT-dependent tumor development remain unclear. Methods: We used WRAP5-based nanoparticles loaded with specifically designed siRNA to selectively silence YAP1 and/or TAZ proteins in KIT-dependent IM-sensitive GIST cell lines. This nucleic acid delivery system enabled efficient and specific knockdown without cytotoxicity. We assessed the impact on cell proliferation, migration, and gene expression, focusing on YAP1/TAZ targets CYR61 and CTGF . Results: TAZ silencing resulted in a substantial reduction in GIST-T1 cell proliferation and migration, whereas YAP1 knockdown was comparatively limited. This finding was consistent with an increased TAZ expression in GIST patients, which was associated with shorter progression-free survival and an increased tendency for metastasis development. A slight additive effect was observed upon a combined YAP1/TAZ silencing in the migration assay, suggesting a more complex regulation between these two proteins. CYR61 and CTGF expressions were predominantly regulated by TAZ, though a stronger downregulation was observed upon dual knockdown in a subset of GIST cell lines with differential YAP1 and TAZ basal expression. Finally, CYR61 seemed to be more implicated in cell proliferation inhibition, which is further supported by the correlation between high CYR61 expression and poor prognosis in the GIST patients. Conclusion: Our results highlight the central regulatory function of TAZ-CYR61 axis in oncogenic processes in KIT-dependent GIST, with a modest contribution from YAP1. Targeting TAZ, alone or in combination with YAP1, may represent a promising therapeutic approach, particularly in the context of tumor heterogeneity.

Cancer remains the leading cause of death, with chemotherapy, radiotherapy, and surgical resection being the primary treatment methods. However, chemotherapy's side effects, surgical limitations, and drug resistance present significant challenges. Small interfering RNA (siRNA) has emerged as a promising tool in cancer therapy due to its ability to silence disease-related genes selectively. Recent advancements in nonviral delivery systems, particularly cell-penetrating peptides (CPPs), have enhanced the efficacy of siRNA delivery. The use of siRNA as a therapeutic tool in cancer treatment has been reported in the literature. However, silencing only one target protein has only a minor effect on tumor cell proliferation, as previously shown for WRAP-based nanoparticles targeting cyclin-dependent kinase 4 (CDK4) in human U87 glioblastoma cells. Here, we designed a more sophisticated approach to enhance therapeutic efficacy, encapsulating multiple siRNAs targeting CDK4, cyclin D1 (CD1), and MCL-1 proteins. The siRNA cocktail, delivered via WRAP5 nanoparticles, effectively silenced these targets and reduced cell proliferation in human U87 glioblastoma cells. Furthermore, the nanoparticles also demonstrated potential therapeutic impact in gastrointestinal stromal tumors (GIST), a rare cancer characterized by its tendency to resist standard treatments. This study highlights the versatility of WRAP5 nanoparticles as a platform for personalized cancer therapy, suggesting that siRNA delivery systems may be tailored to specific cancer types for more effective treatment strategies.

Small interfering RNAs (siRNA) are promising therapeutic molecules that require delivery systems to reach their targets. Several siRNA delivery systems, such as lipid‐ or peptide‐based nanoparticles, are developed for different pathologies. In this context, we previously conceived a cell‐penetrating peptide WRAP5‐forming nanoparticles in the presence of siRNAsand validated the efficiency of this delivery system in inhibiting protein expression. In the pathophysiological context of acute myocardial infarction, which causes a pH drop in the ischemic heart tissue, we optimized the WRAP5‐based nanoparticles for a pH‐sensitive siRNA‐targeted delivery. Therefore, pH‐sensitive acyl hydrazone linkers are used to graft polyethylene (PEG) on the WRAP5 peptide. Proof of concept of the targeted delivery is performed using siRNA silencing the Fas‐associated death domain (FADD)‐containing protein implicated in apoptosis during myocardial ischemia‐reperfusion injury on two human cell models (vascular endothelial cells and hiPSC‐derived cardiomyocytes). The results show that only PEGylated WRAP5 nanoparticles via an appropriate acyl hydrazone linker can induce a specific FADD knockdown at pH 5 compared to naked nanoparticles. These optimized WRAP‐based nanoparticles could be a novel therapeutic tool for treating myocardial infarction by inhibiting apoptosis induced by reperfusion and maximizing local delivery of the nanoparticle content at the site of injured cells.

Cancer remains the leading cause of death, with chemotherapy, radiotherapy, and surgical resection being the primary treatment methods. However, chemotherapy’s side effects, surgical limitations, and drug resistance present significant challenges. Small interfering RNA (siRNA) has emerged as a promising tool in cancer therapy due to its ability to silence disease-related genes selectively. Recent advancements in non-viral delivery systems, particularly cell-penetrating peptides (CPPs), have enhanced the efficacy of siRNA delivery. The use of siRNA as a therapeutic tool in cancer treatment has been reported. However, silencing only one target protein has only minor effects on tumor cell proliferation as previously shown for WRAP-based nanoparticles targeting cyclin-dependent kinase 4 (CDK4) in human glioblastoma cells. Here, we designed a more sophisticated approach to enhance therapeutic efficacy, encapsulating multiple siRNAs targeting CDK4, cyclin D1 (CD1), and Mcl-1 proteins. The siRNA cocktail, delivered via WRAP5 nanoparticles, effectively silenced these targets and reduced cell proliferation in human glioblastoma cells. Furthermore, the nanoparticles also demonstrated potential therapeutic impact in gastrointestinal stromal tumors (GIST), a rare cancer characterized by its tendency to resist standard treatments. This study highlights the versatility of WRAP5 nanoparticles as a platform for personalized cancer therapy, suggesting that siRNA delivery systems may be tailored to specific cancer types for more effective treatment strategies.

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.

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.

Small interfering RNA (siRNA) exhibits a high degree of specificity for targeting selected genes. They are efficient on cells in vitro, but in vivo siRNA therapy remains a challenge for solid tumor treatment as siRNAs display difficulty reaching their intracellular target. The present study was designed to show the in vivo efficiency of a new peptide (WRAP5), able to form peptide-based nanoparticles (PBN) that can deliver siRNA to cancer cells in solid tumors. WRAP5:siRNA nanoparticles targeting firefly luciferase (Fluc) were formulated and assayed on Fluc-expressing U87 glioblastoma cells. The mode of action of WRAP5:siRNA by RNA interference was first confirmed in vitro and then investigated in vivo using a combination of bioluminescent reporter genes. Finally, histological analyses were performed to elucidate the cell specificity of this PBN in the context of brain tumors. In vitro and in vivo results showed efficient knock-down of Fluc expression with no toxicity. WRAP5:siFluc remained in the tumor for at least 10 days in vivo. Messenger RNA (mRNA) analyses indicated a specific decrease in Fluc mRNA without affecting tumor growth. Histological studies identified PBN accumulation in the cytoplasm of tumor cells but also in glial and neuronal cells. Through in vivo molecular imaging, our findings established the proof of concept for specific gene silencing in solid tumors. The evidence generated could be translated into therapy for any specific gene in different types of tumors without cell type specificity but with high molecular specificity.

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.